A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium

Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP-binding proteins (GbpA- GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP-phosphodiesterase activity and a 10-fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA(-)B(-) cells is associated with increased myosin II phosphorylation compared with wild-type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process.     

Activation of Dictyostelium myosin light chain kinase A by phosphorylation of Thr166.

Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.     

Effects of the regulatory light chain phosphorylation of myosin II on mitosis and cytokinesis of mammalian cells

Myosin plays an important role in mitosis, especially during cytokinesis. Although it has been assumed that phosphorylation of regulatory light chain of myosin (RLC) controls motility of mammalian non-muscle cells, the functional significance of RLC phosphorylation remains uninvestigated. To address this problem, we have produced unphosphorylatable RLC (T18A/S19A RLC) and overexpressed it in COS-7 cells and normal rat kidney cells. Overexpression of T18A/S19A RLC but not wild type RLC almost completely abolished concanavalin A-induced receptor cap formation. The results indicate that myosin phosphorylation is critical for concanavalin A-induced gathering of surface receptors. T18A/S19A RLC overexpression resulted in the production of multinucleated cells, suggesting the failure of proper cell division in these cells. Video microscopic observation revealed that cells expressing T18A/S19A RLC showed abnormalities during mitosis in two respects. One is that the cells produced abnormal cleavage furrows, resulting in incomplete cytokinesis, which suggests that myosin phosphorylation is important for the normal recruitment of myosin molecules into the contractile ring structure. The other is that separation of chromosomes from the metaphase plate is disrupted in T18A/S19A RLC expressing cells, thus preventing proper transition from metaphase to anaphase. These results suggest that, in addition to cytokinesis, myosin and myosin phosphorylation play a role in the karyokinetic process.     

Myosin II transport, organization, and phosphorylation: evidence for cortical flow/solation-contraction coupling during cytokinesis and cell locomotion.

The mechanism of cytokinesis has been difficult to define because of the short duration and the temporal-spatial dynamics involved in the formation, activation, force production, and disappearance of the cleavage furrow. We have investigated the structural and chemical dynamics of myosin II in living Swiss 3T3 cells from prometaphase through the separation and migration of daughter cells. The structural and chemical dynamics of myosin II have been defined using the semiautomated, multimode light microscope, together with a fluorescent analogue of myosin II and a fluorescent biosensor of myosin II regulatory light chain (RLC) phosphorylation at serine 19. The correlation of image data from live cells using different modes of light microscopy allowed interpretations not possible from single-mode investigations. Myosin II transported toward the equatorial plane from adjacent regions, forming three-dimensional fibers that spanned the volume of the equator during anaphase and telophase. A global phosphorylation of myosin II at serine 19 of the RLC was initiated at anaphase when cortical myosin II transport started. The phosphorylation of myosin II remained high near the equatorial plane through telophase and into cytokinesis, whereas the phosphorylation of myosin II at serine 19 of the RLC decreased at the poles. The timing and pattern of phosphorylation was the same as the shortening of myosin II-based fibers in the cleavage furrow. Myosin II-based fibers shortened and transported out of the cleavage furrow into the tails of the two daughter cells late in cytokinesis. The patterns of myosin II transport, phosphorylation, and shortening of fibers in the migrating daughter cells were similar to that previously defined for cells migrating in a wound in vitro. The temporal-spatial patterns and dynamics of myosin II transport, phosphorylation at serine 19 of the RLC, and the shortening and disappearance of myosin II-based fibers support the proposal that a combination of the cortical flow hypothesis and the solation-contraction coupling hypothesis explain key aspects of cytokinesis and polarized cell locomotion.     

Phosphorylation of the myosin-II light chain does not regulate the timing of cytokinesis in fission yeast.

Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure that each daughter cell inherits an equivalent set of chromosomes. It has been proposed that one mechanism by which this is achieved is through temporally regulated myosin regulatory light chain (RLC) phosphorylation (Satterwhite, L. L., and Pollard, T. D. (1992) Curr. Opin. Cell Biol. 4, 43-52). A variety of evidence is consistent with this model. A direct test of the importance of RLC phosphorylation in vivo has been done only in Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (Jordan, P., and Karess, R. (1997) J. Cell Biol. 139, 1805-1819) but not essential in Dictyostelium (Ostrow, B. D., Chen, P., and Chisholm, R. L. (1994) J. Cell Biol. 127, 1945-1955). The Schizosaccharomyces pombe myosin light chain Cdc4p is essential for cytokinesis, but it was unknown whether phosphorylation played a role in its regulation. Here we show that the S. pombe myosin light chain Cdc4p is phosphorylated in vivo on either serine 2 or 6 but not both. Mutation of either or both of these sites to alanine did not effect the ability of Cdc4p to bind the type II myosin Myo2p, and cells expressing only these mutated versions of Cdc4p grew and divided normally. Similarly, mutation of Ser-2, Ser-6, or both residues to aspartic acid did not affect growth or division of cells. Thus we conclude that phosphorylation of Cdc4p is not essential in vivo for the function of the protein.     

Regulatory mechanism of Dictyostelium myosin light chain kinase A

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.     

In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis

Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs.     

Myosin phosphatase targeting subunit 1 affects cell migration by regulating myosin phosphorylation and actin assembly

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.     

Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational 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 GTPase-dependent 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.     

