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.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 (1).In line with an adaptor role, cortactin is a multidomain protein, with the individual modules capable of mediating specific protein-protein interactions (1). The N-terminal region mediates binding to the Arp 1 2/3 complex, a highly conserved regulator of the assembly and structure of actin networks (2), and contains a DDW motif characteristic of Arp2/3-interacting proteins such as WASP, Myo3p, and ActA (2, 3). 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 (2). 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 (4 -6)....

Podocyte Mitosis – A Catastrophe

“…Nephrin , a member of the Ig-superfamily (Ig-SF) of transmembrane cell adhesion molecules, is one of the major structural components of the slit diaphragm [15]. The cytoplasmic tail of nephrin, like other transmembrane Ig-SF members, is anchored to the actin cytoskeleton of podocyte by means of CD2-associated protein, CD2AP , a cytoplasmic adaptor protein which interacts with actin [16] and the actin-binding proteins cortactin [17], capping protein Z (CapZ) [18], and the actin polymerization complex Arp2/3 [17]. CD2AP and nephrin interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K), recruit PI3K to the plasma membrane, and stimulate PI3K-dependent AKT signaling in podocytes [19], thus participating in a common signaling pathway necessary to maintain crucial podocyte functions.…”

Prognostic significance of cortactin levels in head and neck squamous cell carcinoma: comparison with epidermal growth factor receptor status

“…A growing list of cortactin-binding partners has been identified, including proteins that regulate receptor clustering, endocytosis, and vesicle transport such as dynamin 2 (McNiven et al, 2000) and CD2AP (CIN85-related protein) (Lynch et al, 2003). Recent results from studies in cultured cells (Lynch et al, 2003;Timpson et al, 2005) indicate that cortactin could participate in receptor-mediated endocytosis of the epidermal growth factor (EGF) receptor (EGFR).…”

“…Recent results from studies in cultured cells (Lynch et al, 2003;Timpson et al, 2005) indicate that cortactin could participate in receptor-mediated endocytosis of the epidermal growth factor (EGF) receptor (EGFR). Thus, cortactin overexpression attenuated ligand-induced downregulation of EGFR and led to sustained activation of EGFR signalling.…”

Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

“…In trafficking events, cortactin has been involved in clathrin-mediated endocyto-sis as well as in pinocytosis, regulation of epidermal growth factor receptor (EGFR) degradation, or endosomal positioning (McNiven et al, 2000;Cao et al, 2003Cao et al, , 2005Lynch et al, 2003;Cabezas et al, 2005;Merrifield et al, 2005;Sauvonnet et al, 2005;Timpson et al, 2005;Zhu et al, 2005;Mettlen et al, 2006;Le Clainche et al, 2007).…”