BCH - Institute of Molecular and Cell Biology

FEBS Letters 586 (2012) 2674–2691

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Review

Functional plasticity of the BNIP-2 and Cdc42GAP Homology (BCH) domain in cell signaling and cell dynamics Catherine Qiurong Pan b, Boon Chuan Low a,b,⇑ a b

Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, 117543 Singapore, Singapore Mechanobiology Institute Singapore, 5A Engineering Drive 1, National University of Singapore, 117411 Singapore, Singapore

a r t i c l e

i n f o

Article history: Received 13 March 2012 Revised 16 April 2012 Accepted 16 April 2012 Available online 21 April 2012 Edited by Marius Sudol, Gianni Cesareni, Giulio Superti-Furga and Wilhelm Just Keywords: BCH domain CRAL–TRIO domain GTPase Kinase Modular Scaffold Signaling Protein–protein interaction Dynamics

a b s t r a c t The BNIP-2 and Cdc42GAP Homology (BCH) domains constitute a new and expanding family of highly conserved scaffold protein domains that regulate Rho, Ras and MAPK signaling, leading to cell growth, apoptosis, morphogenesis, migration and differentiation. Such versatility is achieved via their ability to target small GTPases and their immediate regulators such as GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), their ability to form intramolecular or inter-molecular interaction with itself or with other BCH domains, and also by their ability to bind diverse cellular proteins such as membrane receptors, isomerase, caspases and metabolic enzymes such as glutaminase. The presence of BCH and BCH-like domains in various proteins and their divergence from the ancestral lipid-binding CRAL–TRIO domain warrant the need to examine closely their structural, functional and regulatory plasticity in isolation or in concert with other protein modules present in the same proteins. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction 1.1. Modular protein domains defines specificity, integration and crosstalk of signaling networks Protein domains are the autonomous, minimal modular entities that constitute the basic structural or/and functional units of a polypeptide. They can act as protein docking sites, enzymatic units or regulatory devices and are classified into different families according to their conserved primary sequences. Each family of domain recognizes unique interaction motif at their target sites. As such, proteins exploit different architecture designs of modular domains in order to exert common, overlapping or distinct functions, particularly in mediating protein–protein and protein–lipid interactions that form the basis for intracellular and intercellular signaling [1]. As cells sense both biochemical and physical environments, it is important to define how these domains work in isolation and in ⇑ Corresponding author at: Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, 117543 Singapore, Singapore. Fax: +65 6872 6123. E-mail address: [email protected] (B.C. Low).

combination, and how their potential functions are regulated across molecular, cellular and tissue levels. For example, the adaptor protein, Grb2 contains both Src-homology-2 (SH2) and Src-homology-3 (SH3) domains which bridge interaction between the phosphorylated tyrosine residue on the receptor tyrosine kinase and the core PXXP motif of the RasGEF, SOS, respectively, thereby transducing signals upon binding of ligands at the extracellular space to intracellular milieu [2]. Precise molecular recognition is also important for transcriptional regulation. For example, under the HIPPO signaling which is crucial for the control of organ size and tumor suppression, the LATS kinase via its PPxY motif, targets the WW domain of the transcriptional regulator, Yes associated protein (YAP) to phosphorylate YAP, leading to sequestration of YAP in cytoplasm by protein 14-3-3. This mechanism helps reduce the YAP-induced cell proliferation and tumor suppression [3]. Similarly, in protein trafficking and targeting, interaction between the pleckstrin homology (PH) domain of the molecular motor, dynamin and phosphoinositide is essential for endocytosis [4,5]. To further enhance the efficiency of multi-component signaling networks, cells employ specific scaffold proteins that utilize their multiple protein domains to recruit and organize different targets into higher order macromolecular complexes at specific location

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.04.023

C.Q. Pan, B.C. Low / FEBS Letters 586 (2012) 2674–2691

and to fine-tune the activity and crosstalk among them. For examples, the activity of Raf-Mek-Erk or Mek-Erk nodes downstream of Ras is supported by different scaffold proteins at different locales, namely the KSR (cell membrane), MP1 (late endosome), Sef (Golgi), paxillin (focal adhesions), IQGAP1 (cytoskeleton) and b-arrestin (early endosome) [6]. Such distribution of signaling nodes by distinct hubs ensure localized and more specific control in the activation (and deactivation) of Erk during different cellular processes. Last but not least, coordination of cell adhesion dynamics require the FERM (four-point-one, ezrin, radizin, moesin) domain of FAK (focal adhesion kinase) interacting with phospholipids PtdIns(4,5)P2 at the cell membrane to release the autoinhibition in FAK. This facilitates the recruitment of Src tyrosine kinase and formation of focal adhesion complex via integration of many more scaffold proteins, kinases, phosphatases, GTPases and their immediate regulators [7,8]. Therefore, precise locales and timing for the molecular recognition by different combinations of protein domains in cis and in trans will help define the specificity, connectivity, crosstalk and feedback among various signaling nodes. Acting in concert, they regulate important functions such as cytoskeleton rearrangement, cell–cell adhesion, cell–matrix interaction, protein trafficking, organelle and membrane dynamics, cell metabolism and immune response, gene expression and protein synthesis and stability, leading ultimately to cell growth, cell death, cell differentiation, cell migration and invasion. 2. BCH domain as an emerging regulatory scaffold domain 2.1. Discovery of the prototypical BCH domain from BNIP-2: from receptor tyrosine kinase to small GTPase, MAP kinase and beyond To understand the fundamental mechanism in cell signaling it is important to identify what and how one particular signaling protein forms its protein interaction networks and how this process is regulated in vitro and in vivo. In our effort to decipher how activation of the fibroblast growth factor receptor (FGFR) tyrosine kinase could lead to novel signaling pathway, yeast 2-hybrid was employed and BNIP-2 was identified as a candidate partner for the cytoplasmic tail of the receptor [9]. Interestingly, only the kinase-dead mutants of the FGFR could ‘‘trap’’ and co-immunoprecipitate with BNIP-2, indicating that their interaction was very transient and BNIP-2 was likely to be a substrate of the receptor. Further in vitro kinase assays and detection with phosphotyrosine antibody confirm BNIP-2 as a bone fide substrate of the FGFR both in vitro and inside the cells [9]. BNIP-2 was first identified as one of the three interacting partners for the anti-apoptotic Bcl2 and viral E1B 19 kDa protein, hence named Bcl-2 and Nineteen kilodalton-Interacting Protein-2, BNIP-2 [10]. However, its precise biological functions remained unknown then. Our further sequence analyses revealed 61% similarity between the C-terminus of BNIP-2 with the N-terminus, non-catalytic region of Cdc42GAP (p50RhoGAP or ARHGAP1) and named the region BNIP-2 and Cdc42GAP Homology (BCH) domain (Fig. 1; [9,11,12]). As this domain is associated with the Rho GTPase-activating domain (GAP) domain of Cdc42GAP, we speculated that it could be linked to function and regulation of GTPases signaling. Among key molecular switches, Ras and Rho small GTPases control cell signaling and cytoskeleton networks by cycling between their inactive GDP-bound form and active GTP-bound state [13]. This process is tightly regulated by their guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP [14] or by their GTPase-activating proteins (GAPs) that help hydrolyze the GTP to GDP [15]. Although the biochemical functions of GTPases, GEFs and GAPs are well established and their deregula-

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tion often lead to developmental defects and disease, less is known about how this ‘‘3G’’ (GTPase, GEF, GAP)-signalome itself is regulated by cellular factors and how their functions and regulation are linked to other signaling networks downstream of membrane receptor and MAP kinase modules. We therefore hypothesized that BCH domain in BNIP-2 and Cdc42GAP could serve as a binding platform for GTPases and possibly their immediate regulators, or they could form multimeric form of BNIP-2, as sometimes seen for other homologous domains used in protein dimerization/ oligomerization. Based on structural modeling, site-directed mutagenesis, binding studies and GTPase assays, we revealed that BCH domain of BNIP-2 indeed binds to itself and the homologous BCH domain of Cdc42GAP via a unique motif, 217-RRKMP-221 [11]. Interestingly, BNIP-2 BCH domain also binds Cdc42, a partner of the Cdc42GAP, via a sequence motif similar to the CRIB (Cdc42/Rac1-Interactive Binding) motif commonly present in the effector proteins of Cdc42/Rac1 [16,17] (see Fig. 3A and C for details later). Unlike binding of CRIB motif which is absolutely GTP-dependent (in order to allow activation of effectors pathway), the BCH domain of BNIP-2 does not distinguish between the unloaded, GDP and GTP-bound form of Cdc42 in vitro [9]. However, it binds preferentially to the dominant negative form of Cdc42, T17N but not with the constitutively active form, G12V [12]. Subsequently, we showed that these profiles are also observed in binding of Rho by the other homologous BCH domains of BNIP-S [33], BNIP-XL [18] and Cdc42GAP [19] and are distinct from that conferred by the GTPase-binding domains of GEFs and GAPs [14,15,20]. Furthermore, BNIP-2 appeared to promote the inactivation of Cdc42 by stimulating the apparent GTPase activity of Cdc42 in vitro and such effect is abrogated upon binding by FGFR [9] or negatively regulated by the homophilic interaction of BNIP-2 or its heterophilic interaction with Cdc42GAP [11]. Since then, by using yeast 2-hybrid, proteomics-based mass spectrometry and candidate approaches, we and others have expanded the family of BNIP-2 interactomes. We have further delineated how some of these BCH domains could in fact either activate or inactivate specific Rho GTPases signaling depending on various cellular contexts the proteins are present, and they could directly or indirectly control multiple signaling and metabolic pathways. These processes involve specific members and conformation of Rho and Ras family of small GTPases and selective GEFs and GAPs, growth promoting FGF receptor tyrosine kinase, myogenic Cdo receptor and the associated JLP/p38-MAP kinase, Mek2, microtubule-based kinesin motor, anti-apoptotic Bcl2, peptidyl prolyl-isomerase Pin1 and glutaminase, a metabolic enzyme essential for cell growth and neuronal differentiation (Fig. 1). Moreover, BCH domains can undergo auto-inhibitory intramolecular interaction and form oligomer with identical BCH domains (homophilic interaction) or with other homologous BCH domains (heterophilic interaction). They also collaborate with other protein modules and their interactomes on the same proteins (in cis) or with different proteins (in trans) in order to ensure faithful signaling integration and crosstalk. The basis for such plasticity for BNIP-2 and several BCH-containing proteins will be further highlighted and discussed below in the context of their versatile binding capabilities through their multiple binding motifs, their dynamic disposition within the cells, their synergism (or antagonism) with other protein domains in cis or in trans, their potential regulation by transcription, alternative transcription start sites, alternative RNA splicing and post-translational modifications, and their molecular evolution which could shed further lights on the common and divergent properties of BCH domains with other protein modules.

