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

• Published in: The Journal of biological chemistry 2003-06, Vol.278 (24), p.21805-21813
• Main Authors: Lynch, Danielle K., Winata, Stephanie C., Lyons, Ruth J., Hughes, William E., Lehrbach, Gillian M., Wasinger, Valerie, Corthals, Garry, Cordwell, Stuart, Daly, Roger J.
• Format: Article
• Language: English
• Subjects: Actin-Related Protein 2; Actin-Related Protein 3; Actins - metabolism; Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Binding Sites; Blotting, Western; Carrier Proteins - chemistry; Cell Line; Cortactin; Cytoskeletal Proteins - metabolism; Cytoskeleton - metabolism; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Endocytosis; Epidermal Growth Factor - metabolism; ErbB Receptors - metabolism; Fluorescent Antibody Technique, Indirect; Glutathione Transferase - metabolism; HeLa Cells; Humans; Immunoblotting; Kinetics; Mass Spectrometry; Microfilament Proteins - chemistry; Microscopy, Fluorescence; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Plasmids - metabolism; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Proteins - chemistry; Proteins - metabolism; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; src Homology Domains; Transfection; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M211407200

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.