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CAP1 (Cyclase Associated Protein 1) 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 both CAP1 functions in actin filament disassembly and cell adhesions. Both the phospho-mimetic and non-phosphorylatable CAP1 mutants, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAPl-knockdown HeLa cells. The phosphor mutants also had defects in alleviating the elevated FAK activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphor-regulatory cell signals for CAP1, we found that CDK5 phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors PKA and Epac. No evidence supports involvement of activated protein phosphatase in executing the dephosphorylation downstream of cAMP, whereas preventing CAP1 access to its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for both CAP1 functions in actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge on the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation. [ABSTRACT FROM AUTHOR]