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Phosphorylation of myosin II regulatory light chains (RLC) by Ca2؉ /calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Posttranslational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPasedependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 ؎ 0.21 mol of PO 4 /mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.The PAK 1 family of serine/threonine protein kinases have been implicated in a broad spectrum of signal transduction pathways leading to diverse physiological end points, including cytoskeletal reorganization, apoptosis, and Ras-mediated cell transformation (1, 2). All PAK isoforms are direct effectors of the Rho family GTP-binding proteins Rac and Cdc42, suggesting that cell-specific responses arise from either selective regulation of G protein activation in response to unique agonists or selective phosphorylation of tissue-specific protein kinase substrates.In the initial studies of the relative roles of G protein activation and protein kinase activity in actin reorganization, Sells et al. (3) demonstrated that microinjection of activated PAK1 induced rapid formation of polarized filopodia in Swiss 3T3 cells. However, the relative importance of PAK-dependent phosphorylation of unique substrates during Cdc42/Racdependent cytoskeletal reorganization is incompletely understood. Transient transfection of HeLa cells with constitutively active PAK1 promoted cytoskeletal rearrangement, which was entirely analogous to that observed in Cdc42 and Rac transfected cells (4). 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). In addition, these studies using purified enzymes established that the regulatory nonmuscle and smooth muscle myosin II light chain is a substrate for PAK2 (9, 1...

The S6/H4 kinase purified from human placenta catalyzes phosphorylation of the S6 ribosomal protein, histone H4, and myelin basic protein. The pseudosubstrate domain at site 1 is autophosphorylated and subsequent bimolecular autophosphorylation at site 2 fully opens the catalytic site. Phosphorylation by a regulatory protein kinase may occur at site 2 in vivo.

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.

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The specificity of the histone-H4-specific, protease-activated protein kinase (H4-PK) was examined using two series of synthetic peptides corresponding to the phosphorylation sites in histone H4 and pyruvate kinase. Optimum kinetic constants for phosphorylation were observed using the peptide Val-Lys-Arg-Ile-Ser-Gly-Leu. Peptides in which the Lys was replaced by Arg or the Lys-Arg sequence was transposed were phosphorylated with less favorable kinetics. Peptides with either basic residue deleted did not serve as substrates. Only the H4 peptide, containing an Arg-Arg sequence, was phosphorylated by the cyclic-AMP-dependent protein kinase (CA-PK). Distinct specificity determinants for H4-PK and CA-PK were also observed using the pyruvate kinase peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly). Collectively the data indicated that the primary substrate specificity determinants for H4-PK are Lys-Arg-Xaa-Ser whereas the CA-PK selectively phosphorylates the sequence Arg-Arg-Xaa-Ser.Since the observation that glycogen phosphorylase activity is regulated by covalent phosphorylation catalyzed by phosphorylase kinase [I], the identification of distinct protein phosphotransferases (protein kinases) and the association of these enzymes with unique protein substrates has received widespread attention [2 -41. Initial studies proposed that the specificity determinants which defined a phosphorylation site were determined by higher orders of protein structure [4,5]. However, the observations that denaturation increased the ability of some proteins to serve as protein kinase substrates [6,7] and that small synthetic peptides could be phosphorylated in vitro [8 -121 have indicated that the substrate specificity determinants were dictated, at least in part, by the primary sequence.The observed kinetic constants, determined in experiments utilizing the cyclic-AMP-dependent protein kinase catalytic subunit and synthetic peptides derived from the primary sequence at the phosphorylation site in pyruvate kinase (Leu-Arg-Arg-Ser-Leu-Gly), indicated that multiple basic residues were required in the proximity of, and aminoterminal to, the modified serine [9]. Additional studies [lo, 1 I], utilizing synthetic peptides with further modification in the pyruvate kinase sequence, suggested that the cyclic-AMPdependent protein kinase selectively phosphorylated serine residues in a sequence of the type Arg-Arg-Xaa-Ser or LysArg-Xaa-Yaa-Ser. The positive correlation between the specificity determinants predicted in the peptide studies and the primary sequence at phosphorylation sites in several physiological protein substrates has provided compelling evidence that the primary sequence substantially determines the substrate specificity of the cyclic-AMP-dependent protein kinase [13].

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