c-Cbl-facilitated cytoskeletal effects in v-Abl-transformed fibroblasts are regulated by membrane association of c-Cbl

Abstract

The multi-functional protein c-Cbl is an important modulator of actin cytoskeletal dynamics in diverse biological systems. We had previously reported that c-Cbl facilitates cell spreading and adhesion and suppresses anchorage-independent growth of v-Abl-transformed fibroblasts. To assess the importance of membrane localization of c-Cbl for the observed effects of c-Cbl in v-Abl-3T3 cells, we first mapped the membrane interactive domain(s) of c-Cbl. Our studies indicate that localization of c-Cbl to the membrane is likely to be mediated by the tyrosine kinase binding (TKB) domain and the proline-rich region of c-Cbl, whereas C-terminal tyrosine phosphorylation does not play a role. The association of v-Cbl, which encompasses the TKB domain, with the membrane was unusual as it was not entirely dependent on SH2-phosphotyrosine interactions. Our studies further demonstrate that Src-like adaptor protein (SLAP), which binds to v-Cbl in a tyrosine phosphorylation-independent manner, facilitates membrane association of Cbl. The interaction between c-Cbl and SLAP in v-Abl-3T3 cells positively influenced c-Cbl-mediated spreading and adhesion of these cells. SLAP appears to exert its effects not simply by increasing the amount of c-Cbl in the membrane but by facilitating binding of p85-phosphatidylinositol-3-kinase (PI3K) with membrane-associated c-Cbl.

Myristoylation and Membrane Binding Regulate c-Src Stability and Kinase Activity

“…Moreover, association with and phosphorylation of Cbl by c-SrcG2A were reduced compared to those with c-SrcWT (data not shown). A subpopulation of Cbl proteins has been reported to be membrane bound, and membrane binding enhances the ability of Cbl to ubiquitinate membrane-bound targets (24,54). Our data suggest that the reduced accessibility of nonmyristoylated c-Src to Cbl and other E3 ubiquitin ligases limits the extent of c-SrcG2A ubiquitination and degradation and contributes to the increased expression levels compared to those of c-SrcWT.…”

The Src-like adaptor protein regulates PDGF-induced actin dorsal ruffles in a c-Cbl-dependent manner

“…C-Cbl also inhibits Rac activity, required for dorsal ruffle formation (Scaife et al, 2003;Swaminathan et al, 2007). Accordingly, PDGF-induced Rac activation was also affected by SLAP.…”

“…However, Src coexpression had no rescuing effect, even when expressed at higher level, suggesting that SLAP does not act at the Src level ( Figure 2d). As c-Cbl has been implicated in F-actin assembly (Scaife et al, 2003;Swaminathan et al, 2007), we then addressed the role of SLAP as a negative regulator of F-actin assembly A Sirvent et al this adapter on SLAP morphological activity. To this end, c-Cbl was depleted from cells with a short hairpin RNA that reduced 80% of protein level (Figure 2b).…”

Chapter 20 Retinal Insulin Receptor Signaling In Hyperosmotic Stress

“…Cbl protein has a tyrosine kinase binding (TKB) domain, proline-rich region and five tyrosine phosphorylation sites; Cbl is able to bind to protein tyrosine kinases (PTK), SH3 and SH2 domain containing proteins, respectively (Swaminathan et al, 2007;Swaminathan and Tsygankov, 2006;Meisner et al, 1995);Meisner, 1995 1543 /id;Fukazawa, 1995 3 /id}. Cbl also contains the RING finger domain and a ubiquitin-associated domain which allow for ubiquitination and proteasomal degradation of activated PTK (Levkowitz et al, 1999;Meisner et al, 1997).…”

“…Phosphorylation of Tyr-731 of Cbl is essential for its binding to SH2 domains of p85 (Feshchenko et al, 1998;Swaminathan et al, 2007). .…”