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1

167

BNIP-2

314 1

72

205 439

GTPases, GEFs, GAPs

BCH

glutaminase

FGFR

CDC42GAP (p50RhoGAP / ARHGAP1)

Cdo receptor

Pin1 Bcl2

morphogenesis, motility, differentiation, trafficking, apoptosis

Fig. 1. The BCH domain and its expanding protein interaction networks. Regions in blue depict the conserved domains between the C-terminus of BNIP-2 and the N-terminus of Cdc42GAP, hence named BNIP-2 and Cdc42GAP Homology (BCH) domain. This novel protein module which is present in diverse proteins can interact with Rho and Ras small GTPases, their GEFs and GAPs, membrane receptors, glutaminase, Pin1, E1B-19 kDa protein, Bcl2 and other cellular proteins where they regulate diverse signaling pathways, leading to changes in cell morphology, motility, differentiation, protein trafficking and apoptosis. BCH domain can also interact with itself (homophilic) or with other homologous BCH domains (heterophilic). For clarity, proteins that bind outside the BCH domains (heterophilic) are not shown here although they could co-regulate specific processes with the interactome of BCH domains.

2.2. Multiplicity and versatility of BCH domain-containing proteins 2.2.1. BCH domain defines a distinct functional subclass of the CRAL/ TRIO superfamily Although initially identified as a region of high protein sequence homology between BNIP-2 and Cdc42GAP, BCH domain also shares 14% sequence identity with the CRAL_TRIO domain, the lipophilic domain of the Saccharomyces cerevisiae Sec14p protein, suggesting that both domains could have evolved from a common ancestral sequence. CRAL_TRIO domain was first identified in cellular retinaldehyde binding protein (CRALBP) and Trio (a RhoGEF) [21]. Other proteins such as tyrosine phosphatase [22], atocopherol transfer protein [23], RasGAP neurofibromatosis type1 protein (NF1) and several other RhoGEFs such as Dbl, Duo, Dbs, Kalirin [24], all are thought to possess similar protein domains. However, each member binds diverse small hydrophobic ligands. For examples, the CRAL_TRIO domain of Sec14L group binds phosphotidylinositol, aTTP has affinity for tocopherol and CRALBP binds retinaldehyde [25]. The ligand specificities of the RhoGEFs and RasGAP groups (represented by NF1 proteins) is currently unknown. However, unlike the BCH domains, none of the CRAL–TRIO domains is known to be associated with any specific protein–protein interaction. Likewise, BCH domains are not known to interact with any lipid molecules inside the cells. This observation raises several important questions. How do BCH domains evolve and attain such versatile functions? Do BCH and CRAL–TRIO domains still share some of their ancient features and exhibit lipids binding and protein–protein interaction capabilities? We have recently conducted extensive genome-wide, crossspecies analyses with 100 CRAL_TRIO and similar domains, including the putative BCH sequences, from eight representative species and compared the pattern of their sequence conservation, gene structure, including the exon/intron evolution, and examined the possible structural folds of BCH domain based on computational modeling [26]. The results reveal a clear distinction of BCH domains as a novel subclass within the CRAL_TRIO superfamily and highlight several unique differences between the two. To date, BCH domains have been identified in at least 175 distinctive proteins from the slime molds, plants, yeasts, insects, fish to human,

adding to its evolutionary importance and diverse functionality [26]. BCH domain is completely absent in prokaryotes and the most primitive BCH domain can be identified from slime mold (Dictyostelium), coanoflagellate (Monosiga), alveolates (Plasmodium, Cryptosporidium), green alga (Chlamydomonas) and yeast. Similarly, CRAL_TRIO domains are identified in many lower species of alveolates. This indicates that BCH domains evolved from their ancestors more than 1500 Mya ago with the appearance of Protists [26,27]. These BCH-containing proteins can be divided into three unique subgroups, namely those associated with BNIP-2 type sequence (Group I), with macro-domain (Group II) and with RhoGAP protein domains (Group III) (Fig. 2). And most significantly, all these BCH domains contain a hallmark signature motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs, where ‘h’ refers to any large and hydrophobic residue and ‘s’ is small and weakly polar residue; Ala, Thr, Gly, Ser. This motif is absent from any of the CRAL–TRIO domains, including those from Trio and NF1. Trio and NF1 are two members of the CRAL–TRIO group that are most closely related to the BCH domains (Figs. 2 and 3A and B). Further analyses of the gene-structure and protein domain context indicate that BCH domain-containing genes have evolved through gene duplication, intron insertions and domain swapping events and this divergence could have occurred as early as the appearance of protists during evolution. Interestingly, sequences coding for BCH domains can undergo alternative RNA splicing, leading to splicing variants of BNIP-2, BNIP-2-Similar [28] and BNIP-2 Extra Long [18] from the Group I. At least 4 splicing variants are also identified in BCH domain-containing, proline-rich RhoGAP protein, BPGAP [29] from the Group III. Interestingly, conservation of certain alternatively spliced sequence such as ‘‘YEEEKFKKRQKR’’ in BNIP-2b appears to be highly similar to a sequence present in BNIP-2 Homology (BNIP-H, or Caytaxin; [30,31] and this sequence is somewhat less similar to that of the corresponding region in BNIP-XLa (Fig. 2). Interestingly, in addition to alternative RNA splicing, different transcriptional initiation sites are also used by the gene locus BMCC1/PRUNE2 to encode three more extended isoforms of BNIP-XL (see details later). All these dynamic features are likely to confer different properties


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Fig. 2. Domain architecture and classifications of BCH domain-containing proteins. Domain architecture for the Group I, Group II and Group III type BCH-containing proteins as defined by the presence of single-BCH, or BCH domain that is associated with the macro and RhoGAP domains, respectively. The percentages indicate the extents of amino acid sequence identities compared to the prototypical BNIP-2 BCH domain. Together, they define a distinct subclass of proteins that could have evolved from a common ancestral module shared with primarily lipid-binding CRAL–TRIO protein domain. Shown also are amino acid sequence inserts within the BCH domains that arise from their alternative RNA splicing. Only some of the splicing variants are represented here.

between the isoforms and also between the homologs, further enriching for the functional plasticity among the BCH domains. Further sequence analyses also revealed significant conservation in two GTPase-binding motifs, that are similar to the Rhobinding domain (RBD) and the Cdc42/Rac interactive binding (CRIB) domain commonly found in effector proteins of Rho and

Cdc42/Rac1, respectively (Fig. 3A, C and D). Among various BCH domains, the BCH domain of BNIP-2 has been validated to contain a novel Cdc42-binding motif (285-VPMEYVGI-292) within its CRIBlike region ([32]; Fig. 3C). Likewise, the RBD-like motifs in BNIP-S [33], BNIP-XL [18] and Cdc42GAP [19] have also been experimentally validated (Fig. 3D). Indeed, these GTPase-binding motifs play

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Fig. 3. Multiple sequence alignment and phylogenetic relationship among BCH domains. (A) Multiple sequence alignment of various BCH domains from Homo sapiens BNIP2a (NP_004321), BNIP-XLa (AY439213), BNIP-H (AAO63019), BNIP-Sa (AY078983), BPGAP1 (AF544240), Cdc42GAP/p50RhoGAP (Q07960), GDAP2 (CAI22738), TRIO (AAC34245), NF1 (P21359) and Saccharomyces cerevisiae Sec14p lipid-binding domain (P24280) were generated using ClustalW (http://www.ebi.ac.uk/Tools/services/web/ toolform.ebi?tool=clustalw2) and displayed by BOXSHADE. (B) Phylogenetic tree of the BCH domains from Homo sapiens BNIP-2a (NP_004321), BNIP-XLa (AY439213), BNIPH (AAO63019), BNIP-Sa(AY078983), BPGAP1 (AF544240), Cdc42GAP/p50RhoGAP (Q07960), GDAP2 (CAI22738), TRIO (AAC34245), NF1 (P21359) and Saccharomyces cerevisiae Sec14p lipid-binding domain (P24280), were generated using ClustalW as above. (C, D) Multiple sequence alignment of BCH domains from Homo sapiens BNIP-2a (NP_004321), BNIP-XLa (AY439213), BNIP-H (AAO63019), BNIP-Sa (AY078983), BPGAP1 (AF544240), Cdc42GAP/p50RhoGAP (Q07960), GDAP2 (CAI22738), TRIO (AAC34245) with the CRIB motif (C) of Homo sapiens PAK1 (AAH50377), Saccharomyces cerevisiae STE20 (Q03497), Candida albican (CLA4 AAAB68613) or with the RBD (Rho-binding domain) motif (D) of Rhophilin-1 (NP_443156), PRK1/PKN (NP_002732) and Rhotekin (AAH17727). The requirement for the CRIB-like motif of BNIP-2 (highlighted in green box in Figure A), RBD-like motifs of BNIP-XL, BNIP-Sa and Cdc42GAP (highlighted in red box in Figure A) and the BCH-BCH binding motifs of BNIP-2 and BNIP-S (highlighted in brown box in Figure A) had been validated by experiments [18,19,32,33]. A cryptic motif ‘‘285-VPMEYVGI-292’’ that is critical for binding to Cdc42, is also shown in red box in Figures A and C. All sequence alignments described above were generated using the default setting with protein weight matrix Gonnet, gap open 10, gap extension 0.2, gap distance 5, iteration none and with NJ clustering.


