Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK).
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.
Department of Pathology, St Louis University School of Medicine, Missouri 63104-1028, USA.
T L
TL
Chew
R A
RA
Masaracchia
Z M
ZM
Goeckeler
R B
RB
Wysolmerski
D000199:Actins / Q000032:analysis; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D054462:p21-Activated Kinases
D000199:Actins; D018994:Myosin Light Chains; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D009218:Myosins