Caveolae Internalization Regulates Integrin-Dependent Signaling ...

[Cell Cycle 5:19, 2179-2182, 1 October 2006]; ©2006 Landes Bioscience

Caveolae Internalization Regulates Integrin-Dependent Signaling Pathways Extra View

ABSTRACT

*Correspondence to: Miguel A. Del Pozo; Integrin Signaling Laboratory; Centro Nacional de Investigaciones Cardiovasculares (CNIC); Melchor Fernández Almagro 3; Madrid 28029 Spain; Tel.: +34.914531212; Fax: +34.914531245; Email: madelpozo@ cnic.es

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3264

KEY WORDS

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Original manuscript submitted: 07/28/06 Manuscript accepted: 08/08/06

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Integrin Signaling Laboratory; Centro Nacional de Investigaciones Cardiovasculares (CNIC); Madrid, Spain

Integrin-mediated adhesion regulates trafficking of cholesterol-enriched membrane microdomains (CEMM). Upon cell detachment from the extracellular matrix (ECM), CEMMs undergo rapid internalization and are cleared from the plasma membrane. This pathway regulates integrin-mediated Rac membrane targeting, allowing coupling of Rac to downstream effectors. Internalization of CEMMs is mediated by Dynamin-2, a regulator of caveolae dynamics, and caveolin-1, an essential caveolae coat protein. Translocation of tyrosine phosphorylated caveolin-1 from focal adhesions to caveolae upon cell detachment induces CEMM internalization. Notably, integrin-mediated regulation of Erk, phosphatidylinositol-3-OH kinase (PI3K) and Rac pathways is dependent on caveolin-1. These results describe a novel pathway in which integrins prevent downregulation of Erk, PI3K and Rac-dependent pathways by inhibiting caveolin-1-dependent endocytosis. This pathway define a novel molecular mechanism for regulated cell growth and tumor suppression by caveolin-1.

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Asier Echarri Miguel A. Del Pozo*

INTRODUCTION

ABBREVIATIONS

Integrins regulate multiple pathways that are frequently constitutively activated in cancer cells, including Erk, PI3K, FAK, Src and Rho GTPases.1,2 Normal cells grow in an anchorage-dependent manner in culture and rely on proper integrin- and growth factormediated signaling for proliferation. Normal cells have multiple mechanisms to ensure that signaling is terminated, such as the inactivation of Erk, Rac and PI3K pathways after cell detachment from the extracellular matrix.3 In contrast, cancer cells have lost their anchorage dependence and signaling pathways required for cell growth and survival are constitutively activated, bypassing integrin requirement for regulated activation of these pathways.4 Small Rho GTPase Rac is implicated in cell survival, gene expression, cell cycle progression, cell migration and cell-cell adhesion.5,6 In addition, Rac function contributes to transformation induced by oncogenes, such as Ras, Bcr-Abl, v-Abl and Src.7 Rac function is regulated by factors that regulate its activated state or its localization and effector coupling.8 Rac activity is directly regulated by Rac GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase activating proteins), that activate the GTPase or inactivate it, respectively. We have previously shown that coupling of Rac to its downstream effector Pak is regulated by integrins9,10 and Rho GDP dissociation inhibitor (GDI).11 In particular, integrin engagement induces Rac translocation to the plasma membrane, allowing coupling to downstream effectors.9 Notably, this is achieved by preventing the internalization of CEMMs from the plasma membrane, which contain the Rac-binding sites.12 Caveolae are a subtype of CEMMs that by electromicroscopy appear as an Ω-shape invaginations of the plasma membrane.13 At steady state, caveolae are not highly dynamic structures,14,15 but under certain conditions they are internalized to the interior of the cell by a dynamin-2-dependent mechanism.3,13,16,17 Caveolin-1 is the major protein of caveolae and it was first identified as a v-Src substrate.18 Caveolae are not observed in caveolin-1 null cells,19,20 showing that the caveolar structural component caveolin-1 is absolutely required for caveolae biogenesis. Caveolin-1 regulates multiple signaling pathways and contributes to membrane and cholesterol homeostasis.21-23 Phosphorylation of tyrosine 14 was first discovered in v-Src transformed cells and it has been suggested to play a role in caveolae internalization and Src regulation.18,24-27 Interestingly, Src has also been involved in caveolae internalization.27,28 Several observations indicate that caveolin-1 is a candidate tumor suppressor. Caveolin-1 expression is suppressed in oncogenically transformed

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BIO

PI3K

extracellular matrix p21 activated kinase P21 binding domain of Pak cholesterol enriched membrane microdomains phosphatidylinositol-3-OH kinase

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ECM Pak PBD CEMMs

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integrins, membrane domains, Rho GTPases, signaling, anchorage-dependent growth, caveolin-1

ACKNOWLEDGEMENTS

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This work was conducted as part of a EURYI scheme award (www.esf.org/euryi). The work was supported by MEC (Spanish Ministry of Science and Education) through grants SAF2005-00493 and GEN2003-20239-C0604 to M.A.d.P. and the Ramón y Cajal Program (to A.E. and M.A.d.P.); by EC Marie Curie International Reintegration Grant MIRG-CT- 2005-016427 to M.A.d.P.; and by the EMBO Young Investigator Programme.

