A Cortactin-CD2-associated Protein (CD2AP) Complex Provides a Novel Link between Epidermal Growth Factor Receptor Endocytosis and the Actin Cytoskeleton

Abstract

Growth factor regulation of the cortical actin cytoskeleton is fundamental to a wide variety of cellular processes. The cortical actin-associated protein, cortactin, regulates the formation of dynamic actin networks via the actin-related protein (Arp)2/3 complex and hence is a key mediator of such responses. In order to reveal novel roles for this versatile protein, we used a proteomics-based approach to isolate cortactin-interacting proteins. This identified several proteins, including CD2-associated protein (CD2AP), as targets for the cortactin Src homology 3 domain. Co-immunoprecipitation of CD2AP with cortactin occurred at endogenous expression levels, was transiently induced by epidermal growth factor (EGF) treatment, and required the cortactin Src homology 3 domain. The CD2AP-binding site for cortactin mapped to the second of three proline-rich regions. Because CD2AP is closely related to Cbl-interacting protein of 85 kDa (CIN85), which regulates growth factor receptor down-regulation via complex formation with Cbl and endophilin, we investigated whether the CD2AP-cortactin complex performs a similar function. EGF treatment of cells led to transient association of Cbl and the epidermal growth factor receptor (EGFR) with a constitutive CD2AP-endophilin complex. Cortactin was recruited into this complex with slightly delayed kinetics compared with Cbl and the EGFR. Immunofluorescence analysis revealed that the EGFR, CD2AP, and cortactin co-localized in regions of EGF-induced membrane ruffles. Therefore, by binding both CD2AP and the Arp2/3 complex, cortactin links receptor endocytosis to actin polymerization, which may facilitate the trafficking of internalized growth factor receptors. Growth factor regulation of the cortical actin cytoskeleton is fundamental to a wide variety of cellular processes. The cortical actin-associated protein, cortactin, regulates the formation of dynamic actin networks via the actin-related protein (Arp)2/3 complex and hence is a key mediator of such responses. In order to reveal novel roles for this versatile protein, we used a proteomics-based approach to isolate cortactin-interacting proteins. This identified several proteins, including CD2-associated protein (CD2AP), as targets for the cortactin Src homology 3 domain. Co-immunoprecipitation of CD2AP with cortactin occurred at endogenous expression levels, was transiently induced by epidermal growth factor (EGF) treatment, and required the cortactin Src homology 3 domain. The CD2AP-binding site for cortactin mapped to the second of three proline-rich regions. Because CD2AP is closely related to Cbl-interacting protein of 85 kDa (CIN85), which regulates growth factor receptor down-regulation via complex formation with Cbl and endophilin, we investigated whether the CD2AP-cortactin complex performs a similar function. EGF treatment of cells led to transient association of Cbl and the epidermal growth factor receptor (EGFR) with a constitutive CD2AP-endophilin complex. Cortactin was recruited into this complex with slightly delayed kinetics compared with Cbl and the EGFR. Immunofluorescence analysis revealed that the EGFR, CD2AP, and cortactin co-localized in regions of EGF-induced membrane ruffles. Therefore, by binding both CD2AP and the Arp2/3 complex, cortactin links receptor endocytosis to actin polymerization, which may facilitate the trafficking of internalized growth factor receptors. Subcellular compartmentalization and trafficking of signal transduction complexes and a variety of dynamic cellular responses to extracellular stimuli require regulated interactions between specific components of signaling pathways and the cytoskeleton. These interactions may be direct or mediated by particular adaptor or scaffolding proteins. Among these, cortactin was identified as a v-Src substrate associated with the cortical actin cytoskeleton approximately a decade ago, although insights into its cellular function and the underlying mechanisms have only been obtained recently (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar). In line with an adaptor role, cortactin is a multidomain protein, with the individual modules capable of mediating specific protein-protein interactions (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar). The N-terminal region mediates binding to the Arp 1The abbreviations used are: Arp, actin-related protein; ASAP, ARF