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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.

The cytoplasmic tyrosine kinase Abl is a Src substrate required for platelet-derived growth factor (PDGF) receptor signaling leading to Myc expression and DNA synthesis. Abl targets are, however, ill defined. Here we report that the small GTPase Rac is an important effector of its mitogenic function. PDGF-induced Rac activation was impaired in cells with inactive Abl and active Rac overcame the mitogenic defects found in these cells. Rac function required both a Jun N-terminal kinase (JNK) and a NADPH oxidase (Nox) pathway. Furthermore, co-activation of JNK and Nox were sufficient to mimic the Rac mitogenic rescue. Abl also regulated PDGF-induced JNK and Nox activation. Finally, we found that Myc is an important target of this signaling cascade: Myc induction was sensitive to small inhibitors of JNK and Nox activities and forced expression of Myc overcame the G1 block induced by dominant interfering mutants of mitogen-activated protein kinase kinase 4 (MKK4) and Nox2 activating subunit. We concluded that cytoplasmic Abl operates on a Rac/JNK and a Rac/Nox pathway for PDGF-induced Myc induction and DNA synthesis.