[Cell Cycle 5:7, 678-680, 1 April 2006]; ©2006 Landes Bioscience
Localization of the Translational Guanine Nucleotide Exchange Factor eIF2B Extra View
A Common Theme for GEFs?
Susan G. Campbell Mark P. Ashe*
Original manuscript submitted: 02/10/06 Manuscript accepted: 02/15/06
This work and SGC were supported by a BBSRC project grant BBS/B/07500 to MPA.
Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP for GTP on specific GTP/GDP binding proteins. GEFs function in a host of cellular processes. For instance, they stimulate the dissociation of GDP from small monomeric GTP-binding proteins in response to a whole variety of upstream signals.1 In addition, following ligand binding, G-protein coupled receptors (GPCRs) facilitate guanine nucleotide exchange on heterotrimeric G-proteins.2 In both of these cases the GEF activity serves as a molecular switch and is therefore generally made use of in cell signalling pathways. In addition, GEFs function constitutively in a number of processes such as protein synthesis and nucleocytoplasmic transport of protein/ RNA.3-5 Therefore GEFs play very varied roles in many aspects of cell biology, functioning from highly inducible components of signal transduction pathways to workhorses of essential cellular processes. Given the plethora of functions it is perhaps not surprising that many human diseases are associated with mutation of guanine nucleotide exchange factors (see refs. 6–8 for a more complete discussion). For instance for the monomeric G-proteins, mutations in the Als2 GEF for Rab5 or the Fgd11 GEF for Cdc42 result in amyotrophic lateral sclerosis or X-linked Faciogenital Dysplasia respectively.9-11 In addition, a whole host of diseases from colour blindness through to Blomstrand Chondrodysplasia are associated with mutations in GPCRs.6 Furthermore, mutations to any of the subunits of the translation initiation GEF, eIF2B, lead to leucoencephalopathy with vanishing white matter.8 Classically the guanine nucleotide exchange process for heterotrimeric G-proteins occurs on the plasma membrane, as the G-protein coupled receptors form a huge family of structurally linked seven transmembrane repeat proteins. As a consequence of extracellular ligand or agonist binding, these proteins adopt an activated conformation and catalyze exchange of GDP for GTP on the α subunit of heterotrimeric G-proteins. This leads to the dissociation of Gα from the Gβγ subunits, with both products harboring the potential to act as signalling molecules and generate the appropriate downstream responses.2 Intriguingly, it has also recently been shown that exchange on heterotrimeric G-proteins can occur via a receptor-independent mechanism. For example, critical functions for heterotrimeric G-proteins have been described during asymmetric cell division. The RIC-8 exchange factor is required for these functions and seems to control the association of heterotrimeric G-proteins with the cell cortex or plasma membrane.12-15 Therefore although this exchange factor is not an integral membrane receptor, it does ultimately control the localization of GTP/GDP binding proteins. Monomeric G- proteins in contrast to the heterotrimeric G-proteins, are small single subunit GTP/GDP binding proteins. They are widely distributed throughout mammalian
eIF2B, eIF2, translation initiation, guanine nucleotide exchange factor, G-protein, localization
Previously published as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2607
*Correspondence to: Mark P. Ashe; The University of Manchester; Michael Smith Building; Oxford Road; Manchester, M13 9PT, UK; Tel.: +44.161.306.4164; Fax: +44.161.275.5082; Email: [email protected]
Faculty of Life Sciences; The University of Manchester; Manchester, UK
The eukaryotic initiation factor 2B (eIF2B) serves an essential recycling function in protein synthesis. As the guanine nucleotide exchange factor for eIF2, it recycles eIF2 from a GDP to a GTP bound form that is competent for translation initiation. Stress-dependent controls target this eIF2B-recycling step allowing a reprogramming of the global gene expression profile. In addition, a human disease, leukoencephalopathy with vanishing white matter (VWM), is caused by mutations in the eIF2B subunit genes. Recently, we have found that the eIF2B guanine nucleotide exchange factor resides in a specific cytoplasmic focus in the yeast, Saccharmoyces cerevisiae. eIF2B is a resident feature of this focus, whereas eIF2 shuttles to and fro. Moreover, the in vivo rate of eIF2 shuttling correlates with changes in guanine nucleotide exchange activity implicating this large cytoplasmic focus as a site of guanine nucleotide exchange. In this perspective, we discuss these findings in the wider context of the assortment of guanine nucleotide exchange factors.
