CAP (Cbl associated protein) regulates receptor-mediated endocytosis

FEBS Letters 583 (2009) 293–300

journal homepage: www.FEBSLetters.org

CAP (Cbl associated protein) regulates receptor-mediated endocytosis Daniela Tosoni a, Gianluca Cestra b,* a b

IFOM, Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milan, Italy Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italy

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Article history: Received 26 September 2008 Revised 2 December 2008 Accepted 16 December 2008 Available online 29 December 2008 Edited by Lukas Huber Keywords: Dynamin Receptor trafficking Podosome

a b s t r a c t CAP (c-Cbl associated protein)/ponsin belongs to a family of adaptor proteins implicated in cell adhesion and signaling. Here we show that CAP binds to and co-localizes with the essential endocytic factor dynamin. We demonstrate that CAP promotes the formation of dynamin-decorated tubule like structures, which are also coated with actin filaments. Accordingly, we found that the expression of CAP leads to the inhibition of dynamin-mediated endocytosis and increases EGFR stability. Thus, we suggest that CAP may coordinate the function of dynamin with the regulation of the actin cytoskeleton during endocytosis. Structured summary: MINT-6804322: CAP (uniprotkb:Q9BX66) physically interacts (MI:0218) with Cbl (uniprotkb:Q8K4S7) and dynamin 2 (uniprotkb:P39052) by pull down (MI:0096) MINT-6804285: CAP (uniprotkb:Q9BX66) physically interacts (MI:0218) with FAK (uniprotkb:O35346), vinculin (uniprotkb:P85972) and dynamin 2 (uniprotkb:P39052) by pull down (MI:0096) MINT-6804245, MINT-6804259, MINT-6804272: CAP (uniprotkb:Q9BX66) physically interacts (MI:0218) with dynamin 2 (uniprotkb:P39052) by pull down (MI:0096) MINT-6804344: CAP (uniprotkb:Q9BX66) physically interacts (MI:0218) with dynamin 2 (uniprotkb:P50570) by anti tag coimmunoprecipitation (MI:0007) MINT-6804371: dynamin 1 (uniprotkb:P21575) physically interacts (MI:0218) with CAP (uniprotkb:O35413) by anti bait coimmunoprecipitation (MI:0006) MINT-6804446, MINT-6804464: F-actin (uniprotkb:P60709), CAP (uniprotkb:Q9BX66) and dynamin 2 (uniprotkb:P50570) colocalize (MI:0403) by fluorescence microscopy (MI:0416) Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Vinexin, CAP (c-Cbl associated protein)/ponsin, and ArgBP2 (Arg Binding Protein 2) belong to a family of adaptor proteins characterized by the presence of an NH2-terminal sorbin homology (SoHo) region and three COOH-terminal SH3 (Src homology domain 3) [1]. The sorbin domain has been recently suggested to operate as a protein–protein interaction motif that binds to lipid raft enriched proteins [2] and to cortical cytoskeleton proteins [3]. CAP/ponsin (hereafter referred to as only CAP) was independently identified as a protein associated to the signaling ubiquitin ligase Cbl [4] and as a component of the adherens junctions connected to the nectin–afadin system [5]. Recently, CAP has been implicated in the insulin signaling [6], although its exact role in the pathway is still controversial [7].

* Corresponding author. Fax: +39 06 4456866. E-mail address: [email protected] (G. Cestra).

Here we show that CAP binds to the major endocytic factor dynamin and affects the actin cytoskeleton organization. Accordingly, we found that CAP expression impairs receptor-mediated endocytosis. 2. Materials and methods 2.1. Plasmids The cDNA encoding for mouse FLAG-tagged CAP was a generous gift of Alan R. Saltiel (University of Michigan).