GTPase-activating protein containing Src homology 3, ankyrin repeat, and pleckstrin homology domains; CD2AP, CD2-associated protein; CIN85, Cbl-interacting protein of 85 kDa; CMS, Cas ligand with multiple Src homology 3 domains; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; FCS, fetal calf serum; FISH, five Src homology 3 domains; GFP, green fluorescent protein; GST, glutathione S-transferase; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; μLC-MS/MS, microcapillary liquid chromatography tandem mass spectrometry; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; SH3, Src homology 3; TBS, Tris-buffered saline; WASP, Wiskott-Aldrich syndrome protein.1The abbreviations used are: Arp, actin-related protein; ASAP, ARF GTPase-activating protein containing Src homology 3, ankyrin repeat, and pleckstrin homology domains; CD2AP, CD2-associated protein; CIN85, Cbl-interacting protein of 85 kDa; CMS, Cas ligand with multiple Src homology 3 domains; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; FCS, fetal calf serum; FISH, five Src homology 3 domains; GFP, green fluorescent protein; GST, glutathione S-transferase; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; μLC-MS/MS, microcapillary liquid chromatography tandem mass spectrometry; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; SH3, Src homology 3; TBS, Tris-buffered saline; WASP, Wiskott-Aldrich syndrome protein.2/3 complex, a highly conserved regulator of the assembly and structure of actin networks (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar), and contains a DDW motif characteristic of Arp2/3-interacting proteins such as WASP, Myo3p, and ActA (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar, 3Uruno T. Liu J. Zhang P. Fan Y.-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (454) Google Scholar). This is followed by six and a half copies of a 37-amino acid repeat, with the fourth repeat necessary for binding to F-actin in vitro (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar). Downstream of the repeats is a predicted helical domain and a region rich in serine, threonine, and proline residues. The latter is a target for both tyrosine and serine/threonine phosphorylation (4Huang C. Ni Y. Wang T. Gao Y. Haudenschild C.C. Zhan X. J. Biol. Chem. 1997; 272: 13911-13915Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 5Huang C. Liu J.L. Haudenschild C.C. Zhan X. J. Biol. Chem. 1998; 273: 25770-25776Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 6Campbell D.H. Sutherland R.L. Daly R.J. Cancer Res. 1999; 59: 5376-5385PubMed Google Scholar). The C terminus of cortactin is characterized by an SH3 domain. Several binding partners for this module have been identified, including CortBP1/Shank 2 (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar, 7Du Y. Weed S.A. Xiong W.-C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (219) Google Scholar), ZO-1 (8Katsube T. Takahisa M. Ueda R. Hashimoto N. Kobayashi M. Togashi S. J. Biol. Chem. 1998; 273: 29672-29677Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and dynamin 2 (9McNiven M.A. Kim L. Krueger E.W. Orth J.D. Cao H. Wong T.W. J. Cell Biol. 2000; 151: 187-198Crossref PubMed Scopus (342) Google Scholar). Cortactin localizes with the Arp2/3 complex at sites of dynamic cortical actin assembly such as lamellipodia (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar). Cortactin binding leads to a relatively weak stimulation of the actin nucleation activity of the Arp2/3 complex (10Weaver A.M. Karginov A.V. Kinley A.W. Weed S.A. Li Y. Parsons J.T. Cooper J.A. Curr. Biol. 2001; 11: 370-374Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), but it can enhance actin polymerization induced by co-activators of Arp2/3 such as WASP family proteins (3Uruno T. Liu J. Zhang P. Fan Y.-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (454) Google Scholar, 10Weaver A.M. Karginov A.V. Kinley A.W. Weed S.A. Li Y. Parsons J.T. Cooper J.A. Curr. Biol. 2001; 11: 370-374Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 11Weaver A.M. Heuser J.E. Karginov A.V. Lee W.