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Caveolae Endocytosis and Integrin Signaling

cells, leading to the absence of detectable caveolae.29 Furthermore, reexpression of caveolin-1 in tumor cells restores anchorage-dependent growth.30 Notably, caveolin-1 in mutated in 16% of human breast cancers31 and loss of caveolin-1 sensitizes mice to tumor formation.32,33 The exact mechanism whereby caveolin-1 exerts its tumor suppressor activity remains elusive. We have shown that integrin-mediated internalization of CEMMs is dependent on caveolin-1 and dynamin-2 and independent on inhibitors of clathrin-mediated internalization. Furthermore, tyrosine phosphorylation of caveolin-1 residue 14 is required for internalization of CEMMs. These results show that integrins are negatively regulating caveolae internalization. This pathway regulates the activation extent of integrin-dependent signaling pathways, including Erk, PI3K and Rac, that are frequently hyperactivated in cancer cells and contribute to anchorage-independent cell growth.

CAVEOLIN-1 MEDIATES INTEGRIN-REGULATED CEMM INTERNALIZATION

Detachment of cells from the ECM induces a rapid internalization of membrane domains that are enriched in lipids, such as ganglioside GM1 and cholesterol, that are incorporated into caveolae.12 Several observations strongly suggest that integrins are regulating caveolin-1mediated CEMM internalization. Internalization of the lipid raft marker GM1 in the absence of integrin signaling is not inhibited by either a mutant form of Eps15 that blocks chlatrin-dependent endocytosis, or by inhibition of Cdc42-mediated signaling implicated in internalization of GPI-anchored proteins.3 Dominant negative dynamin-2, an inhibitor of caveolae-mediated endocytosis,16,17 completely inhibits GM1 internalization.3 Taken together, these results indicate that internalization of membrane domains in suspended cells is independent on classic clathrin-mediated endocytosis and Cdc42 signaling, but dependent on dynamin-2-mediated endocytosis. These observations suggest that a caveolae-mediated mechanism is clearing membrane domains from the plasma membrane in an integrin-dependent manner. This hypotheses is supported by direct observation of caveolin-1 in suspended cells. Inhibition of integrin signaling induces rapid clearance of caveolin-1 from the plasma membrane and increased caveolin-1 localization in an internal compartment. In addition, invaginated caveolae observed by electron microscopy are significantly reduced in cells in suspension.3 Strong evidence supporting the role of caveolin-1 in membrane domain clearance after cell detachment comes from studies in cells lacking caveolin-1 expression. M21L melanoma cells do not express caveolin-1, 2 or 3 at detectable levels and cannot internalize GM1 after detachment from the ECM. In contrast, caveolin-1 reconstituted M21L cells are capable of internalization.3 Notably, caveolin-1 knock-out derived mouse embryo fibroblasts (MEFs) behave similar to M21L cells regarding the inhibition of GM1 internalization after cell detachment, while reconstituting caveolin-1 expression in these cells restores GM1 internalization. These results clearly indicate that upon integrin uncoupling from intracellular signaling (i.e., after cell detachment from the ECM), GM1 domains are cleared from the plasma membrane by a caveolae-dependent mechanism.

ROLE OF CAVEOLIN-1 TYROSINE PHOSPHORYLATION IN INTEGRIN-REGULATED CEMM INTERNALIZATION

Internalization of caveolae can be induced by several stimuli.12,27,34,35 Tyrosine phosphorylation of caveolin-1 has been 2180

suggested to be important for caveolin-1-mediated signaling events24,25 and caveolae internalization.26,36,37 While caveolin-1 knock-out cells reconstituted with wild type caveolin-1 internalize GM1 after cell detachment, caveolin-1 Y14F reconstituted knock-out cells cannot internalize this ganglioside, strongly suggesting that tyrosine phosphorylation of Y14 is critical to internalize caveolae in an integrin-dependent manner.3 Furthermore, stimulation of caveolin-1 phosphorylation by sodium pervanadate (a phosphatase inhibitor) induces both tyrosine phosphorylation of caveolin-1 in Cos-7 cells and a concomitant integrin-dependent internalization of GM1 and caveolae. In cells attached to the ECM, tyrosine phosphorylated caveolin-1 is localized to focal ahesions24,26 and out of caveolae-as labeled by caveolin-1 staining.3 Interestingly, tyrosine phosphorylated caveolin-1 and caveolin-1 colocalize in detached cells, suggesting that integrins are regulating focal adhesion localization of phospho-caveolin-1 and preventing caveolae internalization. When cells are detached, phospho-caveolin-1 translocates to caveolae, where it induces internalization of caveolae. This mechanism is not relying in total levels of phospho-caveolin-1 to regulate internalization, but rather it is regulating the localization of phospho-caveolin-1 in an integrindependent manner. Grb7 and C-terminal Src kinase (Csk) interact with caveolin-1 in a Y14 phosphorylated manner, and shear stress-induced caveolin-1 phosphorylation triggers colocalization with Csk at focal adhesions.38 Future studies will determine whether Csk or/and other proteins are recruiting phospho-caveolin-1 to focal adhesions upon integrin activation. Other studies have involved FAK in regulating GM1-rich domains39 and SV40 internalization, a process partially dependent in caveolae.28 However, FAK appears not to be involved in integrin-mediated CEMM internalization, as FAK null MEFs show no differences in phospho-caveolin-1 localization at focal adhesion or integrin-mediated internalization of GPI-linked proteins.3 These findings suggest that different regulatory pathways are contributing to the internalization of CEMMs depending on the stimuli that induces their internalization, or alternatively that different subtypes of CEMMs exist at the plasma membrane and they are regulated by different pathways.