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Fig. 3 (continued)

important roles in mediating cell morphogenesis, cell migration and cell differentiation, as will be discussed in details next. Interestingly, although all BCH domains confer BCH/BCH homophilic and heterophilic interactions, it appears that different sets of motifs are employed for different groups. For example, the motif ‘‘RRKMP’’ used on BNIP-2 [11] is only conserved in BNIP-H and BNIP-XL, but it is absent from BNIP-S, Cdc42GAP and BPGAP1 (Fig. 3A). Indeed, mutating the corresponding region in Cdc42GAP failed to disrupt its ability to form homophilic and heterophilic complex [11]. BNIP-S, on the other hand, utilizes a separate motif 215-ATWYVKA-221 for its homophilic interaction (Fig. 3A) whereas it binds to Cdc42GAP at a unique GAP-Binding Motif (aa 133–147) that overlaps with the Rho-binding region within an extended RBD-like motif (aa 133–177) [33]. Taken together, BCH domains have evolved to acquire multiple functional motifs that contribute to its functional versatility. 2.2.2. The BNIP-2 and BPGAP family proteins and their interactomes – mechanisms of action and regulation To further explore the extents of distribution, function and regulation of BCH domain-containing proteins, several novel sequences encoding multiple homologs of BNIP-2 were identified from human and other species and their biochemical and cellular functions delineated. In the following sections, we will highlight how BNIP-2, BNIP-S, BNIP-XL, BNIP-H, BPGAP1 and Cdc42GAP employ their unique functional motifs in their BCH domains to engage multiple targets and how they act in concert with other protein domains to give rise to emerging functions and properties (Table 1 and Fig. 4). For examples, in the context of 3G-signalome, the BCH domain of BNIP-2 induces cell protrusions by activating Cdc42 via a yet unidentified regulator [32] or acting as a scaffold for the activation of Cdc42 that promotes muscle differentiation upon coupling to the active myogenic Cdo receptor and p38-MAPK activation [34] (Fig. 4A); whereas the BCH domain of Cdc42GAP

sequesters RhoA from inactivation by the adjacent GAP domain, thus preventing cell rounding and retraction [19] (Fig. 4E). In contrast, the BCH domain of BNIP-Sa promotes cell rounding and apoptosis by activating RhoA while simultaneously preventing RhoA from inactivation by Cdc42GAP by forming a heterophilic BCH/BCH complex [33] (Fig. 4D). In comparison, the BCH domain of BNIP-XL inhibits cell transformation by sequestering oncogenic Lbc RhoGEF from activating RhoA [18] (Fig. 4C). Extending this, the BCH domain of the brain-specific BNIP-H/Caytaxin promotes neurites outgrowth by acting in concert with the other non-BCH entity as a scaffold to traffic glutaminase on a kinesin motor and towards the tip of differentiating neurons [30,35] (unpublished data) (Fig. 4B). Last, but not least, the BCH domain of BPGAP1, a homolog of Cdc42GAP, promotes ERK activation and cell migration by collaborating with the RhoGAP domain and its proline-rich motifs that interact with cortactin [36], endophilin-2 [37], Mek2 and Pin1 isomerase [38] (Fig. 4F). The mechanistic action and the possible regulation for these versatile processes are further discussed below. 2.2.2.1. BNIP-2 with morphogenesis, cell growth, apoptosis and differentiation. Since the earlier work that described BNIP-2 as a partner for the pro-survival Bcl2 and E1B 19 kDa [10], we and others have delineated the biochemical and cellular functions of BNIP2 as a core regulatory protein in multiple signaling gateways, including the FGF receptor tyrosine kinase [9], Cdo receptor [34], Cdc42 [9,12,32], Cdc42GAP [9,11,32] which all engage its BCH domains (Fig. 5A). In particular, expression of BNIP-2 in epithelial and fibroblast cells induce extensive changes in cell morphology and membrane protrusions by targeting and activating Cdc42 with its unique Cdc42-binding motif 285-VVMEYVGI-292 present within the core CRIB-like motif of the BCH domain ([32]; Fig. 5A). An adjacent region to this motif is also required for the concerted activation, but the target protein, believed to be a GEF that help

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Table 1 BCH domain as a multifunctional regulatory scaffold domain. BCH domain-containing proteins regulate diverse signaling pathways and functions by utilizing their conserved BCH domains and their interplay with other associated domains. Proteins BNIP-2 family BNIP-2 (Bcl2 and 19 kDa interacting protein-2)

BNIP-H (BNIP-2 Homology; CAYTAXIN)

BNIP-XL (BNIP-2 Extra Long; BMCC1-isoform 4)

Target proteins

Functions

Regulation

Targets of BCH domain: E1B 19 kDa and Bcl2 [10]; Cdc42 [11,12,32], BNIP-2 [11] and Cdc42GAP (p50RhoGAP) [11], Cdo receptor [34]; FGF receptor-1 [9]; Rho and RhoGEF (unpublished data) Targets of other domain: caspases [41], granzyme B [42]

(1) Binding to viral E1B 19 kDa protein and Bcl2 promotes cell survival [10] (2) Substrate of FGFR [9]. Its C-terminus shares high sequence homology with the N-terminus of Cdc42GAP, hence termed BCH domain [11] (3) BCH domain binds Cdc42 via a CRIBlike motif and leads to drastic cell elongation and membrane protrusions where it is localized to [32]. It also forms BCH/BCH complex with itself or Cdc42GAP via a different motif [11] (4) Promotes myogenic and neuronal differentiation by acting as a scaffold that links Cdo receptor to activation of Cdc42, leading to JLP-scaffolded activation of p38a/b [34,40] (5) Human BNIP-2 is cleaved by caspases 3, 6, 8, 10 in vitro, or by caspases 3, 8 and 9 in cells [41] (6) Also subjected to cleavage by granzyme B [42] (7) Contains a putative kinesin-binding motif for intracellular trafficking; possibly acting as scaffold for cargo trafficking [35] (8) Retards MDCK epithelia cell spreading and collective cell migration by activation of Rho (unpublished data) (9) Binding to BPGAP1 enhances its GAP activity towards Rho and reducing cell proliferation (unpublished data)

(1) Homophilic or heterophilic interactions via BCH domains affects Cdc42 activity [11] (2) Phosphorylation by FGFR disrupts its binding to Cdc42 and Cdc42GAP [9] (3) Estrogen treatment reduces BNIP-2 expression and its pro-apoptotic activity [43] (4) Cleavage by caspases and granzyme B might lead to the release of pro-apoptotic BCH-containing fragment [41,42] (5) Splicing within BCH domains generates BNIP-2a and BNIP-2b with potentially different properties and functions (6) Contextual signaling: versatility in engaging different Rho GTPases and their GAPs or GEFs, leading to context-dependent regulation of GTPase signaling

Targets of BCH domain: glutaminase KGA [30], peptidyl–prolyl isomerase Pin1 [55] Targets of other domain: kinesin-1 light chain [35], caspases 3, 7 [57], kinesin-1 heavy chain (unpublished data) ⁄⁄ Binding sites of Mek2 [55] and Ubiquitin E3 ligase CHIP [56] have not been mapped

(1) Specific expression in brain, especially in the cerebellum and hippocampus [30] (2) Mutation in ATCAY gene leads to Cayman ataxia characterized by hypotonia, mental dysfunction and cerebellar ataxia [31] (3) Promotes neuronal differentiation and relocalises glutaminase to neurite terminals and regulates glutamate production [30] (4) Transports mitochnodria on kinesin-1 [35] (5) As an adaptor/scaffold for transporting KGA on kinesin-1 along microtubules of neurites (unpublished data) (6) Binds E3 ligase CHIP and undergoes polyubiquitination [56] (7) As a presynapse substrate of caspases3 and -7. Contains 102-DETD-105 cleavage motif, releasing BCH fragment that might regulate Mek2 signaling [57] (8) Interacts with Pin1 upon NGF stimulation or stimulated by active Mek2; Pin1 disrupts the binding between BNIP-H and glutaminase [55]

(1) Intramolecular inhibition that is released by NGF stimulation or active Mek2 [55] (2) Binding to CHIP (E3 ligase) and polyubiquitination is likely for degradation [56]

Targets of BCH domain: RhoA (strongest binding for T19N, F30L but no binding to G14V and Q63L), RhoC but not RhoB [18]; BCH domain interacts with DH–PH domain of Lbc RhoGEF and possibly p115RhoGEF also [18] Targets of other domain: Multiple caspases [41] Lbc binds to N-terminus and BCH domain of BNIP-XL via its proline region and DH–PH domains, respectively [18]

(1) One of the 4 splicing variants encoded by the BMCC1 gene (also known as PRUNE2). BNIP-XL (BMCC1 isoform4) itself also undergoes alternative splicing within BCH domain, generating BNIP-XLa and BNIP-XLb [18] (2) Associated with neuronal apoptosis and is expressed in brain, spinal cord and dorsal root ganglia; predominantly in the neurons of the cranial nerve motor nuclei and motor neurons of the spinal cord [66]

(1) Alternative splicing encoded from the BMCC1 locus; further splicing within BCH domain generates BNIP-XLa and BNIP-XLb (2) BMCC1 expression is downregulated after NGF-induced differentiation but upregulated during the NGF-depletion-induced apoptosis [66] (3) Subjected to cleavage by caspases [41] Implicated in disease: (1) BMCC1-3 is a good prognosis marker for neuroblastomas in children [66]

Implicated in disease: Mutation in ATCAY gene leads to Cayman ataxia [31] and is also linked to the ‘‘jittery’’, ‘‘hesitant’’ and ‘‘sidewinder’’ ataxia models in mice [46,47]

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C.Q. Pan, B.C. Low / FEBS Letters 586 (2012) 2674–2691 Table 1 (continued) Proteins

BNIP-S (BNIP-2 Similar; BNIPL)

RhoGAP Family Cdc42GAP (p50RhoGAP;ARHGAP1)

BPGAP1 (BCH containing, proline-rich RhoGAP1;ARHGAP8)

Target proteins

Functions

Regulation

BMCC1/PRUNE2 binds GTP, 8-oxoGTP and putative protein targets identified but not yet validated [69]

(3) BNIP-XL restricts binding of RhoA with its RhoGEF Lbc, thus reducing RhoA activation and cellular transformation [18] (4) Also binds to p115RhoGEF but the function is unknown [18] (5) Cleaved by caspases 3,6, 7, 8, 9 and 10 in vitro, or by caspases 8 and 9 in cells [41] (6) Binds GTP and 8-oxo-GTP and several putative protein targets [69], and also microtubule–associated protein, MAP6 [93]

(2) BMCC1-1 is identified as a susceptibility gene for Alzheimer’s disease [71] and BMCC1-2 as a biomarker for leiomyosarcomas [72] (3) BMCC1-3 and BNIP-XL (BMCC1-4) is proapoptotic protein in neuronal cells [66] and an antagonist of Rho-mediated cellular transformation [18], respectively

Targets of BCH domain: RhoA (stronger preferences for T19N but no binding to G14V) [33], Bcl2 [74], BNIP-S [28], BNIP-2 [28], Cdc42GAP [28,33,74], proto-Lbc RhoGEF [18] ⁄⁄ Binding sites of the cellproliferation-related proteins MIF and GFER have not been mapped [73]

(1) Induces apoptosis by targeting Cdc42GAP and RhoA with its BCH domain. This disrupts Cdc42GAP and RhoA association thereby increasing RhoA activation for cell rounding and apoptosis [33] (2) BCH domain of BNIPL-2 interacts with Bcl2 and Cdc42GAP during apoptosis [74] (3) BNIPL-2 promotes invasion and metastasis via Cdc42 activation and upregulation of CD44 in human hepatocellular carcinoma [75]