-I. Parsons J.T. Cooper J.A. Curr. Biol. 2002; 12: 1270-1278Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Furthermore, cortactin promotes the formation of branched actin networks and their stabilization (10Weaver A.M. Karginov A.V. Kinley A.W. Weed S.A. Li Y. Parsons J.T. Cooper J.A. Curr. Biol. 2001; 11: 370-374Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). One function of cortactin is to regulate the organization and subcellular localization of transmembrane complexes. The cortactin SH3 domain target CortBP1/Shank 2 performs a scaffolding function in the organization of receptor complexes at post-synaptic sites of excitatory synapses, whereas a further SH3 binder, ZO-1, interacts with the transmembrane proteins claudin and occludin at epithelial tight junctions (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar). A second functional role for cortactin is regulation of receptor-mediated endocytosis, via its interaction with dynamin 2, a mechanochemical GTPase that participates in the scission of endocytic vesicles at the plasma membrane (9McNiven M.A. Kim L. Krueger E.W. Orth J.D. Cao H. Wong T.W. J. Cell Biol. 2000; 151: 187-198Crossref PubMed Scopus (342) Google Scholar, 12Cao H. Orth J.D. Chen J. Weller S.G. Heuser J.E. McNiven M.A. Mol. Cell. Biol. 2003; 23: 2162-2170Crossref PubMed Scopus (182) Google Scholar). In addition, cortactin localizes with the Arp2/3 complex at cytoplasmic spots associated with endosomal vesicles, suggesting a role in driving vesicle trafficking (13Kaksonen M. Peng H.B. Rauvala H. J. Cell Sci. 2000; 113: 4421-4426Crossref PubMed Google Scholar). Therefore, cortactin is representative of a growing list of proteins that physically link the endocytic machinery with components or regulators of the actin cytoskeleton. These include mAbp1, which binds F-actin and dynamin, and which links proteins to the Arp2/3 complex D.A. Curr. Cell Biol. 2002; PubMed Scopus Google Scholar). the 2 complex is in actin including the growth formation of the that (9McNiven M.A. Kim L. Krueger E.W. Orth J.D. Cao H. Wong T.W. J. Cell Biol. 2000; 151: 187-198Crossref PubMed Scopus (342) Google Scholar, D.A. Weed S.A. Karginov A.V. Parsons J.T. Cooper J.A. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar, E.W. Orth J.D. Cao H. McNiven M.A. Mol. Biol. 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J. 2002; PubMed Scopus Google Scholar). CD2AP or Cbl and 2 The cortactin was and was The was the of T. H. H. H. T. Cancer Res. 2002; Google Scholar). was proteins to the cortactin helical and proline-rich regions and the SH3 domain as D.H. Sutherland R.L. Daly R.J. Cancer Res. 1999; 59: 5376-5385PubMed Google Scholar). expression for cortactin, the and as D.H. Sutherland R.L. Daly R.J. Cancer Res. 1999; 59: 5376-5385PubMed Google Scholar, 2000; PubMed Scopus Google Scholar). The a of the SH3 domain was obtained of expression CD2AP was by of the three proline-rich regions of CD2AP S. H. Sci. S. A. 1999; PubMed Scopus Google Scholar), and and an cortactin J.E. Sci. S. A. PubMed Scopus Google with an site at the and a and an site at the can be and to J. T. A and into the sites of of CD2AP by was CD2AP in Li J. S. N. P. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). and to the The was One was can be in both to that the required been and of used to to proteins by chromatography PubMed Scopus Google Scholar). and proteins to A Scholar). to of with protein, the of was by to of for at proteins by with in for at and three in containing Daly R.J. A. Sutherland R.L. Oncogene. Google Scholar). The of was with the for 2 at and five in by to a and to The was to analysis of by mass by of protein with of the in to analysis by in which proteins by of was with of The in to by the and to the and MALDI-TOF mass was a with a of to the of for protein used to the and via the was by R. T. and Scholar). was a with an for with of SH3 or proteins by with for at The was by of for proteins by to a and for in in with the proteins for and in containing protein was the cells in transiently with EGFR. The cells for to stimulation with in in and in in with CD2AP in containing cells with for These and a with a and of to the SH3 of of is associated with in but R. D.H. Lee Daly R.J. R. Sutherland R.L. Oncogene. 1997; PubMed Scopus Google Scholar). In order to proteins with the cortactin SH3 region in we chromatography This line was it and a H. P. J. E.W. Cancer Res. PubMed Scopus Google Scholar). was a of a protein to investigated the of the cortactin region by the and was as a proteins by and by proteins by the SH3 domain and by the One protein of kDa the The was and proteins by and proteins identified by MALDI-TOF or Among the proteins identified to as CD2AP Li J. S. N. P. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. Sci. S. A. 1999; PubMed Scopus Google Scholar), P. T. S.A. J. 1998; PubMed Scopus Google Scholar), J. H. Cooper J.A. