Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion.

Abstract

Cyclase-associated protein 1 (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for CAP1 function in both actin filament disassembly and cell adhesion. Both the phosphomimetic and the nonphosphorylatable CAP1 mutant, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAP1 knockdown HeLa cells. The phosphorylation mutants also had defects in alleviating the elevated focal adhesion kinase (FAK) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphoregulatory cell signals for CAP1, we found that cyclin-dependent kinase 5 (CDK5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac). No evidence supports an involvement of activated protein phosphatase in executing the dephosphorylation downstream from cAMP, whereas preventing CAP1 from accessing its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for CAP1 functions in both actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge of the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.