(1) Splicing variants, BNIP-Sa and BNIPSb. BNIP-Sa induce cell rounding and apoptosis whereas BNIP-Sb is localized to the nucleus and has no effect on cell morphology [28]

Targets of BCH domain: RhoA [19], Rab5 [89], Rab11 [89], BNIP-Sa [28,33] ⁄⁄ Binding sites of Nudel has not been mapped [92]

(1) GAP for Cdc42 and Rho [77,81,83,85] (2) BCH domain acts as a local modulator to sequester RhoA from inactivation by its adjacent RhoGAP domain [19] (3) Target to endosome via BCH domain and form complex with Rab11 and Rab5 [89] (4) Nudel sequesters Cdc42GAP at the leading edge of the cell to increase active Cdc42 in presence of phosphorylated ERK [92] (5) Promotes cell migration [81,82] (6) Suppress muscle cell differentiation by inactivating Cdc42 [34]

(1) Auto-inhibition [88] (2) Sequestered by BNIP-Sa from inactivating RhoA, leading to cell rounding and caspase-independent apoptosis [33] Implicated in disease: (1) Up-regulated in Waldenstrom macroglobulinemia [79] (2) Homozygous knockout embryo and newborn mice have reduced organ and body size, due to elevated JNKmediated apoptosis [81,83,84]

Targets of BCH domain: RhoA, BNIP-2 BCH domain; K-Ras and SmgGDS (unpublished data) Targets of other domain: Cortactin [36], Endophilin 2 [37], Pin1 [38], Mek2 [38] ⁄⁄ Binding sites of Cdc42 and Mek2 have not been mapped

(1) Induces membrane protrusions and cell migration via interplay of BCH domain, GAP domain and proline-rich region [29] (2) Induces motility by translocating Cortactin to cell periphery [36] (3) Induces EGFR endocytosis and ERK activation by coupling to endophilin2 [37] (4) BCH induces chronic K-Ras/Erk activation and PC12 neuronal differentiation that is suppressed by another Ras modulator, SmgGDS (unpublished data) (5) BNIP-2 promotes BPGAP1’s GAP activity towards RhoA and downregulates cell proliferation (unpublished data)

(1) Splicing variants, BPGAP1–4 [29] (2) Intramolecular inhibition at prolinerich region, released by active Mek2 acting as scaffold to promote interaction between Pin1 and BPGAP1 and suppresses BPGAP1-induced cell motility and ERK activation [38] Implicated in disease: Upregulated in primary colorectal tumors [78] and cervical cancer [80]

activate Cdc42, remains elusive. Although BCH domains are highly conserved across different members, this Cdc42-binding motif for BNIP-2 is only conserved in BNIP-XL and BNIP-H (Fig. 3C). However, no binding of Cdc42 have been observed for these two proteins, indicating that unique structures surrounding this motif could play an important role in the substrate recognition. BNIP-2 utilizes another distinct motif 217-RRKMP-221 to mediate its homophilic and heterophilic BCH/BCH interactions. The homophilic interaction of BNIP-2 or its heterophilic binding with Cdc42GAP could affect their ability to promote the inactivation of Cdc42 in vitro, although such interactions do not affect their binding to Cdc42 [11]. Interestingly, such interaction as well as its

interaction with Cdc42 are abrogated upon binding and phosphorylated by FGF receptor [9]. Although the BCH domain of BNIP-2 appears insensitive towards either the GDP-bound or GTP-bound form of Cdc42, its strong preference for the dominant negative form of Cdc42–T17N and its near-complete loss of binding with the constitutive active form, Cdc42–G12V [12] support the absolute requirement of Gly-12 to promote their interaction, rather than depending on the activity per se. The ability of BCH domains to still recognize either active or inactive form of Cdc42 (and Rho in other examples) appears to be consistent with its scaffold function to bring any forms of GTPase and their immediate regulators in context-dependent manner. The significance of this recurring

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A

B

C

D

F

E

Fig. 4. Functional plasticity and mechanisms of action by the BCH domains. BCH domains are involved in targeting small GTPases, their GAPs, GEFs, signaling receptors, cargo enzymes or other cellular proteins to elicit diverse cellular responses. These are shown by the following models, depicted clockwise. (A) BNIP-2 induces extensive cell elongation and membrane protrusions in single epithelial cells via the BCH domain binding to Cdc42 [32]. It also acts as a scaffold protein that couples the promyogenic receptor, Cdo and activation of Cdc42 with the activation of p38a/b MAPK on the scaffold, JLP. The alliance of these two scaffold proteins promotes myogenic differentiation [34]. (B) BNIP-H (or Caytaxin) promotes neurite outgrowth by engaging its BCH domain as a scaffold/adapter to traffic and localize KGA (kidney-type glutaminase) towards the neurites termini [30]. BNIP-H itself can be trafficked by kinesin motor in hippocampus [35] supporting our observations that BNIP-H can act as an adaptor for transportation of cargo proteins or signaling complexes along neurites (unpublished data). (C) BNIP-XL suppresses stress fiber formation and cellular transformation by engaging its BCH domain to sequester RhoA from activation by RhoGEF, proto-Lbc; The N-terminal region of BNIP-XL interacts with the proline-rich region of Lbc. These concerted interactions reduce the level of active RhoA, leading to disruption of stress fibers and Lbc-induced cell transformation [18]. (D) BNIP-Sa induces cell retraction, cell rounding and apoptosis, all via its BCH domain sequestering RhoA and also by displacing Cdc42GAP/p50RHoGAP from inactivating RhoA via its BCH/BCH interaction with Cdc42GAP. Together, such mechanisms sequester RhoA from immediate inactivation by Cdc42GAP thereby facilitating the activation of RhoA, leading to cell rounding and apoptosis [33]. (E) The BCH domain of Cdc42GAP sequesters RhoA from being inactivated by the adjacent RhoGAP domain, thus reducing GAP-mediated Rho inactivation and preventing cell rounding [19]. (F) BPGAP1 enhances cell motility and ERK1/2 activation. The BCH domain of BPGAP1 induces short pseudopodia in MCF7 cells [29]. The domain interplay of the BCH, PRR (proline-rich region; labelled P) and RhoGAP domains lead to enhanced cell motility and ERK1/2 activation. For example, through the PRR, BPGAP1 mediates translocation of cortactin from cytosol to membrane periphery for cell migration [36] and it also engages EEN/Endophillin II to increase EGF receptor endocytosis and ERK1/2 activation [37]. In addition, BPGAP1 can be negatively regulated by the peptidyl-prolyl cis/trans isomerase Pin1 via targeting both the PRR and the RhoGAP of BPGAP1; thereby suppressing the BPGAP1-induced cell motility and ERK1/2 activation. Intriguingly, such interaction between BPGAP1 and Pin1 is promoted by the active regulatory scaffold, Mek2 by aiding the relief of the auto-inhibited site in BPGAP1 [38].

theme is also seen in the binding of Rho to the BCH domains of BNIP-S, BNIP-XL and Cdc42GAP. Extending its involvement in anchoring membrane receptor signaling, BNIP-2 has also been shown to play a crucial scaffold function to support Cdo receptor activation of Cdc42, in concert with p38a/b-MAPK activation by another scaffold protein, JLP – all triggered under the same Cdo receptor upon activation through N-cadherin ligation. This mechanism brings both scaffold proteins together to activate myogenic differentiation [34,39]. Interestingly, Cdo receptor binding sites have been mapped to encompass aa 261–292, which include the Cdc42-binding motif. The intricate mechanism underlying the activation of Cdc42 by this Cdo-Bnip2-Cdc42 complex remains to be determined. Given the increasing evidence that BCH domains can also interact with GEFs and GAPs, it is tempted to speculate that either one such GEF is to be recruited to activate the pathway, or/and a Cdc42GAP has been inactivated. In the later scenario, overexpression of Cdc42GAP in the same model system of C2C12 myoblasts suppressed the myogenic differentiation. Through the same module of Cdo-Bnip-2-Cdc42, BNIP-2 is also involved in promoting neuronal differentiation

[40]. While providing one novel mechanism for specificity of p38a/b activation during myogenic and neuronal differentiation, forming scaffold alliances is becoming a distinctive mode of cell surface receptor signaling. Adding to its versatility, human BNIP-2 is subjected to cleavage by caspases 3, 6, 8, and 10 in vitro or at least by caspase 3, 8 and 9 inside the cells [41]. It can also be cleaved by granzyme B [42]. All these processing occur outside the BCH domain of BNIP2. Granzyme B triggers apoptosis via the cleavage of a repertoire of cellular proteins, leading to caspase activation and mitochondrial depolarization. As Granzyme B cleaved recombinant BNIP-2 in vitro and endogenous BNIP-2 was cleaved during the natural killer cell-mediated killing of tumor cells [42] these observations raise the possibilities that cleavage of BNIP-2 by caspases and granzyme could lead to well controlled and timely release of the potent pro-apoptotic BCH domains. However, the mechanism by which BNIP-2 causes apoptosis remains unclear, although we have shown that the corresponding BCH domain in BNIP-S lead to apoptosis by activating through a caspase-independent but Rho-dependent pathway [33]. As BNIP-2 harbors a Rho-binding

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BNIP-2

A

BCH domain signature motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs

putative kinesinbinding motif

granzyme cleavage site 35

IEAD

235

94

38

EFEWED 99

250

183

RBD -like

1 167

259

211

BCH

294

CRIB -like

314

EF-hand motif 79 EIDLDGLDTPSE 90 83

217

RRKMP

221 285 VPMEYVGI292

Cdc42-binding motif

BCH-BCH binding motif

caspase 86 cleavage site

• The BCH domain binding sites for FGFR, Bcl2, E1B 19KDa and Cdc42GAP have not been mapped •

Cdo receptor binding site; aa 261-292

B BNIP-H (CAYTAXIN) BCH domain signature motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs 259