Cytokinesis is not controlled by calmodulin or myosin light chain kinase in the Caenorhabditis elegans early embryo

Furrow ingression in animal cell cytokinesis is controlled by phosphorylation of myosin II regulatory light chain (mRLC). In Caenorhabditis elegans embryos, Rho-dependent Kinase (RhoK) is involved in, but not absolutely required for, this phosphorylation. The calmodulin effector myosin light chain kinase (MLCK) can also phosphorylate mRLC and is widely regarded as a candidate for redundant function with RhoK. However, our results show that RNA mediated interference against C. elegans calmodulin and candidate MLCKs had no effect on cytokinesis in wild-type or RhoK mutant embryos, ruling out the calmodulin/MLCK pathway as the missing regulator of cytokinesis in the C. elegans early embryo.     

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Thus far, determining the relative contribution of Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and Ca²⁺-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca²⁺, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO₄/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO₄/mol RLC, respectively, within 2.5 min. Ca²⁺ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation     

The regulation of myosin II in Dictyostelium

Dictyostelium conventional myosin (myosin II) is an abundant protein that plays a role in various cellular processes such as cytokinesis, cell protrusion and development. This review will focus on the signal transduction pathways that regulate myosin II during cell movement. Myosin II appears to have two modes of action in Dictyostelium: local stabilization of the cytoskeleton by myosin filament association to the actin meshwork (structural mode) and force generation by contraction of actin filaments (motor mode). Some processes, such as cell movement under restrictive environment, require only the structural mode of myosin. However, cytokinesis in suspension and uropod retraction depend on motor activity as well. Myosin II can self-assemble into bipolar filaments. The formation of these filaments is negatively regulated by heavy chain phosphorylation through the action of a set of novel alpha kinases and is relatively well understood. However, only recently it has become clear that the formation of bipolar filaments and their translocation to the cortex are separate events. Translocation depends on filamentous actin, and is regulated by a cGMP pathway and possibly also by the cAMP phosphodiesterase RegA and the p21-activated kinase PAKa. Myosin motor activity is regulated by phosphorylation of the regulatory light chain through myosin light chain kinase A. Unlike conventional light chain kinases, this enzyme is not regulated by calcium but is activated by cGMP-induced phosphorylation via an upstream kinase and subsequent autophosphorylation.     

Myosin II regulatory light chain as a novel substrate for AIM-1, an aurora/Ipl1p-related kinase from rat

Previous studies demonstrated that the phosphorylated myosin II regulatory light chain (MRLC) is localized at the cleavage furrow of dividing cells, suggesting that phosphorylation of MRLC plays an important role in cytokinesis. However, it remains unclear which kinase(s) phosphorylate MRLC during cytokinesis. AIM-1, an Aurora/Ipl1p-related kinase from rat, is known as a serine/threonine kinase that is required for cytokinesis. Here we examined the possibility that AIM-1 is a candidate for a kinase that phosphorylates MRLC during cytokinesis. As a result, we showed that AIM-1 monophosphorylated MRLC at Ser19 using two-dimensional phosphopeptide mapping analysis and several MRLC mutants. Furthermore, AIM-1 was colocalized with monophosphorylated MRLC at the cleavage furrow of dividing cells. We propose here that AIM-1 may participate in monophosphorylation of MRLC during cytokinesis.     

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

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 Ca²⁺ 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 light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.     

Multiple regulatory mechanisms for the Dictyostelium Roco protein GbpC

GbpC is a multidomain Roco protein in Dictyostelium, involved in transduction of intracellular cGMP that is produced by chemotactic signals. We have shown previously that cGMP binding to GbpC induces an intramolecular signaling cascade by activating subsequently the GEF, Ras, and kinase domains. In this study, we report on the cellular localization of GbpC. In resting cells, the protein is present in the cytoplasm, but GbpC rapidly translocates to the cell boundary upon stimulation with the chemoattractant cAMP. Also, during the formation of cell-cell streams and osmotic shock, the protein localizes toward the plasma membrane and actin cytoskeleton. The translocation upon cAMP stimulation occurs downstream of heterotrimeric G proteins but is independent of guanylyl cyclases and the previously identified cGMP-induced intramolecular signaling cascade in GbpC. Mutations in the GRAM domain of GbpC lead to disturbed membrane association and inactivation of GbpC function during chemotaxis in vivo. Furthermore, we show that the GRAM domain itself associates with cellular membranes and binds various phospholipids in vitro. Together, the results show that GbpC receives multiple input signals that are both required for functional activity in vivo. cAMP-stimulation induces a cGMP-dependent signaling cascade, leading to activation of kinase activity, and, independently, cAMP induces a GRAM-dependent translocation of GbpC toward the plasma membrane and cell cortex, where it may locally phosphorylate effector proteins, which are needed for proper biological activity.     

Constitutive Phosphorylation of Cardiac Myosin Regulatory Light Chain in Vivo

In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca²⁺ sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.     

Actomyosin adenosine triphosphatase regulation by intramolecular myosin mechanisms. Myosin light chains functions and rod modification effects

Mechanisms of the actomyosin ATPase modulation via the myosin light chains (LC) in various myosin types are discussed. The essential LC increase the stability of the myosin heavy chains (HC) in the myosin heads and, under certain conditions, they can affect the degree of interaction of HC with actin. The regulatory LC (RLC) are sensitive to calcium binding on specific sites or to calcium activated phosphorylation. These factors induce changes of the RLC state followed by changes of the HC state in response to calcium concentration changes during the contractile process. Direct calcium binding or phosphorylation effects in various muscles are mediated by special types of RLC and HC. Several examples of actomyosin ATPase changes induced by modifications of the myosin rod are compared. A common feature of these effects is a possible involvement of certain configurational changes of the myosin molecule. These changes can affect the spatial position of the myosin heads and the myosin-actin interaction.     

N-methyl-D-aspartate receptor subunits are non-myosin targets of myosin regulatory light chain

Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the "GXXXR" portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.     

Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.