ANCHORAGE-DEPENDENT SIGNALING PATHWAYS AND CAVEOLIN-1-MEDIATED CEMM INTERNALIZATION

Endocytic entry routes transport proteins and lipids from the plasma membrane to the interior of the cell in order to regulate signaling intensity.40 Proper coupling of activated proteins with their effectors is critical in order to propagate the signal. Therefore, localization of activated proteins will regulate downstream cascades. Multiple signaling molecules are associated with caveolae, suggesting that caveolae compartmentalize signaling molecules and serve as platforms to regulate their activity, localization and/or effector coupling.3,13 Caveolae internalization serves to transport membrane receptors and ligands to the other side of the cell (transcellular transport), to an internal compartment or back to the same side of the plasma membrane (recycling).13 Similar to classic clathrin endocytic route, caveolin-1-dependent internalization also downregulates signaling.3,41 We have shown that integrin-mediated regulation of Erk and Akt pathways is regulated by caveolin-1. When integrin signaling is completely shut down in cells, Erk and PI3K pathways undergo a rapid downregulation in wild type MEFs. In contrast, caveolin-1 null MEFs retain Erk and Akt signaling intact

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A MODEL FOR INTEGRIN-MEDIATED CAVEOLAE INTERNALIZATION Our data suggest a model that underscores the tight connection between integrins, signaling pathways and caveolae internalization (Fig. 1). When cells are attached to the ECM, integrins negatively regulate caveolae/CEMM internalization, preventing uncoupling of signaling molecules such as Rac disengagement from its effector PAK (Fig. 1). Other pathways, such as Erk and PI3K pathways, are likely to be regulated by a similar mechanism. In addition, integrins regulate the focal adhesion localization of tyrosine phosphorylated caveolin-1, preventing its recruitment to caveolae and internalization (Fig. 1). This model predicts that the absence or inhibition of caveolae internalization would result in an increased signaling, that could participate in cell transformation. This mechanism could explain the cell growth regulatory role of caveolin-1. Interestingly, several oncogenes severely downregulate caveolin-1 expression.29 However, other molecular mechanisms may explain modulation of signaling pathways by caveolin-1, such as the proposed direct interaction with several tyrosine kinases through the scaffolding domain of caveolin-1.23 Elucidating the pathways regulating integrin-dependent caveolae internalization will undoubtedly reveal novel pathways potentially implicated in cell proliferation, cell migration and other cellular processes. References

Figure 1. Model for integrin-dependent regulation of caveolae/CEMM internalization. Under integrin-dependent signaling, i.e., in adherent cells (upper panel), caveolae/CEMMs are at the plasma membrane, allowing the proper plasma membrane targeting of Rac. This integrin-regulated process may be carried out directly by integrins or by an unknown signaling pathway. This pathway is likely to regulate at least the localization of phospho-caveolin-1 at focal adhesions, preventing caveolae internalization. When integrin signaling is interrupted, i.e., in non-adherent cells (lower panel), the pathways inhibiting caveolae/CEMM internalization are shut down, which results in endocytosis of caveolae/CEMMs. Internalization is mediated by translocation of phospho-caveolin-1 to caveolae/CEMMs, triggered by an unknown mechanism. Caveolae/CEMM internalization results in uncoupling of Rac from downstream effectors, resulting in a shut-down of the signal. In the cytosol, Rac is bound by RhoGDI, further blocking downstream signaling.

after integrin disengagement.3 These results show that caveolin-1 is an important player in the downregulation of signaling cascades whose activation depends on integrins. Similarly, TGFβ receptor is routed to caveolar pathway where it is degraded, resulting in an attenuation of the signal.41 These studies suggest that caveolae can regulate different pathways by clearing proteins from the plasma membrane. Interestingly, multiple kinases involved in signaling are regulating internalization of caveolae,28 underscoring the tight interconnection between caveolae and signaling pathways. As expected, not all integrin-regulated pathways are modulated by caveolin-1. Thus, FAK attenuation in cells in suspension is downregulated equally well in wild type or caveolin-1 knock-out cells, suggesting that this mechanism is not universally applied. www.landesbioscience.com

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