2.2. Antibodies The antibodies used in this study are: rabbit polyclonal anti-CAP (Upstate-Millipore, Temecula, CA, USA), mouse monoclonal antivinculin (Sigma–Aldrich, St. Louis, MO, USA), mouse monoclonal anti-FAK (Upstate-Millipore), mouse monoclonal anti-dynamin1

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.12.047

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(Hudy1, Upstate-Millipore) and DG1 (De Camilli, Yale University, Connecticut, USA), goat anti-dynamin2 (C-18, Santa Cruz, CA, USA), rabbit anti-dynamin2 (McNiven, Mayo Clinic, MN, USA), mouse monoclonal anti-FLAG, mouse monoclonal anti-FLAG conjugated beads and rabbit polyclonal anti-FLAG (Sigma–Aldrich), rabbit anti-Cbl (C-15, Santa Cruz), mouse monoclonal anti-EGFR (Millipore, USA), rabbit anti-EGFR (Di Fiore, IFOM, Milan, Italy), anti-phospho-MAPK (p42/44), anti-phospho-AKT (Ser 473), antiAKT (Cell Signaling).

Fluorescence was visualized with Axiophot epifluorescent microscope (Carl Zeiss Inc., Thornwood, NY) using 40 and 63 oil-immersion objectives. 2.4. EGF and transferrin internalization assays

2.3. Cell culture and immunofluorescence microscopy

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HEK293, HeLa and COS-7 cells were purchased from ATCC. Cells were grown in standard Dulbecco’s modified Eagle’s medium (GIBCO-Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (GIBCO-Invitrogen). Primary cultures of osteoclasts were prepared as previously described [8]. Cell transfections were performed with Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Immunofluorescence staining experiments were performed as previously described [9]. Oregon Green and Texas Red-conjugated secondary antibodies and Texas Red-conjugated phalloidin were purchased (Molecular Probes-Invitrogen).

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Internalization assay of fluorescently conjugated ligands was performed in HeLa cells as described [10,11]. Receptor internalization block was measured in two independent experiments, counting at least 100 cells per condition in duplicate. In the biotinylation assay HEK293 cells were starved for 16 h and incubated with reactive NHS-SS-biotin compound (0.5 mg/ ml) (Pierce, Rockford, IL, USA) dissolved in tyrode buffer (10 mM HEPES, 136 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2) for 3 min at 37 °C. After two washes in tyrode buffer, cells were incubated for 10 min at 37 °C in Dulbecco’s modified Eagle’s serum-free medium plus EGF (20 ng/ml) (Upstate-Millipore). Cells were kept on ice for 1 hour in reducing buffer (100 mM DTT, 150 mM NaCl; 100 mM TRIS pH 8,8) and incubated in ice cold blocking buffer [50 mM iodoacetamide (Sigma–Aldrich), 250 mM Tris pH 8] for 30 min. Finally, cells were lysated in ice cold RIPA buffer [Tris 20 mM pH 7.5, NaCl 100 mM, NaF 50 mM, NP40 1%, DOC 0.1%, SDS 0.1%, EDTA

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vinculin dynamin2 Cbl FAK Fig. 1. (A) CAP binds to dynamins. The three SH3 domains of CAP, fused to the GST domain, were used in affinity purification experiments with rat brain extract (BTE, brain Triton X-100 extract). Pulled down proteins and GST fusion SH3 domains incubated with no extract were resolved by SDS–PAGE and stained with Coomassie Blue. Asterisk marks the position of dynamin protein. (B) Anti-dynamin2 rabbit polyclonal antibody was used in western blotting to detect dynamin2 in protein material bound to each CAP GST-SH3 domain (SM, starting material; B bound material; NB, unbound material). (C) The amount of dynamin2 and other CAP interacting proteins, retained by the three GST-SH3 domains and by the GST as control, was compared by western blotting analysis using the indicated antibodies. (D) Immunoprecipitation of FLAG-tagged CAP from HEK293 cells expressing FLAG-CAP and dynamin2-HA. Total mouse immunoglobulins (IgG) were used as a negative control of the immunoprecipitation experiments. Presence of CAP and dynamin2 in the immunoprecipitated proteins retained by the anti-FLAG conjugated beads was detected by western blotting using anti-CAP and antidynamin2 rabbit polyclonal antibodies. (E) Immunoprecipitation of endogenously expressed dynamin1 from rat cerebellum extract, utilizing the anti-dynamin1 mouse monoclonal antibody DG1. Mouse total immunoglobulins were used as a negative control of the immunoprecipitation experiments. Immunoprecipitated protein material was assayed by western blotting using anti-CAP rabbit polyclonal antibody.

