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J. Biol. Chem., Vol. 283, Issue 50, 35199-35211, December 12, 2008

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

Paula B. Deming¹, Shirley L. Campbell¹, Linda C. Baldor, and Alan K. Howe²

From the Departments of Medical Laboratory and Radiation Sciences and Pharmacology, and the Vermont Cancer Center, the University of Vermont, Burlington, Vermont 05405

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 (PIP₃) 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 PIP₃-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 PIP₃ following PDGF stimulation, without effects on PI3-kinase (PI3K) activity. The deficits in PIP₃ 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 PIP₃ marker to membrane ruffles in PKA-inhibited cells, even in the absence of PI3K activity. These data demonstrate that, like Rac, PKA contributes to PIP₃ and membrane dynamics independently of direct regulation of PI3K activity and suggest that modulation of PIP₃/3-phosphatidylinositol (3-PI) lipids represents a major target for PKA in the regulation of PDGF-induced chemotactic events.

Received for publication, June 10, 2008 , and in revised form, September 29, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM-074204 (to A. K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3, Movies 1-10, and RuffleQuant.txt.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pharmacology, 149 Beaumont Ave., HSRF 322, Burlington, VT 05405-0075. Tel.: 802-656-9521; E-mail: alan.howe{at}uvm.edu.

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