Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK)

J Muscle Res Cell Motil. 1998 Nov;19(8):839-54. doi: 10.1023/a:1005417926585.

Abstract

Myosin regulatory light chain (RLC) phosphorylation has been implicated in Rho-mediated stress fibre formation. The recent observation that Rho kinase phosphorylates RLC in vitro suggests that serine/threonine kinases other than those in the myosin light chain kinase (MLCK) family have the potential to activate myosin II. In this study we report that gamma-PAK, which is activated by the GTP-binding proteins Cdc42 and Rac, catalyses phosphorylation of intact non-muscle myosin II and isolated recombinant RLC. gamma-PAK phosphorylated endothelial cell myosin II to 0.85 +/- 0.02 mol PO4 per mol RLC. Phosphorylation is Ca2+/calmodulin-independent and the enzyme has a K(m) and Vmax for myosin II regulatory light chain of 12 microM and 180 nmol/min/mg respectively. No myosin II heavy chain phosphorylation was detected. Phosphopeptide maps and phosphoamino acid analysis revealed that gamma-PAK phosphorylates Ser-19 but does not phosphorylate Thr-18. A panel of recombinant RLC mutants was used to confirm that Ser-19 is the only phosphorylation site modified by gamma-PAK. On substitution of both Ser-19 and Thr-18 with Ala or Glu, no phosphorylation of other Ser/Thr residues in the RLC was detected. Similar to MLCK, Arg-16 is required for interaction of gamma-PAK with the substrate, since converting Arg-16 to Ala significantly reduced RLC phosphorylation. Endothelial cell monolayers permeabilized with saponin retract upon exposure to either Cdc42 or trypsin-activated gamma-PAK and ATP. Activation of gamma-PAK is required to initiate Ca2+/calmodulin-independent cell retraction and actin rearrangement. Taken together, these data suggest that myosin II activation by the p21-activated family of kinases may be physiologically important in regulating cytoskeletal organization.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / analysis
  • Animals
  • Cattle
  • Cells, Cultured
  • Endothelium, Vascular / chemistry
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / enzymology*
  • Enzyme Activation / physiology
  • Gene Expression / physiology
  • Mutagenesis, Site-Directed / physiology
  • Myosin Light Chains / genetics
  • Myosin Light Chains / metabolism*
  • Myosins / genetics
  • Myosins / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Pulmonary Artery / cytology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • p21-Activated Kinases

Substances

  • Actins
  • Myosin Light Chains
  • Recombinant Proteins
  • Protein-Serine-Threonine Kinases
  • p21-Activated Kinases
  • Myosins