Essential light chain of myosin II
The Dictyostelium myosin essential light chain (MELC) encodes a polypeptide with 66% amino acid sequence identity with the Physarum polycephalum EMLC, and approximately 30% identity with mammalian MELC sequences. The Dictyostelium EMLC gene contains a single intron located immediately 3' of the translation initiation codon, encoding a product most similar to the MLC3 isoform of vertebrates. Based on comparison with published EMLC sequences from a variety of sources, the Dictyostelium MELC shows slightly higher similarity to vertebrate MELCs from striated muscle sources than nonmuscle sources. While Dictyostelium and human nonmuscle sequences display only 28% identity over their entire sequence, the region from residue 88 to 108 shows much higher identity (67%). The high evolutionary conservation of this region of the MELC suggests it may play an important role in MELC function (Pollenz, 1991).

A Dictyostelium mutant (7-11) that expresses less than 0.5% of wild-type levels of the myosin essential light chain has been created by overexpression of antisense RNA. Cells from 7-11 contain wild-type levels of the myosin heavy chain (MHC) and regulatory light chain (MRLC). Myosin isolated from 7-11 cells consists of the MHC with the RMLC associated in reduced stoichiometry, and binds to purified actin in an ATP-sensitive fashion. Purified 7-11 myosin displays calcium-activated ATPase activity with a reduced Vmax , and a reduced Km for ATP. 7-11 myosin displays greatly reduced actin-activated ATPase activity. Phenotypically, 7-11 cells resemble MHC mutants, growing poorly in suspension and becoming large and multinucleate. When starved for multicellular development, 7-11 cells take several hours longer than wild-type cells to aggregate. Although multicellular aggregates eventually form, they fail to develop further. The cells are also unable to cap receptors in response to Con A treatment. Since cells expressing the MELC are phenotypically similar to MHC null mutants, the MELC appears necessary for myosin function, at least in part because it is required for normal actin-activated ATPase activity (Pollenz, 1992).

Dictyostelium Myosin essential light chain mutants (mlcE- mutants), when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants. Myosin purified from the mlcE- cells exhibits significant calcium ATPase activity, but the actin-activated ATPase activity is greatly reduced. MELC is critical for myosin function. The proper localization of myosin in mlcE- cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization. The enzymatic defect of myosin in mlcE- cells also suggests a possible mechanism for the observed chemotactic defect of mlcE- cells. While mlcE- cells are able to respond to chemoattractant with proper directionality, their rate of movement is reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. The localization of prespore cells is more susceptible to the loss of MELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild-type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin (Chen, 1995).

Myosin deficient in the essential light chain of Dictyostelium does not function normally either in vivo or in vitro. Deleting the NH2-terminal 11 or 28 amino acid residues (delta N11 or delta N28) or the COOH-terminal 15 amino acid residues (delta C15) of MELC abolishes binding of the MELC to the MHC. In contrast, the MELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment binds the MHC, although with reduced affinity. When these mutants are expressed in MELC-minus (mlcE-) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous MELC, delta N28 and delta N11 bind to the MHC at lower levels and delta C15 does not bind to a significant degree. I4 and D4, however, bind with normal stoichiometry. These data indicate that residues at both termini of the MELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants are analyzed for their ability to complement the cytokinesis defect displayed by mlcE- cells, a correlation to the level of ELC carried by the MHC is observed, indicating that a stoichiometric MELC-MHC association is necessary for normal myosin function in vivo (Ho, 1995).

Antibody specific for Dictyostelium discoideum myosin was used to screen a lambda gt11 cDNA expression library to obtain cDNA clones that encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone shows 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, show distinct regions of homology. Much of the observed homology is localized to regions corresponding to consensus Ca2+-binding of E-F hand domains. Southern blot analysis suggests that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression shows that a significant increase in EMLC message levels occurs during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity (Chisholm, 1988).

