Abstract. The phosphorylation of regulatory myosin light chains by the Ca2÷/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B., and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. . We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2-to 2.5-fold within 5 min and remains elevated for at least 60 min.The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation.Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.NDOTHELIAL cells lining most vessels form a continuous layer that normally constrains proteins as well as formed blood elements to the vascular lumen. The loss of continuity of the endothelial sheet leads to increased permeability and the development of edema (30,47,51,58,71,75). The increase in microvascular permeability by inflammatory and chemical mediators has been correlated with the presence of small gaps between adjacent endothelial cells. Furthermore, mediators that disrupt actin filaments and chelate extracellular calcium have been implicated in changes in macromolecular flux. The factors that may play a role in altering the barrier function of the endothelium leading to edema include: (a) loss of Address all correspondence t...

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 mechanism or mechanisms by which polymorphonuclear leukocytes (PMN) penetrate junctions between neighboring endothelial cells (EC) to traverse endothelial barriers remain unresolved. We report that chemoattractant-stimulated PMN induce a coordinate increase in both phosphorylation of serine 19 and threonine 18 of EC myosin regulatory light chains and isometric tension generation by EC monolayers. Unstimulated PMN had no effect on either parameter. These findings, coupled with our previous report (Huang et al., J. Cell Biol. 120: 1371-1380, 1993) that chemoattractant-stimulated PMN cause a rise in EC cytosolic free Ca2+, provide strong presumptive evidence that myosin light chain kinase is the EC enzyme responsible for initiating myosin light chain phosphorylation, EC contraction, and isometric tension generation in response to chemoattractant-stimulated PMN. We suggest that, by inducing phosphorylation of EC cytoskeletal proteins, chemoattractant-stimulated PMN induce EC to open their intercellular junctions, thereby facilitating transendothelial movement of these leukocytes.

To better understand the distinct functional roles of the 220- and 130-kDa forms of myosin light chain kinase (MLCK), expression and intracellular localization were determined during development and in adult mouse tissues. Northern blot, Western blot, and histochemical studies show that the 220-kDa MLCK is widely expressed during development as well as in several adult smooth muscle and nonmuscle tissues. The 130-kDa MLCK is highly expressed in all adult tissues examined and is also detectable during embryonic development. Colocalization studies examining the distribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDa MLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB or F-actin. In contrast, the 220-kDa MLCK did not colocalize with either nonmuscle myosin II isoform but instead colocalizes with thick interconnected bundles of F-actin. These results suggest that in vivo, the physiological functions of the 220- and 130-kDa MLCKs are likely to be regulated by their intracellular trafficking and distribution.

Goeckeler ZM, Bridgman PC, Wysolmerski RB. Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity. Am J Physiol Cell Physiol 295: C994 -C1006, 2008. First published August 13, 2008; doi:10.1152/ajpcell.00318.2008.-Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca 2ϩ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity.actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions MYOSIN II IS THE MAJOR CYTOSKELETAL protein in muscle and nonmuscle cells with the ability to self-assemble into bipolar filaments and convert chemical energy of ATP into mechanical work. Myosin II-based contractile activity has been shown to play important roles in a host of cellular functions such as cell spreading, motility, and cell division. In addition, it has been implicated in the maintenance of cell shape and generation of basal and agonist-induced cytoskeletal tension (15,25,31,91). While myosin II was initially considered a passive structural participant in cytoskeletal organization and maintenance of cell shape in nonmuscle cells, more recent immunolocalization studies have shown that it is arranged in stress fibers in a sarcomeric-like organization similar to muscle (31,85,86). This morphological organization has led to the hypothesis that stress fibers decorated with myosin II are contractile structures assembled for the generation of cellular tension (10, 33). Permeabilized cell preparations validated this hypothesis, demonstrating that cell contraction and tension generation are dependent on myosin II activation. Furthermore, the network contraction model now proposes that myosin II is responsible for both tension generation and the rearrangement of actin filaments into appr...

Nerve growth factor (NGF) stimulation of embryonic mouse sensory axon outgrowth is MII dependent. NGF regulates two actomyosin processes: transverse actin bundling and peripheral retrograde (radial) network actin flow. These two processes oppose microtubule advance and differentially involve MIIA and MIIB, respectively.

(20,21,23,33,64), neurite outgrowth (1, 8, 57), endothelial cell contraction (23,69,70), and resistance to mechanical perturbations (8,11,31). These myosin II-based cellular events have been considered to be regulated solely by an increase in cytosolic Ca 2ϩ according to the classic smooth muscle model of Ca 2ϩ -coupled contraction. However, within the last 10 years myosin II motor activity has been shown to be activated by Ca 2ϩ -independent pathways (39,41,63,68) in addition to Ca 2ϩ -dependent pathways (7,23,32,70). Ca 2ϩ -dependent cell contraction results from an influx of Ca 2ϩ from the extracellular space or the release of Ca 2ϩ from sequestered internal stores. Ca 2ϩ binds to calmodulin (CaM); the Ca 2ϩ /CaM complex in turn binds to and activates the serine/threonine protein kinase, myosin light chain kinase (MLCK). The active MLCK complex catalyzes myosin II regulatory light chain (RLC) phosphorylation at two sites, Ser-19 and Thr-18. Phosphorylation at these sites is required for myosin II filament formation (6), myosin II interaction with F-actin (23), and an increase in myosin II ATPase activity (7). These phosphorylation driven events are essential for initiation and maintenance of myosin II-based contraction. MLCK, the enzyme responsible for initiating Ca 2ϩ -dependent contraction, exists in several isoforms, resulting from splice variants with distinct intracellular localizations and tissue distributions (3,5,32,52). To date, all MLCK isoforms have been shown biochemically to be strictly dependent on Ca 2ϩ /CaM for activation and are known to catalyze phosphorylation of a single substrate, myosin II RLC.The discovery of the Rho family of proteins initiated study of possible Ca 2ϩ -independent regulation of myosin II motor activity and contraction. A range of protein kinases have now been shown to activate myosin II motor activity by catalyzing phosphorylation of myosin II RLC in vitro or in vivo, including Rho-kinase (2), p21-associated protein kinase (PAK) (9, 73), and integrin-linked kinase (ILK) (10). The most intensively studied of the Ca 2ϩ -independent activators of myosin II motor activity and contraction are those regulated by the Rho family of proteins. Rho is a member of the Ras superfamily of small GTPases, which functions as a molecular switch cycling between the active GTP-bound and inactive GDP-bound state. In the active GTP state, RhoA interacts with its effector molecules to initiate downstream responses. Of several possible Rho effector proteins (4, 17) that bind to GTP-bound Rho, the serine/threonine kinase, Rho-kinase, is the one that has been implicated in the regulation of myosin II and contraction.Rho-kinase exists in two isoforms, ROK␣/ROCK-II (43, 46) and ROK␤/ROCK-I/p160 ROCK (29, 42); both are ubiquitously expressed in various tissues and cells. Rho-kinase isoforms range in mass from 150 to 160 kDa and contain a kinase domain in their NH 2 -terminal domains, a central coil-coiled domain, and a pleckstrin homology domain in the COOH-terminal domains. The coil-coiled domai...

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.