Two distinct pools of Src family tyrosine kinases regulate PDGF-induced DNA synthesis and actin dorsal ruffles

Abstract

The mechanism by which the Src family of protein-tyrosine kinases (SFKs) regulate mitogenesis and morphological changes induced by platelet-derived growth factor (PDGF) is not well known. The cholesterol-enriched membrane microdomains, caveolae, regulate PDGF receptor signalling in fibroblasts and we examined their role in SFK functions. Here we show that caveolae disruption by membrane cholesterol depletion or expression of the dominant-negative caveolin-3 DGV mutant impaired Src mitogenic signalling including kinase activation, Myc gene induction and DNA synthesis. The impact of caveolae on SFK function was underscored by the capacity of Myc to overcome mitogenic inhibition as a result of caveolae disruption. Using biochemical fractionation we show that caveolae-enriched subcellular membranes regulate the formation of PDGF-receptor-SFK complexes. An additional pool of PDGF-activated SFKs that was insensitive to membrane cholesterol depletion was characterised in non-caveolae fractions. SFK activation outside caveolae was linked to the capacity of PDGF to induce F-actin rearrangements leading to dorsal ruffle formation. Inhibition of phospholipase C γ (PLCγ), sphingosine kinase and heterotrimeric Gi proteins implicates a PLC γ–sphingosine-1-phosphate–Gi pathway for PDGF-induced SFK activation outside caveolae and actin assembly. In addition, the cytoplasmic tyrosine kinase Abl was identified as an important effector of this signalling cascade. We conclude that PDGF may stimulate two spatially distinct pools of SFKs leading to two different biological outcomes: DNA synthesis and dorsal ruffle formation.

Growth Factors Outside of the Platelet-derived Growth Factor (PDGF) Family Employ Reactive Oxygen Species/Src Family Kinases to Activate PDGF Receptor α and Thereby Promote Proliferation and Survival of Cells

“…These data show that all agents activated Src. Although the magnitude of the increase in phosphorylation was modest, it is consistent with what other groups report using this approach (30,64) and what we have found using a different approach to monitor activation of SFKs (43). B, mouse embryo fibroblasts that lacked all SFKs (39) were reconstituted with either an empty expression vector (Ϫ/Ϫ), or wild type c-Src (Src) (40) and stimulated with the indicated agents as described in panel A.…”

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

“…Ras and Rab5 pathways have also been documented (Lanzetti et al, 2004), and we have recently shown that this response implicates Src family tyrosine kinases (SFK). In addition, our results suggest that in contrast to Src mitogenic function, SFK signal transduction promoting actin assembly does not require receptor association (Veracini et al, 2006). Rather, it involves the lipid second messenger sphingosine-1-phosphate, which may activate SFK through a seven-transmembrane receptor of the EDG family and an associated heterotrimeric Gi protein.…”

“…This potentiating effect was specific to dorsal ruffles as lateral ruffles were not (Manes et al, 2000) were subcloned into pBABE. Cell culture, transfection, infection, immunofluorescence analysis, Q-PCR and biochemistry have been described by Veracini et al (2006). White arrows indicate cells with dorsal ruffles.…”

Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization

“…Moreover, Csk relocalization to these membranes induced, at most, a 50% reduction of SFK activity, confirming that it does not target all pools of SFK present in these cells. Accordingly, spatial regulation has been considered as an important feature of signal specificity induced by SFK in nontransformed cells (Veracini et al, 2006(Veracini et al, , 2008, and this may apply for transformed cells as well. For example, when localized at focal adhesions, SFK has important roles in cell growth and survival (Frame, 2004).…”

“…Purified FynK298M (FynK À ) was described in Koegl et al (1994). Anti-SFK (cst1) was described in Veracini et al (2006); anti-Src specific (2.17) antibody was a generous gift from Dr S Parsons (University of Virginia, VA, USA). Anti-Csk was described in Be´nistant et al (2001), anti-PAG in Veracini et al (2008) and anti-Cbp in Oneyama et al (2008).…”