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1 mM, and protease inhibitor cocktail (Roche, Mannheim, Germany)]. After centrifugation of cell extract at 9500g for 10 min at 4 °C, the supernatant was incubated with streptavidin-conjugated beads (Sigma–Aldrich) for 2 h at 4 °C under gently rocking. Proteins retained by the beads were resuspended in Laemmli buffer, boiled and subjected to SDS–PAGE and western blotting analysis. 2.5. EGFR stability assay HeLa cells were transfected with Lipofectamine according to the manufacturer’s instructions (Invitrogen) and starved in serum-free and antibiotic-free medium for 16–18 h. After treatment with EGF (100 ng/ml) at 37 °C for the indicated time, cells were lysated in ice cold RIPA buffer (see above) and processed for western blotting analysis. 2.6. Immunoprecipation and pull-down experiments Tissue homogenization and GST pull-down experiments were performed as previously indicated [12]. Immunoprecipitations were performed using RIPA buffer cell lysates or homogenized tissues. 3. Results 3.1. The SH3 domains of CAP bind to dynamins We set out to search for new interactors of CAP using an affinity purification approach. GST fusion proteins of each of the three SH3 domains of CAP (SH3-1, SH3-2 and SH3-3) were utilized as baits in pull-down assays using rat brain protein extracts. Proteins bound to the GST-fusions were analyzed by SDS–PAGE and by western blotting. From its molecular weight, its abundance in the rat brain extract (Fig. 1A, asterisk) and its well characterize binding to SH3 containing proteins [3] we supposed that the more prominent

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binding partner of CAP in our pull-down experiment was the endocytic protein dynamin (Fig. 1A). Indeed we confirmed by western blotting that the major interactor of CAP SH3 domains was dynamin (both dynamin1 and dynamin2) (Fig. 1B). Dynamin GTPase is a key player in the regulation of actin dynamics [13–17] and in the fission of a variety of endocytic buds, including clathrin-coated pits [18,19]. The interaction of the three SH3 domains of CAP with dynamin was compared to their interaction with well-known CAP binding partners, such as Cbl, FAK and vinculin [4,5,20]. The apparent binding affinity of each SH3 domain of CAP for dynamin was comparable and even stronger to its affinity for Cbl and FAK (Fig. 1C) [4,5,20]. 3.2. Dynamins co-precipitates with CAP To further confirm the interaction between CAP and Dynamin, we performed immunoprecipitation experiments using HEK293 cells exogenously transfected with plasmids encoding FLAG-tagged CAP and HA-tagged dynamin2. Protein extracts from transfected cells were used to immunoprecipitate FLAG-CAP with an anti-FLAG antibody. As a control, cell extracts were subjected to immunoprecipitation with mouse immunoglobulins. HA-dynamin2 was detected in CAP immunoprecipitates, while it was absent from control immunoprecipitates (Fig. 1D). Paralleling results obtained with exogenously transfected HEK293 cells the endogenous CAP protein was specifically recovered from rat cerebellum extracts subjected to immunoprecipitation with an anti-dynamin1 antibody (Fig. 1E). Cerebellum was chosen as a source of proteins because of the good level of CAP expression in this tissue [21]. 3.3. CAP co-localizes with dynamin Podosomes are adherent points formed by a dense central core of actin filaments surrounded by adhesion and scaffolding proteins. The dynamic assemble and disassemble of a great number of podosome units within a single cell can generate specific patterns such

Fig. 2. CAP and dynamin2 co-localize to podosomes of osteoclast cells. Endogenous localization of CAP, dynamin2 and F-actin in primary culture of osteoclasts was studied by immunofluorescence using rabbit anti-CAP polyclonal antibody, goat anti-dynamin2 antibody and fluorescently labeled phalloidin. At low magnification (main fields) CAP, dynamin and F-actin localize together at the peripheral belt of podosomes (Fig. 2, main fields) (Scale bar, 20 lm). At higher magnification (Fig. 2, insets), CAP and dynamin2 localize together around the central core of F-actin, which identifies each single podosome unit (Fig. 2, insets) (Scale bar, 4 lm in the inset).