GEFs and Their Localization
cells and over 100 small G-proteins have been identified from yeast to mammals. These can be classified into at least five families; Ras, Rho, Rab, Sar1/Arf and Ran, each of which have been extensively studied and characterized. In broad terms the Ras G-proteins have been identified in the regulation of gene expression, Rho G-proteins regulate both cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf members regulate intracellular vesicle trafficking and the Ran proteins regulate nucleocytoplasmic transport during the cell cycle and are also involved in microtubule organization during M phase.1 Although monomeric GFigure 1. Localization of the translation initiation GEF and G-protein to discrete cytoplasmic proteins are widely distributed among many cell types their foci. (A) Live cell confocal images of yeast strain YMK880 bearing a chromosomally correct localization within the cell is crucial for their activa- integrated C-terminal eGFP tagged for the eIF2Bε subunit (GEF).37 (B) Live cell confocal tion. In many cases, this localization is dependent upon the images of yeast strain YMK883 bearing a chromosomally integrated C-terminal eGFP localization of the appropriate guanine nucleotide exchange tagged for the eIF2α subunit (G-protein).37 (C) Model representing the different pools factor (GEF). For example, the plasma membrane localization of eIF2B GEF activity. of Ras proteins is achieved only after the correct localization of the GEF SOS to the plasma membrane via adaptor proteins have also been shown to facilitate the localization of GEFs specific GRB2 and SHC/GRB2.16,17 Rho proteins involved in actin reor- for alternative downstream G-proteins, thus generating G-protein ganization are predominately cytosolic until their activation. This cascades.29 For example, in yeast activation of GTP bound Ypt1p activation is dependent upon the correct localization of the corre- (located on Golgi membranes and required for ER to cis-golgi sponding GEF. For example, the GEF frabin, which activates the G- protein transport) by its GEF TRAPP, recruits the GEF for the Gprotein Cdc42 involved in filopodium formation, harbors an protein Ypt31/32p (involved in exit from the trans Golgi).30,31 actin-binding domain in its N terminal region.18 This localization of Activation of Ypt31/32p recruits another GEF Sec4p to the membranes frabin to actin facilitates the recruitment and activation of Cdc42 of secretory vesicles and results in the generation of the GTP-bound allowing subsequent Cdc42 dependent actin reorganization. The Sec2p.32,33 Therefore, a theme that appears to be emerging for this diverse Tiam1 GEF for Rac proteins contains domains in the N terminal region of the protein that are involved in its localization to plasma functional category of factors is that of cellular localization. The membranes thus recruiting Rac proteins involved in cell membrane GEFs for hetrotrimeric G-proteins (e.g., GPCRs and RIC-8) are ruffling.19 For the Sar1 G-proteins, the localization of the Sar1 GEF localized to the plasma membrane, whereas the GEFs for monomeric Sec12p to the endoplasmic reticulum facilitates the recruitment and G-proteins can be localized to almost any organelle or structure activation of the GDP bound Sar1 to its GTP bound form on the within the cell (e.g., SOS, Tiam1, fabrin, Sec12 and RCC1). The cytoplasmic surface of the ER.20,21 Therefore, there are many examples classical view of translational GEFs is that specific localization within of GEFs for monomeric G-proteins, which are specifically localized the cell would not play a role in their activity therefore it is generally assumed they would display a broadly cytoplasmic localization. within the cell. The tight Ran GTP gradient, which exists across the nuclear However, even early data suggested that the process of protein envelope is crucial for Ran G-protein function in nucleocytoplasmic synthesis might be intimately associated with the cytoskeleton in transport, nuclear envelope formation and spindle assembly and is eukaryotes.34,35 In particular studies by Howe and Hershey revealed facilitated by the defined localization of the Ran GTPase regulators.22 an association between the cytoskeleton and eIF2.36 More recently The nuclear specificity of the GTP bound Ran protein is dependent we have found that a pool of the translational GEF eIF2B is localized upon the localization of the Ran GEF RCC1, to chromatin in the to a large cytoplasmic foci (Fig. 1A).37 The recycling of eIF2 from a GDP bound form to the translanucleus.23 After the nucleotide exchange reaction, (which occurs on the chromosome) Ran GTP associates with exportin and proteins tionally active GTP-bound form represents one of the key controlled requiring nucleocytoplasmic transport.1,22 Once in the cytoplasm steps in eukaryotic protein synthesis. This exchange reaction is Ran GTP is hydrolysed, the cargo is released and the GDP bound achieved via a heteropentameric GEF complex eIF2B.38 Our recent studies have revealed that both the GEF and the G-protein colocalize form reenters the nucleus. Whole cascades of GEF/G-protein localization and regulation to a discrete cytoplasmic region in yeast.37 This region is highly have been identified for families of small G-proteins, examples can dynamic with the G-protein eIF2 shuttling at a very rapid rate be found in budding process of S. cerevisiae and the regulation of the between the cytoplasm and the concentrated focus. Intriguingly, secretory pathway.24 In yeast, the G-protein Rsr1/Bud1p which is however, the GEF, eIF2B, remains a constant component of this normally located randomly along the plasma membrane is