The crystal structure of subfragment 1 of the Myosin heavy chain indicates that both the regulatory light chains (RLCs) and the essential light chains stabilize an extended alpha-helical segment of the heavy chain. A motility assay has shown that removal of either light chain markedly reduces actin filament sliding velocity without a significant loss in actin-activated ATPase activity. It can be demonstrated by single actin filament force measurements that RLC removal has little effect on isometric force, whereas MELC removal reduces isometric force by over 50%. These data are interpreted with a simple mechanical model where subfragment 1 behaves as a torque motor whose lever arm length is sensitive to light-chain removal. Although the effect of removing RLCs fits within the confines of this model, altered crossbridge kinetics, as reflected in a reduced unloaded duty cycle, probably contributes to the reduced velocity and force production of MELC-deficient myosins (VanBuren, 1994).

Regulatory light chain of myosin II

Caenorhabditis elegans embryonic elongation is driven by cell shape changes that cause a contraction of the epidermal cell layer enclosing the embryo. This process requires a Rho-associated kinase (LET-502) and is opposed by the activity of a myosin phosphatase regulatory subunit (MEL-11). Myosin phosphatase affects the phosphorylation state of myosin regulatory light chain and thereby regulates the level of actin-activated myosin Mg-ATPase activity. Myosin phosphatase holoenzyme consists of three subunits: a type 1 catalytic subunit that belongs to the PP1c subfamily of Ser/Thr phosphatases, a combined regulatory and targeting subunit, and a smaller subunit of unknown function. The holoenzyme dephosphorylates Ser19 of myosin regulatory light chain thus counteracting phosphorylation of this residue by myosin light chain kinase. Myosin phosphatase activity itself is regulated by phosphorylation of the regulatory subunit, which decreases activity of the holoenzyme. This inhibition is linked to GTP levels and thus suggests a role for the small GTPases, especially Rho. The recently identified Rho-binding kinase is activated by RhoGTP and subsequently inactivates the myosin phosphatase holoenzyme by phosphorylation of the regulatory subunit. mel-11 activity is required both in the epidermis during embryonic elongation and in the spermatheca of the adult somatic gonad. let-502 and mel-11 reporter gene constructs show reciprocal expression patterns in the embryonic epidermis and the spermatheca, and mutations of the two genes have opposite effects in these two tissues. These results are consistent with let-502 and mel-11 mediating tissue contraction and relaxation, respectively. mel-11 embryonic inviability is genetically enhanced by mutations in a Rac signaling pathway, suggesting that Rac potentiates or acts in parallel with the activity of the myosin phosphatase complex. Since Rho has been implicated in promoting cellular contraction, these results support a mechanism by which epithelial morphogenesis is regulated by the counteracting activities of Rho and Rac (Wissmann, 1999).

Scallop adductor myosin is regulated by its subunits: the regulatory light chain (R-LC) and essential light chain (E-LC). Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation. The binding of Ca2+ to myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains. To map the functional domains of the R-LC, mutagenesis was carried out followed by bacterial expression. Wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin. A point mutation of the sixth Ca2(+)-liganding position of domain I results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site (but not regulation of the actin-activated Mg2+ ATPase). A second mutation restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity. The divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site. An intact domain I and the COOH terminus are required to suppress the myosin ATPase activity. The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable; therefore, the R-LC has distinct functional regions (Goodwin, 1990).

Dictyostelium myosin II is known to be regulated in vitro by phosphorylation of the RLC. Cells in which the wild-type myosin II heavy chain was replaced with a recombinant form that lacks the binding site for RLC carries out cytokinesis and almost normal development, processes known to be dependent on functional myosin II. Characterization of the purified recombinant protein suggests that a complex of RLC and the RLC binding site of the heavy chain plays an inhibitory role for adenosine triphosphatase activity and a structural role for the movement of myosin along actin (Uyeda, 1993).