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as clusters, or belts [22]. Since CAP and dynamin are highly expressed in cells forming podosome belts such primary cultures of osteoclasts [23–25] we used this cell system to study their endogenous localization. At low magnification, CAP showed a high degree of co-localization with dynamin2 and actin in podosome belts (Fig. 2, main fields). At higher magnification, CAP and dynamin2 immunoreactivities were both distributed around the central core of F-actin, which identifies each podosome unit (Fig. 2, insets).

endogenous dynamin2 on CAP-induced tubule like structures (Fig. 3B) and to cause a significant reduction of its cytoplasmic pool. Similar tubule like structures are typically observed when the membrane fission activity of dynamin is blocked or strongly impaired [26]. In fact, inhibition of the pinching off activity required for the resolution of membrane pits in membrane vesicles usually results in the formation of elongated membrane tubules [26].

3.4. Ectopic expressed CAP co-localizes with dynamin2 and causes the formation of dynamin2-containing tubule like structures

3.5. CAP over-expression inhibits receptors internalization

The concomitant over-expression of FLAG-CAP and dynamin2GFP in Cos-7 or HeLa (data not shown) cells causes the formation of a prominent mesh of dynamin-coated membrane tubule like structures (Fig. 3A), which are never observed with the sole expression of dynamin2-GFP (Fig. 4B and D). Moreover, the over-expression of FLAG-CAP in HeLa cells is sufficient to re-localize

Interfering with dynamin function through the expression of specific binding partners [27] or through the introduction of specific mutations in the dynamin GTPase domain [28–30] induces the formation of specific dynamin membrane tubules similar to those induced by the over-expression of CAP. This suggests an inhibitor action of CAP on dynamin function.

Fig. 3. Effect of CAP expression on dynamin2 localization. (A) The expression of FLAG-CAP in Cos-7 cells promotes the formation of elongated dynamin2-containing tubule like structures, which are enriched in F-actin. Cos-7 cells were co-transfected FLAG-CAP and dynamin2-GFP plasmids and processed for immunofluorescence with anti-FLAG rabbit polyclonal antibody and fluorescently labeled phalloidin. Both CAP and dynamin2 proteins localize with F-actin to tubule like structures (Scale bar, 10 lm). (B) The expression of FLAG-CAP in HeLa cells promotes the re-localization of endogenous dynamin2 on CAP mediated tubule like structures, with a significant reduction of the dynamin2 cytoplasmatic pool. FLAG-CAP transfected HeLa cells were processed for immunofluorescence with anti-FLAG mouse monoclonal antibody and anti-dynamin2 rabbit polyclonal antibody (Scale bar, 20 lm).

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To directly address this hypothesis, given the well established role of dynamin in both ligand-induced (e.g. EGF) and constitutive (e.g. transferrin) receptor-mediated endocytosis, we analyzed the internalization of EGF and transferrin receptors in cells ectopically expressing CAP. We found that the expression of FLAG-CAP reduced the internalization of both transferrin and EGF receptors (Figs. 4 and 5). Finally, we analyzed the effect of CAP on EGFR internalization using an in vivo biotinylation assay [31], which allows to study intracellular trafficking of EGF receptor from the very initial steps of internalization. In HEK293 cells expressing GFP as a control, treatment with EGF caused internalization of EGFR (Fig. 5B and C). By contrast, no increase in EGFR internalization was observed in FLAG-CAP over-expressing cells stimulated with EGF. This block was very similar to the inhibition of EGFR internalization caused by over-expression of a mutated form of dynamin2 (K44A) (Fig. 5B and C). Dynamin2 K44A in fact is a very well characterized dominant negative mutant, which is defective in both