274

kinesin-binding WED motif 115

ELEWED

207

120

1

283

235

318

191

RBD -like 102

DETD

BCH

CRIB -like

371

105

caspase-3 cleavage site

site I 242

site II RRRMP

245

putative BCH-BCH binding motif

KGA binding site: a) Site I –residues 191-235 b) Site II –residues 288-331

Pin1 binding site: a) Site I –residues 191-206 b) Site II –residues 286-332

•BCH domain binding sites for Mek2 and CHIP have not been mapped Fig. 5. Linguistics of BCH domains. Schematic diagrams of (A) BNIP-2, (B) BNIP-H, (C) BNIP-XL with BMCC1-1/PRUNE2 and (D) BPGAP1, as the representative members of BCH domain-containing proteins, highlighted with their known or putative functional motifs, including the Rho-binding domain, CRIB-like motif, BCH signature motif, BCH/BCH interaction motif, caspase or granzyme cleavage sites and kinesin-targeting motifs. Note: indicates the putative Rho binding domain based on close homology to known RBD in BNIP-XL, BNIP-S and Cdc42GAP (see Fig. 3D) while indicates putative Cdc42/Rac interactive binding domain based on close homology to known CRIB-like motif in BNIP-2 (see Fig. 3C). For BMCC1/PRUNE2, the sequence used is from accession number BAJ08045.1 [69].

domain (RBD)-like sequence similar to those in BNIP-S and Cdc42GAP (Fig. 3A and D), the released fragment of BCH with RBD might activate Rho towards apoptosis. Consistent with its role in regulating apoptosis, expression of BNIP-2 mRNA has been shown to be downregulated upon estrogen treatment, a regime thought to confer neuroprotection against neurodegenerative diseases such as Alzheimer’s disease [43]. Its expression is also downregulated in the heart upon coxsackievirus B3 infection in mice [44], but up-regulated in lung cancers [45], further indicating its complex nature of regulation in cell growth and cell death. We have further observed that BNIP-2 retards MDCK epithelial cells spreading and collective cell migration by activating Rho/ROCK/myosin signaling cascade (unpublished data) whereas in fibroblasts, BNIP-2 enhances the ability of BPGAP1

to inactivate Rho, leading to greater loss of stress fibers and reduced cell proliferation (unpublished data). Taken together, these findings support the notion that both BNIP-2 and BNIP-S are likely to act as tumor suppressor instead of being proto-oncogenic. To explore the multifunctional nature of BNIP-2, we recently employed fluorescent reporter construct coupled to Total Internal Reflection Fluorescent (TIRF) microscopy that measured the activity of BNIP-2 especially its dynamics near the membrane. We observed that BNIP-2 undergoes dynamics distribution between the endosomes and cell protrusions along the microtubules, but with major concentrations being detected at the protrusive tips. And, this dynamics is recapitulated by the expression of the BCH domain alone (unpublished data). In this regard, the N-terminus of BNIP-2 carries a putative kinesin-binding WED motif 94-

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BNIP-XL

C

BCH domain signature motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs 668

caspase cleavage site 170

ETDNSDL 176

683

P-loop

332

DSGPGWS 339

616

1

600

692

644

RBD -like

650

BCH

RRRMP

727 769

CRIB -like

654

putative BCH-BCH binding motif putative kinesinbinding motif 1139-DLDWDD-1144

coiled-coil sequence

P-loop

1283 / 1311

2654 / 2660

1

2922

BCH proline-rich sequence

617

D

BMCC1 (PRUNE2)

2745

BNIP-2 Homology

1916 / 1921

PRUNE2 Homology

3062

BPGAP1 BCH domain signature motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs 99

1

34

114

51

75

RBD -like

123

BCH

158

GAP

P

CRIB -like 176

R233

167

PPPTKTPPPRPPLP

189

433

256 Pin1 PPI- 260

binding motif DDYGD •Residues P184 and P186 (underlined) are critical for binding to the SH3 domain of Cortactin and Endophilin-2. •186-PPLP-189 motif is the auto-inhibited region which is relieved upon binding to active Mek2. This motif is also targeted by WW domain of Pin1. The peptidyl-prolyl isomerase (PPI) domain of Pin1 targets the GAP domain. •Residue R232 is the arginine finger motif for the RhoGAP domain where Rho inactivation is required for endophilin 2-mediated endocytosis of EGFR •The BCH domain binding sites for Rho, Ras and SmgGDS have not been mapped

Fig. 5 (continued)

EFEWED-99 similar to that identified on BNIP-H (Caytaxin) (Fig. 5A and B) that is required for its trafficking inside the cells [35]. This observation suggests that BNIP-2 could itself translocate towards protrusive membranes to exert its scaffold functions there or it actively recruits and targets cargos (possibly signaling complexes) to the right cellular compartments. The physiological significance and the mechanism of this function remain to be explored. Furthermore, it remains to be determined how alternatively spliced variants of BNIP-2 could be generated and regulated to impact on any functions that have been described so far. Taken together, BNIP-2 regulates diverse cellular functions by engaging multiple partners and is likely to be activated under different cellular context, pending on the biochemical signals and their impending target proteins. 2.2.2.2. BNIP-H (Caytaxin) with neuronal differentiation, neurotransmission and ataxia. Unlike BNIP-2 which is ubiquitously expressed, expression of BNIP-H (or Caytaxin) is highly specific to the brain,

especially in the cerebellum and hippocampus [30]. Several forms of mutations at the human and mouse BNIP-H loci have been linked to a recessive congenital ataxia in human Cayman cerebellar ataxia (thus named ‘‘Caytaxin’’) [31], and also linked to the ‘‘jittery’’, ‘‘hesitant’’ and ‘‘sidewinder’’ ataxia models in mice [46,47]. This disease is associated with hypotonia, variable psychomotor retardation, truncal ataxia and intention tremor, scoliosis, and ocular abnormalities. Interestingly, the two types of BNIP-H gene mutation associated with Cayman ataxia were predicted to cause point mutation (e.g. S310R) or truncation within the BCH domain [31]. BNIP-H deficiency in rats also leads to generalized dystonia [48], another neurologic movement disorder characterized by sustained muscle contractions that produce abnormal movements or postures. Furthermore, expression of BNIP-H is regulated in a developmental and spatial-specific manner and present in presynaptic cytosol [49] while its deficiency could disrupt signaling in cerebellar cortex [50]. While all these genetics studies firmly establish that BNIP-H is required for proper brain function, nothing


is known about how BNIP-H executes its function(s) and regulated at the molecular, cellular and physiological levels. To achieve this objective, we employed proteomics/MALDI-TOF and identified a kidney-type phosphate-activated glutaminase (KGA) as a binding partner for BNIP-H. KGA is a metabolic enzyme responsible for the production of the glutamate which can be used as a form of neurotransmitter. We showed that BNIP-H expression is upregulated in retinoic acid-induced neuronal differentiation in embryonic carcinoma cell P19 and BNIP-H could relocalise KGA to the tips of neurons and control the steady-state level of glutamate [30]. Our model suggests that deregulated level of glutamate in BNIP-H-deficient individuals could render glutamate excitotoxicity or/and deregulated glutamatergic activation. This could then lead to ataxia and dystonia and possibly other excitotoxicity-based neuronal damage [51–54]. Subsequently, we have also identified several more novel protein targets for BNIP-H, among them are the heavy chain of kinesin-1 (previously named KIF5B) motor, Rab small GTPases, Mek and Pin1 isomerase. We showed that nerve growth factor stimulates the interaction of BNIP-H with peptidyl–prolyl isomerase Pin1, a process reproduced by the presence of a constitutive active form of Mek2 such that it involves the release of an auto-inhibitory intramolecular interaction on BNIP-H. Consequently, two binding sites for Pin1 and KGA have been mapped and shown to be overlapping (Fig. 5B) such that Pin1 disrupts the interaction between BNIP-H and KGA [55]. Interestingly, the mutation S310R found in a mutant form of BNIP-H lies within one of the binding sites overlapping with those of Pin1 and KGA. Its precise impacts on BNIP-H binding to all known cellular partners still remain unclear. BNIP-H has been shown to act as an adaptor to transport mitochondria on the kinesin-1 light chain along the neurites [35]. A kinesin-binding motif (115-ELLEWED-120) has been identified. How BNIP-H functions to transport KGA and regulated by differentiation signals and whether there exist cargo proteins other than KGA, all remain to be further investigated. Interestingly, unlike BNIP-2 which targets Cdc42, BNIP-H interacts predominantly with Rab GTPases and co-localized with them in endosomes and along neurites, reflecting their tight regulation by vesicular-based transport system (unpublished data). Furthermore, BNIP-H undergoes polyubiquitylation by CHIP, a E3 ligase protein, probably for its degradation [56]. Similar to BNIP-2 and BNIP-XL (see later), BNIP-H is also subjected to cleavage by caspases. Interestingly, BNIP-H is shown to be a presynapse substrate for caspase-3 and caspase-7 that target the highly conserved 102-DETD-105 motif, releasing the BCH domain that appears to regulate Mek2 signaling through a yet unknown mechanism [57]. Since caspases activity is essential for many other non-apoptotic functions in cell signaling, learning and memory [58–60], differentiation of neural stem cells [61], dendritic [62]/axon [63] pruning and cell migration [64,65], it remains to be seen how these processes are linked to the control of neuronal differentiation mediated by BNIP-H. As dysregulation of BNIP-H function lead to cerebellar ataxia, dystonia, and possibly other related neurological disorders, our current attempt to understand the mechanistic function of BNIP-H and its checkpoints at cellular and molecular levels could pave way to our future understanding on the neuronal remodeling, plasticity and neurotransmission at synapses, and with a long term objective of identifying key strategic target(s) for intervention in such diseases. 2.2.2.3. BNIP-XL with tumor suppression and neuronal differentiation. BNIP-XL initially represents the longest form of the BNIP-2 family proteins, with its sequence homology extended beyond the highly conserved BCH domain, hence we named it BNIP-2 Extra Long [18]. To date, it is shown to be one of the four major isoforms encoded via differential initiation sites from the complex BMCC1 gene (for BCH motif-containing molecule at the carboxyl terminal