Smooth muscle myosin acts as a molecular motor only if the regulatory light chain (RLC) is phosphorylated. This subunit can be removed from myosin by a novel method involving the use of trifluoperazine. The motility of RLC-deficient myosin is very slow, but native properties are restored when RLC is rebound. Truncating 6 residues from the COOH terminus of the RLC has no effect on phosphorylated myosin's motor properties, while removal of the last 12 residues reduces velocity by approximately 30%. Very slow movement is observed once 26 residues are deleted, or with myosin containing only the COOH-terminal RLC domain. These two mutants thus mimicked the behavior of RLC-deficient myosin, with the important difference that the mutant myosins are monodisperse when assayed by sedimentation velocity and electron microscopy. The decreased motility therefore cannot be caused by aggregation. A common feature of RLC-deficient myosin and the mutant myosins that moved actin slowly is an increased myosin ATPase compared with dephosphorylated myosin, and a lower actin-activated ATPase than obtained with phosphorylated myosin. These results suggest that the COOH-terminal portion of an intact RLC is involved in interactions that regulate myosin's "on-off" switch, both in terms of completely inhibiting and completely activating the molecule (Trybus, 1994).

Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells. To examine how such phosphorylation is regulated in space and time within cells during coordinated cell movements, including cell locomotion and cell division, a phosphorylation-specific antibody was generated. Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated myosin along the direction of cell movement. The level of myosin phosphorylation is high in an anterior region near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involved in cell locomotion. Phosphorylated myosin is also concentrated in cortical microfilament bundles, indicating that cortical filaments are under tension. The enrichment of phosphorylated myosin in the moving edge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated myosin. In contrast, the phosphorylation level of circumferential microfilament bundles in cell-cell contacts is low. These observations suggest that peripheral microfilaments at the edge are involved in force production to drive the cell margin forward while microfilaments in cell-cell contacts play a structural role. During cell division, both fibroblastic and epithelial cells exhibit an increased level of myosin phosphorylation upon cytokinesis. In the case of the NRK epithelial cells, phosphorylated myosin first appears in the midzones of the separating chromosomes during late anaphase, but apparently before the formation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis (Matsumura, 1998).

Lethal(2)giant larvae homologs

A human homologue to the Drosophila l(2)gl gene is designated as hugl. The hugl cDNA detects a locus spanning at least 25 kilobases (kb) in human chromosome band 17p11.2-12, which is centromeric to the p53 gene and recognizes a 4.5 kb RNA transcript. The hugl gene is expressed in brain, kidney and muscle but is barely seen in heart and placenta. Sequence analysis of the hugl cDNA demonstrates a long open reading frame, which has the potential to encode a protein of 1057 amino acids with a predicted molecular weight of 115 kDaltons (kD). To further substantiate and identify the HUGL protein, rabbit antibodies were prepared against synthetic peptides corresponding to the amino and carboxyl termini of the conceptual translation product of the hugl gene. The affinity-purified anti-HUGL antibodies recognize a single protein with an apparent molecular weight of approximately 115 kD. Similar to the Drosophila protein, HUGL is part of a cytoskeletal network and, is associated with nonmuscle myosin II heavy chain and a kinase that specifically phosphorylates HUGL at serine residues (Strand, 1995).

Myosin Light Chain Kinase

In vertebrates, a nonmuscle myosin light chain kinase (nmMLCK) phosphorylates and regulates the activity of the regulatory light chain. The first primary structure nmMLCK has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) (see Drosophila Calmodulin) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, with possible effects on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences. Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrating a direct link between nmMLCK and non-muscle cell function, and providing a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation (Shoemaker, 1990).

Phosphorylation of the regulatory light chain of myosin by the Ca2+/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca2+/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence increases the Ca2+ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca2+ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca2+ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation (Stull, 1993).

Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part of the regulation of smooth muscle and mammalian nonmuscle myosin II. A protein-based optical biosensor was designed to monitor this phosphorylation "switch." The myosin II optical biosensor exhibits nearly control levels of the rate of phosphorylation, K+ATPase activity, and in vitro motility. The optical biosensor light chain was exchanged into the A-bands of chicken pectoralis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Exchanged myofibrils express a phosphorylation-dependent fluorescence change. Labeled light chains are also incorporated into stress fibers of living fibroblasts and smooth muscle cells (Post, 1994). Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers (Post, 1995).

