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

Mol Cell Biol. 2020 Jan 30;40(4):e00282-19. doi: 10.1128/MCB.00282-19. Print 2020 Jan 30.

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.

Keywords: CAP1; CDK5; actin cytoskeleton; cell adhesion; cyclic AMP; protein phosphorylation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Actin Cytoskeleton / metabolism*
  • Actin Depolymerizing Factors / metabolism
  • Actins / metabolism
  • Bridged Bicyclo Compounds, Heterocyclic / metabolism
  • Cell Adhesion / physiology*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Line, Tumor
  • Cyclic AMP / metabolism*
  • Cyclin-Dependent Kinase 5 / metabolism*
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • Focal Adhesion Protein-Tyrosine Kinases / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Microfilament Proteins / metabolism
  • Phosphorylation
  • Signal Transduction / physiology
  • Thiazolidines / metabolism

Substances

  • Actin Depolymerizing Factors
  • Actins
  • Bridged Bicyclo Compounds, Heterocyclic
  • CAP1 protein, human
  • Cell Cycle Proteins
  • Cytoskeletal Proteins
  • Microfilament Proteins
  • Thiazolidines
  • Cyclic AMP
  • Focal Adhesion Protein-Tyrosine Kinases
  • Cyclin-Dependent Kinase 5
  • CDK5 protein, human
  • latrunculin A