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region 1) located at the human chromosome 9 (9q21.2) [66–69]. This gene, spanning 295 kbp, is also known as PRUNE2 as it encodes the largest isoform (BMCC1 isoform-1/BMCC1-1/PRUNE2-1/ NM_015225, 3088aa; or BAJ08045.1, 3062aa) that harbors a region of homology with the PRUNE, a His-Asp-Asp super family proteins at its N-terminus. It also harbors a coiled-coil region, a proline-rich region and a putative P-loop sequence (Fig. 5C). A slightly shorter isoform consisting of 3062aa may result from an alternative splicing [69]. Interestingly, the Prostate Cancer Antigen 3 gene (PCA3), a highly specific biomarker upregulated in prostate cancer, is located within the intron 6 of BMCC1 but in the opposite orientation [67]. In contrast, BMCC1 isoform-2 (BMCC1-2/PRUNE2-2/C9orf65/ BC019095/NM_138818) comprises the first 6 exons of BMCC1 but it terminates immediately upstream of the PCA3 gene. The isoform-3 (BMCC1-3/BMCC1/ABO50197; 2724aa) does not overlap with BMCC1-2 but it comprises 13 distinct exons (exons 7–19) positioned immediately downstream of PCA3 [66]. BNIP-XL is the isoform-4 (BMCC1-4/KIAA0367/AY43213; 769aa) which has the start site located still further downstream within the second exon of BMCC1-3. All three isoforms-1, -3 and -4 encode the highly conserved BCH protein domain. Interestingly, BNIP-XL mRNA itself can undergo alternative RNA splicing, generating a 769 amino acidlong protein (BNIP-XLa) and a 732 amino acid-long protein (BNIP-XLb) (Fig. 2). The shorter variant results from alternative splicing of exons 11 and 12 (with respect to exon numbering in BNIP-XL), which introduces an in-frame stop codon. Multiple sequence analyses of BNIP-XL with BNIP-2, BNIP-Sa, BNIP-H/Caytaxin and BPGAP1 indicates the closest homology to BNIP-2 across the entire protein (58% identity, 74% similarity) whereas its BCH domain is most similar to that of BNIP-H/Caytaxin (76% identity, 90% similarity). All isoforms of BMCC1 have been linked to, or identified as possible biomarker for, various human pathologies. For example, BMCC1-1 was initially thought to offer an improved diagnosis for prostate cancer because of its upregulation with PCA3 gene in prostate cancers and in response to androgen [67]. However, both genes may not appear to be tightly co-regulated [70]. BMCC1-1 has also been identified as a susceptibility gene for Alzheimer’s disease [71] while BMCC1-2 as a biomarker for leiomyosarcomas [72]. BMCC1-3 and BNIP-XL (BMCC1-4) have been shown to be a proapoptotic protein in neuronal cells [66] and an antagonist of Rho-mediated cellular transformation [18], respectively. Expression of BMCC1-3 enhances neuronal apoptosis upon NGF depletion and is expressed in brain, spinal cord and dorsal root ganglia (DRG). It also offers a good prognostic marker for childhood neuroblastomas [66]. Overall, BMCC1-1 mRNA and proteins are predominantly expressed in the neurons of the cranial nerve motor nuclei and motor neurons of the spinal cord and to lesser extent, in other nerve tissues [68,69]. DRG neurons express higher levels of BMCC1-1 in their soma compared with adjacent cells. Moreover, their expression is most profound in adult nerve tissues than those in fetal or neonatal nerve tissues. These expression profiles suggest that various BMCC1 isoforms, including BNIP-XL, may contribute to the maintenance of mature nervous systems and inhibition of unwarranted cell growth and proliferation. However, the underlying mechanisms for their action remain unknown. In contrast to the positive regulation of BNIP-2 and BNIP-S on their cognate GTPases inside the cells, i.e. activating Cdc42 and RhoA, respectively, BNIP-XL affects actin cytoskeletal reorganization and suppresses cellular transformation by inhibiting Rho signaling instead [18]. At the molecular levels, the BCH domain of BNIP-XL interacts with RhoA and RhoC but not with RhoB. Furthermore, it binds to specific conformers of RhoA and also mediates association with the catalytic DH–PH domains of Lbc, a RhoA-specific guanine nucleotide exchange factor (RhoGEF). Consistent with the observations made by other members, BNIP-XL

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does not recognize the constitutive active G14V and Q63L mutants of RhoA but it targets the fast-cycling F30L and the dominant-negative T19N mutants. Whereas overexpression of BNIP-XL reduces active RhoA levels without changing its level of expression, knockdown of BNIP-XL expression has the reverse effect. The constitutive active G14V and Q63L are locked in an active conformation mimicking GTP-RhoA in vitro, because residues for GAP recognition in the P-loop (residues 12–19) and switch II (residues 62–68) regions have been mutated. By contrast, the ‘fast-cycling’ F30L is spontaneously active and demonstrates enhanced GDP to GTP exchange, while retaining regular GTP hydrolysis rates catalyzed by GAPs. The dominant negative T19N mutant on the other hand, sequesters and inhibits multiple endogenous RhoGEFs, preventing RhoA activation leading to elevated GDP-RhoA levels in vivo. Collectively, these results suggest that BNIP-XL inhibits distinct RhoA pathways, including stress fiber assembly and transformation. Such result is consistent with its function as an antagonist for cell growth in fibroblasts or as a pro-apoptotic factor in neuronal cells. Further mutagenesis studies revealed that BNIP-XL utilizes both its BCH domain and the N-terminus to target the DH–PH moiety and the proline-rich region of Lbc, respectively [18]. Consequently, BNIPXL inhibits Lbc-induced oncogenic transformation. Given the importance of RhoA and RhoGEF signaling in tumorigenesis, BNIP-XL could suppress cellular transformation by preventing sustained Rho activation in concert with restricting RhoA and Lbc binding via its BCH domain. This could provide a general mechanism for regulating RhoGEFs and their target GTPases. The identification of a specific BNIP-XL mutant that uncouples GEF interactions while retaining RhoA binding should address the specific contributions of BNIP-XL in linking actin cytoskeleton rearrangements and oncogenic transformation. Interestingly, some BMCC1 isoforms have been shown to bind GTP, its oxidized form 8-oxo-GTP (an agent known to cause mutagenesis and cell death), the T-cell activation RhoGAP, TAGAP [69] and microtubule–associated protein MAP6 [93]. While their mechanism of binding and function remain to be established, these observations imply a complex regulatory scaffold function by BNIP-XL and possibly other BMCC1 isoforms in regulating GTPase signaling and other cell growth/death signaling pathways. Adding to the dynamic roles of these proteins in neuronal cell growth and development, BNIPXL, similar to BNIP-2 and BNIP-H, can also undergo cleavage by multiple caspases-3, 6, 7, 8, 9 and 10 in vitro and at least by caspases 8 and 9 inside the cells, albeit at site distinct from that of BNIP-2 [41] (Fig. 5C). Such actions could result in the release of the BCH domain or its smaller fragments that are crucial in regulating apoptosis or engaging small GTPases signaling in cell dynamics control. Surprisingly, unlike BNIP-2, BNIP-H and BNIP-S which all carry a highly conserved ‘‘E(L/F)EWED’’ kinesin-1 light chain binding motif, BNIP-XL do not posses such a motif. Instead, a similar motif ‘‘DLDWDD’’ is found in its longer isoform BMCC1-1/PRUNE2 (aa 1139–1144) and BMCC1-3 (aa 777–782; [35]), which are located outside the region of homology to the BNIP-2 Family proteins (Fig. 5C). It remains to be seen whether BNIP-XL traffics in the cell via other motor system or it could ‘‘piggyback’’ on these longer isoforms via their BCH/BCH interaction motif. 2.2.2.4. BNIP-S with apoptosis. Comparing the functions of BNIP-2, BNIP-H and BNIP-XL, BNIP-S (or BNIPL for BNIP-2-Like) represents the most potent form of pro-apoptotic BCH containing protein despite sharing high sequence homology (86% similarity) with the BCH domain of BNIP-2 or 72% similar across the whole protein [28]. Phylogenetic analyses suggest that BNIP-S could have appeared earlier than the others during the evolution [26]. BNIP-S can undergo alternative RNA splicing with the retention of an intron that introduces a non-sense mutation. This results in a truncated variant, BNIP-Sb, that is devoid of the last 71 amino acids

of the BCH domain. Full-length BNIP-S is present in the cytosol and its overexpression cause extensive cell rounding, leading to caspase-independent apoptosis. However, BNIP-Sb is localized to the nucleus and fail to exert any changes in cell morphologies or cell fates [28]. The transcripts for both variants can be detected in different cancer lines. It appears that the expression for the full-length version is more abundant in the male reproductive organ of mice [28], or in the human placenta and lung [73]. Most significantly, this apoptotic effect is induced by the BCH domain which triggers an initial phase of extension followed by extensive retraction. And this effect can only be blocked by co-expressing dominant negative mutant form of RhoA, indicating that this induction is likely to involve activation of Rho [33]. BNIP-S does not bind Rac1 or Cdc42. However, BNIP-S BCH domain binds to the unloaded or GDP-bound form of RhoA but not to the GTPbound RhoA. And when co-expressed inside the cells, BNIP-S interacts strongly with the dominant negative mutant of RhoA, T19N, but not with the constitutively active RhoA-G14V [33]. Sequence comparison between its BCH domain and REM Class 1 Rho-binding domains reveals a putative RBD-like motif which when deleted lead to the loss of RhoA binding and failure to execute apoptosis. All these observations indicate that BNIP-S binds to inactive RhoA and that could lead to its activation, leading to cell rounding and apoptosis. Interestingly, unlike BNIP-2 which uses the same BCH/BCH binding motif ‘‘RRKMP’’ for its both homophilic and heterophilic interaction with BCH domains, BNIP-S does not possess such motif. Instead, it uses a separate motif 215-ATWYVKA-221 for its homophilic BCH/BCH interaction [33] (Fig. 3A), and this interaction is required for its apoptotic activity [28]. The binding site for Cdc42GAP is instead located at the region proximal to the BCH domain (aa 133–147), overlapping part of the extended Rhobinding region of the BCH domain. Consequently, overexpression of BNIP-S can capture RhoA for further activation while separate pools of BNIP-S sequester Cdc42GAP via their heterophilic interaction. Acting in concert, this mechanism leads to RhoA activation, cell rounding and apoptosis (Fig. 4D). However, it remains to be seen how BNIP-S binding to RhoA could lead to activation of RhoA in the absence of negative regulation by Cdc42GAP. Perhaps, BNIPS could recruit a RhoGEF in their close proximity since dominant negative mutant of RhoA could inhibit the pro-apoptotic effect of BNIP-S. In this regard, BNIP-S has been shown to interact with proto-Lbc RhoGEF [18] but it remains to be confirmed by means of genetic knockdown and mutant studies to see if this binding is specific and linked to this process. In addition to involvement of Rho signaling in its pro-apoptotic effect, BNIP-S has also been shown to interact with two cell-proliferation related proteins, MIF (macrophage migration inhibitory factor) and GFER (growth factor erv1 (Saccharomyces cerevisiae)like) and help suppress colony formation and cell proliferation [73]. Another variant of BNIP-S (BNIPL-2), also interacts with Bcl2 and Cdc42GAP via its BCH domain [74]. Similar to the proposed function of BNIP-2, BNIPL-2 is thought to suppress the anti-apoptotic effects of Bcl2 and the activity of Cdc42GAP towards Cdc42. Furthermore, BNIPL-2 can also promote invasion and metastasis via Cdc42 activation and upregulation of CD44 in human hepatocellular caricinoma [75]. However, none of these studies had completely delineated their curical binding sites on the BCH domains in order to establish their molecular interaction and functional relationship. 2.2.2.5. Cdc42GAP and BPGAP1 with cell morphogenesis, migration, invasion and neuronal differentiation. RhoGAPs function as negative regulators by activating the intrinsic Rho GTPase activity, converting their active GTP-bound state to the inactive GDP-bound state. The human genome encodes more than 80 RhoGAPs with distinctive arrays of protein domain/motifs and exerts diverse physiological outcomes [76]. These domain/motifs could potentially act as


