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

Abstract

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...

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

“…Protein Purification-Recombinant rabbit smooth muscle MLCK (MLCK 155 ) was expressed in SF-9 cells (23,41); recombinant nonmuscle myosin II RLC was expressed in BL21 (DE3) bacteria (41) and purified as described previously (42).…”

“…Control and treated monolayers were flooded with 1 ml of ice-cold 10% trichloroacetic acid containing 10 mM DTT and processed as described previously (23,45). The pellets were dissolved in 80 l of 9.5 M urea, 10 mM DTT, 20 mM Tris, 23 mM glycine, and 0.04% bromphenol blue, pH 8.8.…”

“…Phosphorylation at these sites results in desensitization of MLCK to activation by Ca 2ϩ / CaM. In addition, inhibition of MLCK activity by p21-activated kinase has been reported (23,24). However, little information is available documenting the effects of phosphorylation on the nonmuscle form of MLCK 206 -220 .…”

Regulation of myosin phosphorylation and myofilament Ca²⁺ sensitivity in vascular smooth muscle

“…The p21-activated protein kinases have been shown to phosphorylate not only MLC (Chew et al, 1998;Van Eyk et al, 1998), MYPT1 (Takizawa et al, 2002) and CPI-17 (Takizawa et al, 2002) (Fig. 1), but also MLCK (Sanders et al, 1999;Goeckeler et al, 2000;Wirth et al, 2003) and caldesmon (Van Eyk et al, 1998). The muscarinic acetylcholine receptor m2 has been shown to activate p21-activated protein kinase 1 via Gi3, rac1/cdc42, and thereby inhibit the MLCK activity in intestinal smooth muscle (Murthy et al, 2003).…”

Expression of Myosin Light Chain Kinase in Kidney of Streptozotocin-Induced Diabetic Rats

“…Phosphorylation of MLC-20 by smooth muscle MLCK is a key event initiate to smooth muscle contraction. Although the roles of MLCK in non-muscle cells are not well defined, a variety of morphological changes such as cellular motility and organelle movement occur concurrently with increasing in cytoplasmic Ca 2+ levels, light chain phosphorylation and activation of MLCK [21,22]. Myosin, one of the major contractile proteins in muscle cells as well as nonmuscle cells, consists of two heavy chains and two light chains.…”