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar), and M. H. T. 1997; PubMed Scopus Google Scholar). The mass obtained for CD2AP is in The specific binding of proteins to the cortactin SH3 domain was by of of cortactin SH3 domain with or proteins. proteins by with specific proteins identified by mass of CD2AP with Cortactin in FISH, and have been in regulation of and the three proteins of Src family S. H. Sci. S. A. 1999; PubMed Scopus Google Scholar, P. T. S.A. J. 1998; PubMed Scopus Google Scholar, J. H. Cooper J.A. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar, Curr. Cell Biol. 2002; PubMed Scopus Google Scholar). These that their as cortactin SH3 targets a functional with we have been to or with cortactin at endogenous expression of the binding although was recently to with cortactin in in an SH3 H. H. H. T. Cancer Res. 2002; Google Scholar). In order to the association of cortactin with CD2AP in analysis was of or of cortactin cells with a and the revealed of the proteins, that at expression The for the cortactin SH3 domain in CD2AP association was investigated by a of cortactin the SH3 domain in with an expression for cortactin as a The cortactin, but the be in CD2AP that the cortactin SH3 domain is for interaction of proteins in with the association of the endogenous proteins, was by EGF The in of and homology that CD2AP and of the of adaptor proteins H. S. N. S. Res. 2000; PubMed Scopus Google Scholar). P. 2002; PubMed Scopus Google Scholar, A. S. N. S. 2002; PubMed Scopus Google have a role for CIN85, in a complex with and in receptor-mediated In this complex, the SH3 to whereas the SH3 region binds a specific proline-rich motif in This the that CD2AP may in growth factor receptor this we investigated the protein-protein interactions CD2AP EGF stimulation and their cells and with EGF for Because CD2AP association with Cbl is Cbl tyrosine phosphorylation T. H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), we investigated the tyrosine phosphorylation of proteins with with revealed three proteins of and kDa and a tight of which between and of EGF stimulation A protein of kDa was only at 2 and with specific identified the and as the and CD2AP association with was constitutive and by EGF CIN85, CD2AP with both Cbl and with a revealed a of association in EGF was a but transient in complex formation that at and to the of of kinetics and that the protein in CD2AP at 2 and EGF treatment is to be These with of cortactin to the endocytic complex, but association of cortactin with CD2AP is slightly delayed compared with that of for is that complexes CD2AP-cortactin and this we whether Cbl and the be in cortactin of cortactin with an revealed an of identified as Cbl A of cortactin was and is to the EGFR. Because we have the of CD2AP in cortactin that cortactin is recruited to the complex. to that the of cortactin and CD2AP was by their SH3 to the proline-rich region of dynamin 2, we investigated whether dynamin 2 be in protein complexes. dynamin 2 was in it was CD2AP This that the association between CD2AP and cortactin is to be of the for the Cortactin SH3 contains three proline-rich regions sites S. H. Sci. S. A. 1999; PubMed Scopus Google The binding for the cortactin SH3 domain been characterized as and and acid J.E. Sci. S. A. PubMed Scopus Google Scholar). of the CD2AP acid reveal an with this In order to the binding of the three proline-rich regions of CD2AP was as a A protein to the binding for the cortactin SH3 domain was as a These proteins, and as a to a with or binding of the SH3 domain to was but binding to and to was These that the cortactin SH3 domain can to proline-rich and Because both cortactin and CD2AP via SH3 it was of to the binding of the SH3 domain to with that of the cortactin SH3 The SH3 domain to and relatively to and This that in may whereas cortactin is recruited to In order to the cortactin SH3 domain binding site in proline to into in CD2AP to the cortactin SH3 domain binding and proteins transiently in cells and binding to the cortactin SH3 domain was by protein analysis of the motif binding to that of the protein, whereas of a of in a which was binding of the was compared with that of the whether association of the proteins in cells followed this binding the CD2AP proteins and for cortactin In with the was identified as the interaction with a the a role for be is that the and in their to binding of the of the protein to endogenous this site the in with the for the cortactin SH3 domain for association with CD2AP and the of the cortactin SH3 domain to to that association of the proteins in cells is mediated by direct binding of the cortactin SH3 domain to the region of the binding activity in