regulator for either their GAP activity, subcellular localization or connecting to various signaling networks via protein–protein interactions. The Cdc42GAP (p50RhoGAP or ARHGAP1; [77]) and its homolog BPGAP1 (or ARHGAP8; [29,78]) are two BCH domain-containing RhoGAPs (Fig. 2) that functions biochemically as a GAP for Cdc42 and Rho. Expression of Cdc42GAP is up-regulated in Waldenstrom Macroglobulinemia [79] whereas expression of BPGAP1 is elevated in primary colorectal tumors [78] and cervical cancer [80]. However, the molecular bases and cellular outcomes for their elevated expression remain unclear. The Cdc42GAP regulates diverse functions, including cell migration [81,82] and muscle cell differentiation by inactivating Cdc42 [34]. Its homozygous knockout embryos/new born mice displayed reduced organ and body size, owing to increased spontaneous JNK-mediated apoptosis [81,83,84]. Although the biochemical and structural properties of the GAP domain of Cdc42GAP are already well defined [77,85– 87], little is known on how their GAP functions inside the cells are regulated. In particular, the impact of the BCH domain that is located N-terminus proximal to the GAP domain remains largely undetermined. We recently showed that the BCH domain on Cdc42GAP could serves as a local modulator to sequester RhoA from being inactivated by its adjacent GAP domain ([19]; Fig. 4E). This BCH domain cannot distinguish between the GDP-bound and GTP-bound form of RhoA. Further mutagenesis revealed a novel RhoA-binding motif (residues 85–120) within the BCH domain (Fig. 3A and D). Deletion of this motif significantly reduced BCH inhibition on GAP-mediated cell rounding in human cervical epithelial cells, whereas its full suppression also required an intramolecular interaction motif (residues 169–197). Activation of RhoA can lead to distinct cellular and physiological outcomes, depending on the cell types and the environment they are in, the types of processes (e.g. cell spreading, cell adhesion versus cell motility) and the involvement of different constituent proteins such as upstream regulators or downstream effectors that propagate the signals at different locales and timing. Cdc42GAP expression (hence inactivation of Rho) would lead to cell protrusion in epithelial breast cancer MCF7 cells. When BNIP-S is overexpressed, this protein induces cell rounding and apoptosis by displacing Cdc42GAP from inactivating RhoA. Separate pools of BNIP-S would then bind RhoA and that lead to RhoA activation possibly by engaging a RhoGEF. It is likely that, while preventing RhoA from being inactivated by the GAP domain of Cdc42GAP, formation of BNIP-S/RhoA complex could trigger additional signaling pathway(s) that couples the activation of Rho to apoptosis. In contrast, when Cdc42GAP is expressed in cervical epithelial HeLa cells, this protein posseses minimal GAP activity towards RhoA owing to sequestration of RhoA by the adjacent BCH domain in cis [19], hence RhoA is not inactivated and HeLa cells remain cuboidal. However, when this sequestration is lost (evidenced by deleting the RhoA-binding motif in the BCH domain), the GAP will inactivate Rho. Under this condition, inactivation of Rho leads to collapse of stress fibers and cell adhesion, and cells become rounded up instead of rendering protrusion. These observations therefore highlight the plasticity and contextual signaling of Rho on cell morphogenesis under the influence of BCH domains. Recent studies also showed that the BCH domain undergoes autoinhibition in vitro [88] and is required for its endosomal localization and binding to Rab5 and Rab11 [89]. Interestingly, the other plant RhoGAPs commonly referred to as RopGAPs (Rop, Rho of plants) are associated with a Cdc42/Rac interactive binding (CRIB) motif at their N-terminus [90]. This CRIB motif can help regulate the local GAP activity by forming high affinity complexes with specific Rho proteins and GAP domains and acts as lid for binding and releasing Rho of plants [91]. Such a sequestration of substrate in cis by the tandem Rho-binding domains therefore provides a novel

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mechanism for regulating the local activity of Cdc42GAP towards Rho. This mechanism could have important bearings on previously unappreciated function of BCH domains or the ‘‘BCH-like’’ CRAL– TRIO domains in other RhoGAPs or RhoGEFs. In a mechanism similar to the activation of RhoA by BNIP-S which blocks Cdc42GAP from acting on Rho through their heterophilic BCH/BCH binding [33], the function of Cdc42GAP can also be suppressed at the leading edge of cells through its sequestration by Nudel, a cytoplasmic regulator for dynein that is activated by active Erk [92]. Consequently, more Cdc42 is activated. In comparison, we have identified BPGAP1 (or ARHGAP8), a close homolog of Cdc42GAP, and showed that it activates cell protrusions and cell migration via the interplay of its BCH domain, a proline-rich region (PRR) and a GAP domain [29]. At least 4 alternative RNA splicing variants have been described, including those present in the BCH domain (Fig. 2), thus adding to the versatility and functional diversity in the BPGAP family proteins [29]. Specifically, the BCH domain of BPGAP1 induces Cdc42/Rac-dependent membrane protrusions that are necessary for its physical and functional coupling to cortactin and endophilin-2 which bind to its proline-rich region (PRR). Cortactin helps form branching actin network [36] whereas endophilin-2 promotes EGF receptor endocytosis for enhanced ERK signaling [37]. This process is also augmented by the activity of the RhoGAP domain that inactivates Rho. Furthermore, we showed that active Mek2 stimulates the release of an auto inhibition on the PRR, thus promoting binding of the peptidyl–prolyl isomerase Pin1 to the PRR and the RhoGAP domain to suppress BPGAP1-induced acute Erk activation and cell motility (Figs. 4F and 5D; [38]). Interestingly, such mechanism is also employed by the BCH domain of BNIP-H where Mek2 promotes Pin1 binding and disruption of the binding of its cargo, glutaminase KGA [55]. It remains to be investigated whether this mechanism would affect the versatility of the BCH domain in BPGAP1. We had earlier reported on the role of BCH domain in stimulating ERK signaling, through a yet unknown mechanism [37]. Using proteomics pulldown and candidate approaches, we have since identified several novel partners, including Ras and one of its regulators, SmgGDS. We show that BCH domain binds to K-Ras and that leads to enhanced Ras/ERK signaling necessary for the neurite outgrowth and differentiation. Knockdown of SmgGDS potentiates K-Ras/BCH-mediated Erk activation further, revealing a novel tripartite regulation of Ras/Erk by the BCH domain (unpublished data). And through its heterophilic BCH/BCH interaction of BNIP2, we are examining how BNIP-2 and BPGAP1 would co-regulate Rho, Ras and Cdc42 signaling.

3. Conclusions and future perspective Protein domains evolve to change their biochemical and biophysical properties to confer different roles in recognizing either specific or diverse targets and to function at unique or multiple locales. They usually do not work in isolation but they interact with other molecule(s) that carry the same or with distinct protein modules or other non-peptide ligands – all defined by their unique sequence motifs. Many domains also co-exist by various copy numbers and permutation in one particular protein. Such designs help thrive for their optimal signaling capabilities, forming more sophisticated yet well coordinated network of signaling hubs. The properties and functions of these domains can also be modified by biochemical and mechanical signals which cells receive from the external environments. Therefore, optimal design and engineering of domain architecture equipped with single or multiple motifs allow them to execute as well as receiving feedback for better integration of cell signaling.

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domains of GEFs, GAPs and GDIs? Do other BCH-like domains also possess some of the binding properties such as GTPase binding? This is of special interest, as the CRAL– TRIO domains present in the RasGAP (e.g. NF1) and RhoGEF (e.g. Trio, DBS) could indeed engage some of these GTPases to regulate cell morphogenesis, migration, apoptosis and differentiation. Identification and systematic studies of other BCH-domain-containing proteins will also help to provide further insights into the mechanistic functions of this emerging class of protein module. (2) How are signaling pathways connected by their binding to BCH domains? Are these pathways merely operating in parallel by different pools of BCH domain proteins or are they physiologically or physically linked? To date, we have observed the significant impact of BCH domains either directly or indirectly via other domains they are associated with, on various signaling nodes, especially in the FGF and Cdo receptor signaling, various Rho, Ras, Rab GTPases, some kinase, isomerase, caspases, Bcl2 and even the metabolic pathway. Under what conditions are they functionally linked? And given that some BCH domains are already targets of kinases, ligase and isomerase, what are the signals that modify their properties through phosphorylation, ubiquitination and isomerization and what are the impacts on their functions? (3) What are the dynamic features of BCH domains that help them achieve their multifunctional roles? And how are these functions regulated in space and time at the cellular to tissue/organismal levels. For examples, BNIP-H and BNIP-2 are actively transported along the microtubules during neu-

In this review, we have highlighted how BCH domains have acquired many of these key features that enable them function as versatile regulatory scaffold modules. Through their multiple functional motifs, some of which obvious but some are cryptic, they interact with a broad spectrum of targets, ranging from membrane receptors, different GTPases and their immediate regulators such as GEFs and GAPs, kinase such as Mek2, isomerase Pin1, anti-apoptosis protein Bcl2 and key metabolic enzymes such as glutaminase, impacting ultimately on cell growth, apoptosis, morphogenesis, migration, metabolism and differentiation. While all these observations point to the functional plasticity of BCH domains and further underscore the importance of BNIP-2 family and BPGAP family proteins in cell dynamics and cell fates control, the precise mechanisms underlying these dynamic processes and their physiological impacts remain to be further elucidated. Here, we reflect on several interconnected issues related to the structural, functional, mechanical, evolutionary, physiological aspects of BCH domains and the cellular environments these proteins are exposed to, and aim to understand them at the molecular, cellular and tissue levels (Fig. 6). Some of these are summarized below: (1) What are the structural bases underlying the plasticity versus specificity of BCH domains in recognizing multiple and diverse targets? In particular, within the same class of partner, such as Rho, how do they distinguish between the active G14V which they do not bind versus inactive mutant T19N which they bind best, despite that the same BCH domain binds both GDP-bound and GTP-bound form of Rho equally well? What are the atomic and molecular bases that distinguish BCH domain from other conventional GTPase-binding

