Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75)

Abstract

The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity.

The Cdk5 activator P39 specifically links muskelin to myosin II and regulates stress fiber formation and actin organization in lens

“…Binding of Cdk5 to the muskelin-p39-myosin complex is also consistent with the previous demonstration that Cdk5 binds p39 at a site distinct from the binding sites for muskelin and MLC [2, 21]. Binding of p39 to Cdk5 activates the kinase activity of Cdk5, which regulates the Rho-ROCK pathway of MRLC phosphorylation by controlling Src-dependent phosphorylation of the upstream inhibitor of Rho, p190RhoGAP [12, 24, 25]. This Cdk5-dependent Rho-ROCK pathway is by far the major regulator of MRLC phosphorylation in the lens, with little or no contribution from myosin light chain kinase (MLCK) [12].…”

Cullin5 destabilizes Cas to inhibit Src-dependent cell transformation

“…It has been unclear which ubiquitin ligase is involved. We and others have found that knockdown of Cul5 in mouse fibroblasts stabilizes active Src, suggesting that Cul5-CRLs are required for Src turnover in these cells (Laszlo and Cooper, 2009;Pan et al, 2011). Moreover, Cul5 knockdown induces transformation of fibroblasts in which Src is also genetically activated, either by SRC gene mutation or by deletion of the CSK gene, which encodes a Src-inhibitory kinase (Laszlo and Cooper, 2009).…”

“…Cul5 is required for the degradation of activated but not inhibited Src in fibroblasts (Laszlo and Cooper, 2009;Pan et al, 2011). We tested whether endogenous Cul5 also regulates Src in MCF10A cells.…”

Cullin-RING Ligases as Attractive Anti-cancer Targets

“…4E ). Progress on identification and characterization of specific endogenous CRL5 substrates is lacking, although it has been suggested that active Src oncoprotein might be subjected to degradation by CRL5 [78,79], On the other hand, it has been relatively well established that several viral proteins can hijack cellular CRL5 E3 to promote degradation of several key host proteins. Examples include HIV-1 viral infectivity factor (Vif), which contains a SOCS-box domain, assembles a cellular CRL5 E3 to promote ubiquitination and degradation of the host anti-viral factor APOBEC3G [80].…”