interactions a to complex CD2AP, and the in EGF CD2AP been to lamellipodia and cytoplasmic S. H. Sci. S. A. 1999; PubMed Scopus Google Scholar, T. N. J. 2001; PubMed Google Scholar). The association between cortactin and CD2AP and the of the in this complex and led to the subcellular of proteins by cells transiently with an expression EGFR, and for or with the plasma of EGF treatment, the in membrane and by into the Cortactin and CD2AP in the cells by CD2AP and cortactin to cytoplasmic in and transiently in membrane of EGF In cells CD2AP and cortactin with the EGFR. of EGF three proteins to the membrane as as a of cytoplasmic This in was transient and to the of cells of EGF These with the transient of complex formation by analysis and the the formation of an EGF-induced complex containing both CD2AP and cortactin at membrane that is to regulate trafficking of internalized the role of cortactin as an adaptor transmembrane or protein with the Arp2/3 complex and hence sites of dynamic actin we it that binding partners for the cortactin SH3 domain may to this by that cortactin may be in cellular roles for T. N. J. 2001; PubMed Google CD2AP in cytoplasmic spots in and that for cortactin, and the Arp2/3 complex. in an of CD2AP and cortactin to the including F-actin and the of cells D.A. C. Cooper J.A. 2000; PubMed Scopus Google Scholar). in this we the that the scaffolding protein CD2AP with the cortactin SH3 and we a functional role for this complex in of cortactin to CD2AP the cortactin SH3 as by and and the cortactin The of dynamin 2 in CD2AP the that the association is to of CD2AP and cortactin to a dynamin 2 The association between cortactin and CD2AP is to be the CD2AP and required for binding in both and and the cortactin SH3 domain binds the region in a as for P. 2002; PubMed Scopus Google Scholar, A. S. N. S. 2002; PubMed Scopus Google Scholar), CD2AP with the SH3 domain to which contains the SH3 binding L. L. A. N. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and relatively to and Therefore, it is that in binds to whereas cortactin binds to and to a a association of cortactin with CD2AP was in a but transient in of the proteins was EGF treatment Because SH3 domain binding was phosphorylation of the proline-rich binding motif J.E. Sci. S. A. PubMed Scopus Google Scholar), the for this is a in cortactin CD2AP, which complex formation by the of the cortactin SH3 domain its target CD2AP is in cells or EGF treatment of cells to CD2AP tyrosine phosphorylation S. H. Sci. S. A. 1999; PubMed Scopus Google Scholar). phosphorylation of the related been The of a protein of kDa in CD2AP of EGF stimulation is with EGF-induced cortactin tyrosine phosphorylation with similar kinetics to a transient 2 of stimulation M. L. J. R. Parsons J. Parsons S. Oncogene. Google Scholar, H. H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, is that cortactin tyrosine phosphorylation of its SH3 domain. A further which is is the of of proteins into a complex. In this it is that the association of CD2AP with Cbl is tyrosine phosphorylation of This is to be to a in Cbl to binding of proline-rich to the second SH3 domain of CD2AP T. H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). EGF-induced complex formation between and Cbl the and transient tyrosine phosphorylation of Cbl H. S. N. S. Res. 2000; PubMed Scopus Google Scholar). Therefore, it is that binding of CD2AP to Cbl of the region of CD2AP to the cortactin SH3 domain. CD2AP been to a role in the formation of or tight junctions in both the in and in In the complex is the the transmembrane Li J. S. N. P. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas in the the tight is the CD2AP a complex with and Li J. R. P. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. M.A. C. P. 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S. 2002; PubMed Scopus Google The binding of cortactin to this complex a link to F-actin assembly via Because the of cortactin to CD2AP is slightly delayed compared with that of it is that cortactin regulate endocytic membrane but vesicle the the in CD2AP both cortactin and This with the association of with the EGFR, which leads to of both proteins in the N. Sci. S. A. 2002; PubMed Scopus Google Scholar). or between the of CD2AP and is that the receptor and Cbl and the J. H. J. J. Cell Sci. 2001; PubMed Google Scholar). In we have identified CD2AP as a cortactin binding a functional between CD2AP and CIN85, and revealed a novel link between the endocytic machinery and the actin cytoskeleton to an role in the trafficking of for with