recruited focus. Three different strategies, which all reduce the eIF2B GEF to the incipient bud site and activated by its GEF, Bud5p and the activity, also reduce the rate of eIF2 shuttling. Most striking amongst GAP Bud2p.25,26 Activation of GTP bound Bud1p recruits the GEF these is a mutation in the catalytic ε subunit of eIF2B that displays Cdc24p to the bud site and subsequently results in the activation of dramatically reduced eIF2 shuttling.37 The fact that a localized GEF is involved in a process as GTP-bound Cdc42p allowing the reorganization of the actin cytoskeleton toward the bud tip.27,28 Activation of Cdc42p also commonplace as translation initiation, begs the question do GEFs results in the recruitment of additional G-proteins such as Rho1 and need to be somehow concentrated at specific locations to function Sec4 to the site of bud selection facilitating further actin reorganization maximally as guanine nucleotide exchange factors? It is clear from by Rho1 and secretory vesicle movement necessary for the developing the discussion above that many factors with GEF function are localized bud.24 Rab proteins, key players in intracellular vesicle trafficking and that in many cases this localization is a prerequisite of their 679
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GEFs and Their Localization
individual function. For instance, a GPCR transmembrane receptor needs to span the plasma membrane in order to sense ligand and generate an intracellular signal via its GEF activity. However, in other cases the precise physiological reason for the localization of the GEF protein is less clear-cut. Indeed for some Rho family GEFs allosteric regulation by phospholipids has been noted such that a defined membrane localization may make the GEF subject to control.39-42 This example may provide a clue as to why a translational GEF might be localized in a specific focus within the cytoplasm. The eIF2B guanine nucleotide exchange step represents one of the key control points of translation initiation in all eukaryotic cells. Indeed the exchange reaction kinetics for eIF2B are delicately poised to allow maximal rate while maintaining the potential for instant shutdown. Perhaps the cytoplasmic focus of eIF2B is a necessary prerequisite for this degree of fine-tuning (Fig. 1C). References 1. Takai Y, Saski T, Matozaki T. Small GTP-binding proteins. Physiological Reviews 2001; 81:153-208. 2. Bhattacharya M, Babwah AV, Ferguson SS. Small GTP-binding protein-coupled receptors. Biochem Soc Trans 2004; 32:1040-4. 3. Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: A conserved switch for diverse cell functions. Nature 1990; 348:125-32. 4. Li HY, Wirtz D, Zheng Y. A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo. J Cell Biol 2003; 160:635-44. 5. Pavitt GD. eIF2B, a mediator of general and gene-specific translational control. Biochem Soc Trans 2005; 33:1487-92. 6. Spiegel AM, Weinstein LS. Inherited diseases involving g proteins and g protein-coupled receptors. Annu Rev Med 2004; 55:27-39. 7. Rossman KL, Der CJ, Sondek J. GEF means go: Turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 2005; 6:167-80. 8. Fogli A, Boespflug-Tanguy O. The large spectrum of eIF2B-related diseases. Biochem Soc Trans 2006; 34:22-9. 9. Hadano S, Hand CK, Osuga H, Yanagisawa Y, Otomo A, Devon RS, Miyamoto N, Showguchi-Miyata J, Okada Y, Singaraja R, Figlewicz DA, Kwiatkowski T, Hosler BA, Sagie T, Skaug J, Nasir J, Brown Jr RH, Scherer SW, Rouleau GA, Hayden MR, Ikeda JE. A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat Genet 2001; 29:166-73. 10. Yang Y, Hentati A, Deng HX, Dabbagh O, Sasaki T, Hirano M, Hung WY, Ouahchi K, Yan J, Azim AC, Cole N, Gascon G, Yagmour A, Ben-Hamida M, Pericak-Vance M, Hentati F, Siddique T. The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat Genet 2001; 29:160-5. 11. Pasteris NG, Cadle A, Logie LJ, Porteous ME, Schwartz CE, Stevenson RE, Glover TW, Wilroy RS, Gorski JL. Isolation and characterization of the faciogenital dysplasia (Aarskog-Scott syndrome) gene: A putative Rho/Rac guanine nucleotide exchange factor. Cell 1994; 79:669-78. 12. Afshar K, Willard FS, Colombo K, Johnston CA, McCudden CR, Siderovski DP, Gonczy P. RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos. Cell 2004; 119:219-30. 13. Hampoelz B, Hoeller O, Bowman SK, Dunican D, Knoblichl JA. Drosophila Ric-8 is essential for plasma-membrane localization of heterotrimeric G-proteins. Nat Cell Biol 2005; 7:1099-105. 14. Tall GG, Krumins AM, Gilman AG. Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor. J Biol Chem 2003; 278:8356-62. 15. Wang H, Ng KH, Qian H, Siderovski DP, Chia W, Yu F. Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G-proteins. Nat Cell Biol 2005; 7:1091-8. 16. Buday L, Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 1993; 73:611-20. 17. Byrne JL, Paterson HF, Marshall CJ. p21Ras activation by the guanine nucleotide exchange factor Sos, requires the Sos/Grb2 interaction and a second ligand-dependent signal involving the Sos N-terminus. Oncogene 1996; 13:2055-65. 18. Umikawa M, Obaishi H, Nakanishi H, Satoh-Horikawa K, Takahashi K, Hotta I, Matsuura Y, Takai Y. Association of frabin with the actin cytoskeleton is essential for microspike formation through activation of Cdc42 small G-protein. J Biol Chem 1999; 274:25197-200. 19. Michiels F, Stam JC, Hordijk PL, van der Kammen RA, Ruuls-Van Stalle L, Feltkamp CA, Collard JG. Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation. J Cell Biol 1997; 137:387-98. 20. Barlowe C, Schekman R. SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER. Nature 1993; 365:347-9.
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