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GTP binding and hydrolysis, thereby exerting a potent inhibitory effect in dynamin-dependent endocytosis [30]. Furthermore, blocking EGFR internalization with either CAP or dynamin2 K44A expression did not affect significantly the activation of mitogenactivated protein kinase signaling as indicated by the level of the anti-phospho-p42/44 mitogen-activated protein kinase (Fig. 5C). 3.6. CAP over-expression increases EGFR stability The internalized receptors can follow two different trafficking routes: being recycled to the cell surface while they are keeping signaling along their way to the plasma membrane or being trafficked to lysosomes where they are degraded [32]. In order to investigate the effect of CAP on EGFR receptor degradation and signaling we performed a time course of EGF stimulation in HeLa cells expressing CAP. As shown in Fig. 5E the expression of CAP significantly decreases degradation rate of EGFR, especially at long time

Fig. 4. The expression of CAP blocks both EGF and transferrin endocytosis. The internalization of fluorescent-labeled transferrin in HeLa is reduced by the expression of FLAGCAP (A), while the expression of dynamin2-GFP has no effect (B) (Scale bar, 10 lm). The expression of FLAG-CAP in HeLa strongly impairs the internalization of fluorescently labeled EGF (C), while the expression of dynamin2-GFP has no effect (D) (Scale bar, 10 lm).

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AKT Fig. 5. Effect of CAP expression on receptors internalization and stability. (A) HeLa cells were transfected with plasmids encoding GFP, dynamin2-GFP, FLAG-CAP, and dynamin2-K44A-GFP. Transfected cells were assayed for their ability to internalize fluorescently labeled EGF and transferrin (Tf). Cells showing less then 20% of the fluorescent ligand internalized by the control cells were scored as ‘no internalizing’. (B) In vivo biotinylation assay. Surface receptors of HEK293 cells expressing FLAG-CAP, dynamin2-K44A-GFP and GFP were in vivo labeled with biotin. Receptors internalized upon EGF treatment were isolated on streptavidin beads. Internalized EGF receptors retained by the streptavidin beads were detected by western blotting. The expression of CAP produces a block of EGFR internalization comparable to the effect of dynamin2K44A-GFP expression (a.u.: arbitrary unit) (B and D). (C) Control of the expression level of GFP, dynamin2-GFP, FLAG-CAP, and dynamin2-K44A-GFP proteins in the starting materials performed by western blotting using rabbit anti-GFP polyclonal antibody and mouse monoclonal anti-FLAG. The EGFR level in the starting materials was used as a loading control. Western blotting with anti-phospho-p38 mitogen-activated protein kinase was used to address the activation of EGF signaling pathway. (E) HeLa cells transfected with FLAG-CAP expressing vector or with empty vector (Control) were stimulate with EGF for the indicated time. Western blotting with anti-vinculin antibody was used as loading control. Western blotting with anti-phospho-AKT compared to that with anti-AKT was used to address the signaling pathway downstream EGFR activation.

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of EGF stimulation (180 min) (Fig. 5E). Additionally, the expression of CAP determined a moderate but significant reduction of the activation of AKT, as indicated by the level of phospho-AKT (Fig. 5E).

