Protein Kinase A Regulates 3-Phosphatidylinositide Dynamics during Platelet-derived Growth Factor-induced Membrane Ruffling and ChemotaxisS

Spatial regulation of the cAMP-dependent protein kinase (PKA) is required for chemotaxis in fibroblasts; however, the mechanism(s) by which PKA regulates the cell migration machinery remain largely unknown. Here we report that one function of PKA during platelet-derived growth factor (PDGF)-induced chemotaxis was to promote membrane ruffling by regulating phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) dynamics. Inhibition of PKA activity dramatically altered membrane dynamics and attenuated formation of peripheral membrane ruffles in response to PDGF. PKA inhibition also significantly decreased the number and size of PIP 3 -rich membrane ruffles in response to uniform stimulation and to gradients of PDGF. This ruffling defect was quantified using a newly developed method, based on computer vision edge-detection algorithms. PKA inhibition caused a marked attenuation in the bulk accumulation of PIP 3 following PDGF stimulation, without effects on PI3-kinase (PI3K) activity. The deficits in PIP 3 dynamics correlated with a significant inhibition of growth factor-induced membrane recruitment of endogenous Akt and Rac activation in PKA-inhibited cells. Simultaneous inhibition of PKA and Rac had an additive inhibitory effect on growth factor-induced ruffling dynamics. Conversely, the expression of a constitutively active Rac allele was able to rescue the defect in membrane ruffling and restore the localization of a fluorescent PIP 3 marker to membrane ruffles in PKA-inhibited cells, even in the absence of PI3K activity. These data demonstrate that, like Rac, PKA contributes to PIP 3 and membrane dynamics independently of direct regulation of PI3K activity and suggest that modulation of PIP 3 /3-phosphatidylinositol (3-PI) lipids represents a major target for PKA in the regulation of PDGF-induced chemotactic events.