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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.

p21-activated Kinase Regulates Endothelial Permeability through Modulation of Contractility

“…This idea is consistent with a number of previous studies indicating that PAK can positively regulate myosin phosphorylation (18,19,35,43). They are also fit well with a large body of data implicating myosin phosphorylation and contractility in regulating endothelial barrier function (reviewed in Ref.…”

“…In considering possible explanations for this result, we recalled that myosin-dependent contractility is well known to regulate cell-cell junctions and permeability across endothelial or epithelial monolayers (29 -32). Although both negative or positive effects of PAK on myosin light chain phosphorylation have been reported (18,19,(33)(34)(35), in endothelial cells, PAK clearly promotes myosin phosphorylation and cell contractility (23,26). Staining of treated cells with rhodamine-phalloidin to visualize F-actin showed first that VEGF addition to BAEC monolayers induced rapid formation of spaces or pores between the cells within 15 min, followed at later times by more general separation of adjacent cells (Fig.…”

Traction forces of fibroblasts are regulated by the Rho-dependent kinase but not by the myosin light chain kinase

“…The former is mediated by the MLCK and represents the primary mechanism of activation in smooth muscles. The latter involves a number of kinases including ROCK [41], DAPK [42], PAK [43], and the ZIP kinase [44]. In addition, ROCK is known to enhance MRLC phosphorylation by inhibiting a myosin light chain phosphatase [35].…”

Phosphorylation of Myosin Light Chain Kinase by p21-activated Kinase PAK2

“…In subsequent studies, a peptide that inhibited kinase activity in PAK blocked Cdc42, Rac, and constitutively active PAK-induced morphological changes in transfected HeLa cells (5). An important role for PAK-mediated phosphorylation in actin reorganization has also been supported by studies in permeabilized (6) and intact (7) endothelial cells as well as skinned smooth muscle cells (8). Permeabilized endothelial cells incubated with Cdc42-activated PAK2 or the catalytic domain of PAK2 underwent ATP-dependent retraction and actin reorganization (6).…”