Structures

Unique GTPases binding

Signaling crosstalk

Rho, Cdc42, Ras, Rab, active vs inactive conformers; binding by other BCH or BCH-like domains

Rho, Ras, RTK, Mek, MAPK (p38/ERK), caspases and metabolic pathways

Protein modification

complex with targets homophilic or heterophilic (BCH-BCH) complex

phosphorylation by RTK, Mek, other kinases ubiquitination isomerization

Cytoskeletal network

Spatiotemporal dynamic trafficking with cargo and signaling proteins

actin and microtubule dynamics linked to morphogenesis and migration

Lipidomics Tissue / Organogenesis

lipids and phosphoinositides targeting or regulatory

zebrafish, mice , rats as disease models

Gene regulation transcription microRNA, translation

Splicing variants, redundancy, tissue specificity physiological and hormonal regulation

Fig. 6. Future perspective: elucidating the functional plasticity of BCH scaffold protein domains across molecular, cellular and physiological levels. To further understand the molecular and cellular bases for the physiological functions and regulation by the BCH domains, future works will aim at (i) solving the structures of BCH domains with their target proteins including those that confer unique GTPase-binding profile (e.g. Rho, Cdc42, Ras, Rab) and their dependence on specific conformation (e.g. dominant negative mutant T17N is most preferred but constitutive G12V is the least or no binding) and the BCH/BCH interactions; (ii) exploring their diverse and dynamic signaling crosstalk between cell growth, proliferation, differentiation regimes (e.g. receptor tyrosine kinases, GTPases, p38 and Erk) and key metabolic pathways (e.g. glutaminase) under the influence of biochemical or/and mechanical cues such as substrate rigidity; (iii) examining their roles on actin and microtubule dynamics, leading to cell morphogenesis and cell migration; (iv) deciphering their post-translational protein modifications (via phosphorylation, ubiquitination and isomerization) and intracellular trafficking as well as identification of cargo proteins such as metabolic enzymes and signaling complexes; (v) examining their possible binding and regulation by lipids and phosphoinositides; (vi) exploring the significance of alternative RNA splicing variants in physiological and hormonal regulation; (vii) identifying their modes of gene regulation and expression by transcription, micro-RNA and translation, and (viii) elucidating their distinct roles in tissue/organogenesis using various vertebrate models. All these issues will be systematically examined and their precise functions/steps elucidated. For clarity, the model presented here depicts the complex between BNIP-2 and Cdc42GAP via their BCH domains (orientation is hypothetical) while each could still act as distinctive scaffold module to regulate binding and activity of 3G-Signalome, i.e. GTPases, GEFs (+ sign) and GAPs ( sign).


(4)

(5)

(6)

(7)

ronal maturation and differentiation. Are they trafficking and delivering any cargos or are they being recruited to the neurites tips before exerting their scaffold functions there? What are those cargos? Are they metabolic enzymes or signaling complexes? Moreover, with their ability to recognize multiple GTPases on the same domains, e.g. via their tandem RBD-like and CRIB-like motifs present in BNIP-2 BCH domain, it is tempted to speculate that this BCH domain can also regulate Rho in addition to Cdc42 signaling. If so, what are the consequences on the spatial and temporal activities of these GTPases? As cells sense both biochemical and mechanical cues under various conditions, it will be important to address whether function(s) of BCH domains, that is usually linked to receptor and Rho signaling, is itself subjected to both the biochemical and mechanical triggers. And, how those signals influence the dynamic distribution of BCH domain proteins and their ability to regulate the actin and microtubule network associated with extensive morphogenesis and motility. Are BCH domains certified ‘‘lipid-free?’’ Despite relatively low sequence identity between the BCH domain and CRAL–TRIO and other lipids binding domains, several studies including ours have not excluded entirely the ability of BCH domain to recognize certain lipids for their function or regulation. It is still plausible that certain species of lipids including the phosphoinositides, could act as ligands to help regulate functions of the BCH domains instead of serving as the substrates for transportation. However, the authenticity of their binding would require further confirmation in vivo or cell-based system. As BCH domains confer functional diversity, the proteins and the genes from which they are encoded are expected to be under tight physiological regulation. This could take in the forms of gene transcription, protein expression and stability, and of course their ability to undergo alternative RNA splicing. The unique pattern of RNA splicing, as we frequently observe in BNIP-2, BNIP-S, BNIP-XL and BPGAP1 calls for attention on their changes in the splicing pattern in response to hormonal and stress signals. Similarly, their regulation by microRNA and tissue specificity factors should also be examined in the right context of cell signaling; Last but not least, as functionalities and plasticity of BCH domain containing proteins are being examined, we should consider developing various vertebrate models to mimic and interrogate the actual physiological response associated with the signaling pathways. These model systems of mice to zebrafish allow us establish necessary platforms to detect their impacts on developmental processes as well as for drug screening. Coupled with genetic manipulations of knockdown and functional rescues with pathway/partner-specific mutants in the cell culture and knockout/knockdown or knock-in for the in vivo models system, can we clearly decipher some of the true meaning behind the multiplexing roles of BCH domain containing proteins.

With the advent of high- and super-resolution live microscopy and the availability of relevant biosensors for signaling proteins, one could trace the spatiotemporal dynamics of BCH domain containing proteins or the BCH domains alone and their physical localization with their partners under different cellular contexts and genetic backgrounds. And with various biophysical and structural determination tools such as NMR to map conformational shifts and X-ray crystallography for detailed binding sites, one can start generating more precise mutants to interrogate their functional plasticity and dynamics distribution in the cells and in tissues. Such versatile properties not only underlie their central roles in

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controlling normal physiology but they also offer us exciting prospects of exploiting them as potential targets of therapeutic intervention. Furthermore, it is plausible now to think about creating novel cellular processes and functions by re-wiring specific networks or modifying their responses using synthetic scaffold proteins. It is with these motivations that we should continue to define the form (structure), the meaning (interactomes) and the context (when, where and regulation) of such linguistic designs of BCH domains that their functional plasticity and biological significance can be fully appreciated. Acknowledgments We thank all the members and collaborators of BCL Lab, past and present, and also colleagues who are working on this subject for their dedication. This work is supported in part by the Biomedical Research Council of Singapore (07/1/21/19/506), by the Ministry of Education (Tier-2 Grant T208A3121) and by the Mechanobiology Institute, co-funded by National Research Foundation and the Ministry of Education of Singapore. References [1] Pawson, T. and Nash, P. (2003) Assembly of cell regulatory systems through protein interaction domains. Science 300, 445–452. [2] Li, N. et al. (1993) Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature 363, 85–88. [3] Sudol, M. (2010) Newcomers to the WW Domain-Mediated Network of the Hippo Tumor Suppressor Pathway. Genes Cancer 1, 1115–1118. [4] Achiriloaie, M., Barylko, B. and Albanesi, J.P. (1999) Essential role of the dynamin pleckstrin homology domain in receptor-mediated endocytosis. Mol. Cell. Biol. 19, 1410–1415. [5] Kenniston, J.A. and Lemmon, M.A. (2010) Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients. EMBO J. 29, 3054–3067. [6] Brown, M.D. and Sacks, D.B. (2009) Protein scaffolds in MAP kinase signalling. Cell. Signal. 21, 462–469. [7] Frame, M.C., Patel, H., Serrels, B., Lietha, D. and Eck, M.J. (2010) The FERM domain: organizing the structure and function of FAK. Nat. Rev. Mol. Cell Biol. 11, 802–814. [8] Lietha, D., Cai, X., Ceccarelli, D.F., Li, Y., Schaller, M.D. and Eck, M.J. (2007) Structural basis for the autoinhibition of focal adhesion kinase. Cell 129, 1177– 1187. [9] Low, B.C., Lim, Y.P., Lim, J., Wong, E.S. and Guy, G.R. (1999) Tyrosine phosphorylation of the Bcl-2-associated protein BNIP-2 by fibroblast growth factor receptor-1 prevents its binding to Cdc42GAP and Cdc42. J. Biol. Chem. 274, 33123–33130. [10] Boyd (1994) Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79, 1121. [11] Low, B.C., Seow, K.T. and Guy, G.R. (2000) The BNIP-2 and Cdc42GAP homology domain of BNIP-2 mediates its homophilic association and heterophilic interaction with Cdc42GAP. J. Biol. Chem. 275, 37742–37751. [12] Low, B.C., Seow, K.T. and Guy, G.R. (2000) Evidence for a novel Cdc42GAP domain at the carboxyl terminus of BNIP-2. J. Biol. Chem. 275, 14415–14422. [13] Wennerberg, K., Rossman, K.L. and Der, C.J. (2005) The Ras superfamily at a glance. J. Cell Sci. 118, 843–846. [14] Schmidt, A. and Hall, A. (2002) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev. 16, 1587–1609. [15] Bernards, A. and Settleman, J. (2004) GAP control: regulating the regulators of small GTPases. Trends Cell Biol. 14, 377–385. [16] Hoffman, G.R. and Cerione, R.A. (2000) Flipping the switch: the structural basis for signaling through the CRIB motif. Cell 102, 403–406. [17] Bishop, A.L. and Hall, A. (2000) Rho GTPases and their effector proteins. Biochem. J. 348 (Pt. 2), 241–255. [18] Soh, U.J. and Low, B.C. (2008) BNIP2 extra long inhibits RhoA and cellular transformation by Lbc RhoGEF via its BCH domain. J. Cell Sci. 121, 1739–1749. [19] Zhou, Y.T., Chew, L.L., Lin, S.C. and Low, B.C. (2010) The BNIP-2 and Cdc42GAP homology (BCH) domain of p50RhoGAP/Cdc42GAP sequesters RhoA from inactivation by the adjacent GTPase-activating protein domain. Mol. Biol. Cell 21, 3232–3246. [20] Bourne, H.R., Sanders, D.A. and McCormick, F. (1991) The GTPase superfamily: conserved structure and molecular mechanism. Nature 349, 117–127. [21] Bankaitis, V.A., Mousley, C.J. and Schaaf, G. (2010) The Sec14 superfamily and mechanisms for crosstalk between lipid metabolism and lipid signaling. Trends Biochem. Sci. 35, 150–160. [22] Gu, M.X., York, J.D., Warshawsky, I. and Majerus, P.W. (1991) Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosinephosphatase with sequence homology to cytoskeletal protein 4.1. Proc. Natl. Acad. Sci. 88, 5867–5871.

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