4. Discussion Using several independent approaches, we have demonstrated that CAP and dynamin physically interact. We have observed that all the three SH3 domains of CAP bind the dynamin isoforms (Fig. 1A–C) and that exogenously expressed CAP and dynamin2 form complexes in intact cells (Fig. 1D). Importantly, we have been able to immunoprecipitate an endogenous CAP–dynamin protein complex from rat cerebellum extracts, suggesting a physiological relevance of the interaction (Fig. 1E). Moreover, a strong degree of co-localization of endogenously expressed CAP and dynamin proteins was also observed in podosome structures exhibited by osteoclasts (Fig. 2). Podosome is a special type of cellular adhesion, probably involved in cell motility [22–24], which is present only in a few cell types such as cells transformed by the Rous sarcoma virus, osteoclasts and macrophages [22–24]. It comprises a central core of actin filaments surrounded by a donut-shaped area enriched in focal adhesion proteins. Podosomes have the peculiar property of deforming and tubulate the plasma membrane. This is probably due to the presence of endocytic and membrane deforming proteins, such as amphiphysins, endophilins and dynamins [9,33]. Interestingly, the striking co-localization of CAP and dynamin2 in the specific region surrounding the central core of actin (Fig. 2, insets) suggests that these two proteins could also cooperate in modifying the plasma membrane curvature of podosomes. Furthermore, in different cell lines such as Cos-7 or HeLa cells the expression of FLAG-CAP causes the formation of a peculiar mesh of tubule like structures where both dynamin2-GFP (Fig. 3A) or endogenous dynamin2 markedly re-localize (Fig. 3B). Since such dynamin-containing tubule like structures, observed only in CAP over-expressing cells, are a strong reminiscence of the ‘‘long necks” structures seen upon the blockage of dynamin fission activity [10,27,29], we hypothesize a role of CAP in controlling dynamin function. Accordingly, we observed a remarkable reduction of the dynamin-dependent internalization of EGF and transferrin receptors, upon the expression of CAP (Figs. 4 and 5A). It has been recently demonstrated that after a treatment with low dose of EGF (1–5 ng/ml) the EGF receptor is primarily internalized through clathrin-mediated endocytosis and it is not ubiquitinated [32,34]. Such new evidence suggests also that clathrinmediated trafficking is essential to recycle EGFR to the plasma membrane and to sustain downstream signaling [32]. Conversely, at high doses of the ligand (20–100 ng/ml) a relevant fraction of EGFR is internalized through a clathrin-independent way, it is ubiquitinated by the ubiquitin ligase Cbl [34,35] and it is trafficked to lysosomes for degradation [32]. Since both the routes are dependent on dynamin function [34], blocking dynamin through the expression of CAP may result in a composite phenotype. Actually, the expression of CAP significantly increases EGFR stability, especially after long stimulations (180 min) with high doses of EGF (100 ng/ml) (Fig. 5E). At high dose of EGF, when EGFR is internalized in a clathrin-independent way, CAP mediated blockage of dynamin affect primarily the ubiquitin dependent internalization and the degradation of the EGFR, thus increasing EGFR stability. Furthermore, as CAP it is also a major Cbl binding partner [4], we can not rule out the possibility that the stabilization effect of CAP on EGFR may be also due to a regulation Cbl function. Since CAP mediated blockage of dynamin may also affect clathrin-mediated endocytosis, which sustains EGFR downstream sig-

