Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein

摘要

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton. CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton. CD2-associated protein (CD2AP) 2The abbreviations used are: CD2AP, CD2-associated protein; GST, glutathione S-transferase; SH, Src homology; HEK, human embryonic kidney; HPLC, high pressure liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl; SPR, surface plasmon resonance. 2The abbreviations used are: CD2AP, CD2-associated protein; GST, glutathione S-transferase; SH, Src homology; HEK, human embryonic kidney; HPLC, high pressure liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl; SPR, surface plasmon resonance. is a 70-kDa protein that was originally cloned as a protein that interacts with the cytoplasmic tail of CD2, a T lymphocyte and natural killer cell transmembrane protein (1Dustin M.L. Olszowy M.W. Holdorf A.D. Li J. Bromley S. Desai N. Widder P. Rosenberger F. van der Merwe P.A. Allen P.M. Shaw A.S. Cell. 1998; 94: 667-677Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). It is composed of three Src homology 3 (SH3) domains at the NH2 terminus followed by proline-rich sequences and a coiled-coil domain at the extreme COOH terminus. It is expressed in all tissues except brain. Interestingly, CD2AP-deficient animals die of renal failure ∼6 weeks of age (2Shih N.Y. Li J. Karpitskii V. Nguyen A. Dustin M.L. Kanagawa O. Miner J.H. Shaw A.S. Science. 1999; 286: 312-315Crossref PubMed Scopus (694) Google Scholar). In the kidney, CD2AP is highly expressed in the glomerular epithelial cell, and it is implicated to play a role in a specialized cell junction known as a slit diaphragm (3Shih N.Y. Li J. Cotran R. Mundel P. Miner J.H. Shaw A.S. Am. J. Pathol. 2001; 159: 2303-2308Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). A homolog of CD2AP, Cin85, was cloned as an interacting protein with the E3 ubiquitin ligase c-cbl (4Take H. Watanabe S. Takeda K. Yu Z.X. Iwata N. Kajigaya S. Biochem. Biophys. Res. Commun. 2000; 268: 321-328Crossref PubMed Scopus (137) Google Scholar) and as an inhibitor of phosphatidylinositol 3-kinase (5Gout I. Middleton G. Adu J. Ninkina N.N. Drobot L.B. Filonenko V. Matsuka G. Davies A.M. Waterfield M. Buchman V.L. EMBO J. 2000; 19: 4015-4025Crossref PubMed Scopus (121) Google Scholar). Recently, several endocytic and actin-associated molecules have been reported to interact wi