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naling [32], it is conceivable to expect an effect of CAP expression on the signaling downstream EGFR activation. Although we did not observe any effect of CAP expression on the MAPK signaling (Fig. 5C), we demonstrated a moderate but significant effect of CAP on the activation of AKT (Fig. 5E). Actually, this result was not surprising because it has been already shown that while MAPK signaling is quite insensitive to the alteration of EGFR recycling and stability [32,36] the AKT signaling responds promptly to modification of EGFR trafficking [32]. In conclusion with this study we have identified a novel function for CAP as adaptor protein involved in regulating receptors internalization through its effects on dynamin and on actin cytoskeleton. References [1] Kioka, N., Ueda, K. and Amachi, T. (2002) Vinexin, CAP/ponsin, ArgBP2: a novel adaptor protein family regulating cytoskeletal organization and signal transduction. Cell Struct. Funct. 27, 1–7. [2] Kimura, A., Baumann, C.A., Chiang, S.H. and Saltiel, A.R. (2001) The sorbin homology domain: a motif for the targeting of proteins to lipid rafts. Proc. Natl. Acad. Sci. USA 98, 9098–9103. [3] Cestra, G., Toomre, D., Chang, S. and De Camilli, P. (2005) The Abl/Arg substrate ArgBP2/nArgBP2 coordinates the function of multiple regulatory mechanisms converging on the actin cytoskeleton. Proc. Natl. Acad. Sci. USA 102, 1731– 1736. [4] Ribon, V., Printen, J.A., Hoffman, N.G., Kay, B.K. and Saltiel, A.R. (1998) A novel, multifunctional c-Cbl binding protein in insulin receptor signaling in 3T3–L1 adipocytes. Mol. Cell Biol. 18, 872–879. [5] Mandai, K., Nakanishi, H., Satoh, A., Takahashi, K., Satoh, K., Nishioka, H., Mizoguchi, A. and Takai, Y. (1999) Ponsin/SH3P12: an l-afadin- and vinculinbinding protein localized at cell–cell and cell–matrix adherens junctions. J. Cell Biol. 144, 1001–1017. [6] Baumann, C.A. et al. (2000) CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature 407, 202–207. [7] Mitra, P., Zheng, X. and Czech, M.P. (2004) RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin. J. Biol. Chem. 279, 37431– 37435. [8] Destaing, O., Saltel, F., Gilquin, B., Chabadel, A., Khochbin, S., Ory, S. and Jurdic, P. (2005) A novel Rho-mDia 2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts. J. Cell Sci. 118, 2901–2911. [9] Ochoa, G.C. et al. (2000) A functional link between dynamin and the actin cytoskeleton at podosomes. J. Cell Biol. 150, 377–389. [10] Tosoni, D., Puri, C., Confalonieri, S., Salcini, A.E., De Camilli, P., Tacchetti, C. and Di Fiore, P.P. (2005) TTP specifically regulates the internalization of the transferrin receptor. Cell 123, 875–888. [11] Haglund, K., Sigismund, S., Polo, S., Szymkiewicz, I., Di Fiore, P.P. and Dikic, I. (2003) Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat. Cell Biol. 5, 461–466. [12] Slepnev, V.I. and De Camilli, P. (2000) Accessory factors in clathrin-dependent synaptic vesicle endocytosis. Nat. Rev. Neurosci. 1, 161–172. [13] Lee, E. and De Camilli, P. (2002) Dynamin at actin tails. Proc. Natl. Acad. Sci. USA 99, 161–166. [14] Baldassarre, M., Pompeo, A., Beznoussenko, G., Castaldi, C., Cortellino, S., McNiven, M.A., Luini, A. and Buccione, R. (2003) Dynamin participates in focal extracellular matrix degradation by invasive cells. Mol. Biol. Cell 14, 1074– 1084. [15] Krueger, E.W., Orth, J.D., Cao, H. and McNiven, M.A. (2003) A dynamincortactin-Arp2/3 complex mediates actin reorganization in growth factorstimulated cells. Mol. Biol. Cell 14, 1085–1096. [16] Orth, J.D., Krueger, E.W., Cao, H. and McNiven, M.A. (2002) The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc. Natl. Acad. Sci. USA 99, 167–172. [17] Qualmann, B. and Kessels, M.M. (2002) Endocytosis and the cytoskeleton. Int. Rev. Cytol. 220, 93–144. [18] Brodin, L., Low, P. and Shupliakov, O. (2000) Sequential steps in clathrinmediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10, 312–320. [19] Danino, D. and Hinshaw, J.E. (2001) Dynamin family of mechanoenzymes. Curr. Opin. Cell Biol. 13, 454–460. [20] Ribon, V., Herrera, R., Kay, B.K. and Saltiel, A.R. (1998) A role for CAP, a novel, multifunctional Src homology 3 domain-containing protein in formation of actin stress fibers and focal adhesions. J. Biol. Chem. 273, 4073–4080. [21] Lebre, A.S. et al. (2001) Ataxin-7 interacts with a Cbl-associated protein that it recruits into neuronal intranuclear inclusions. Human Mol. Genet. 10, 1201– 1213. [22] Buccione, R., Orth, J.D. and McNiven, M.A. (2004) Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat. Rev. Mol. Cell Biol. 5, 647–657. 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