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation are decreased, and cell spreading is inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation (Sanders, 1999).

Myosin light chain phosphatase

Inhibition of myosin phosphatase (Drosophila homolog: Flapwing) is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. This study addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogen-targeting subunit (G(M)) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. It is proposed that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1.PP1C.P-CPI-17, leading to an increase in smooth muscle contraction (Eto, 2004).

The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes. The human genome encodes a far greater number of Ser/Thr protein kinases than of phosphatases. Protein phosphatase 1 (PP1), in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell-cycle progression and muscle relaxation. PP1 has evolved effective catalytic machinery but lacks substrate specificity. Substrate specificity is conferred upon PP1 through interactions with a large number of regulatory subunits. The regulatory subunits are generally unrelated, but most possess the RVxF motif, a canonical PP1-binding sequence. This study revealed the crystal structure at 2.7 Å resolution of the complex between PP1 and a 34-kDa N-terminal domain of the myosin phosphatase targeting subunit MYPT1. MYPT1 is the protein that regulates PP1 function in smooth muscle relaxation. Structural elements amino- and carboxy-terminal to the RVxF motif of MYPT1 are positioned in a way that leads to a pronounced reshaping of the catalytic cleft of PP1, contributing to the increased myosin specificity of this complex. The structure has general implications for the control of PP1 activity by other regulatory subunits (Terrak, 2004).

Phosphorylation of myosin light chain (MLC) and contraction of differentiated smooth muscle cells in vascular walls are regulated by Ca2+ -dependent activation of MLC kinase, and by Rho-kinase- or protein-kinases-C-dependent inhibition of MLC phosphatase (MLCP). This study examined regulatory pathways for MLC kinase and MLCP in cultured vascular smooth muscle cells (VSMCs), and for isometric force generation of VSMCs reconstituted in collagen fibers. Protein levels of RhoA, Rho-kinase and MYPT1 (a regulatory subunit of MLCP) were upregulated in cultured VSMCs, whereas a MLCP inhibitor protein, CPI-17, was downregulated. Endothelin-1 evoked a steady rise in levels of Ca2+, MLC phosphorylation and the contractile force of VSMCs, whereas angiotensin-II induced transient signals. Also, Thr853 phosphorylation of MYPT1 occurred in response to stimuli, but neither agonist induced phosphorylation of MYPT1 at Thr696. Unlike fresh aortic tissues, removal of Ca2+ or addition of voltage-dependent Ca2+ -channel blocker did not inhibit contractions of reconstituted VSMC fibers induced by agonists or even high concentrations of extracellular K+ ions. Inhibitors of Ins(1,4,5)P3-receptor and Rho-kinase antagonized agonist-induced or high-K+ -induced contraction in both reconstituted fibers and fresh tissues. These results indicate that both Ins(1,4,5)P3-induced Ca2+ release and Rho-kinase-induced MYPT1 phosphorylation at Thr853 play pivotal roles in MLC phosphorylation of cultured VSMCs where either Ca2+ -influx or CPI-17-MLCP signaling is downregulated (Woodsome, 2006).

Nitric oxide induces vasodilation by elevating the production of cGMP, an activator of cGMP-dependent protein kinase (PKG). PKG subsequently causes smooth muscle relaxation in part via activation of myosin light chain phosphatase (MLCP). To date, the interaction between PKG and the targeting subunit of MLCP (MYPT1) is not fully understood. Earlier studies by one group of workers showed that the binding of PKG to MYPT1 is mediated by the leucine-zipper motifs at the N and C termini, respectively, of the two proteins. Another group, however, reported that binding of PKG to MYPT1 did not require the leucine-zipper motif of MYPT1. This work fully characterizes the interaction between PKG and MYPT1 using biophysical techniques. For this purpose a recombinant PKG peptide was constructed corresponding to a predicted coiled coil region that contains the leucine-zipper motif. Various C-terminal MYPT1 peptides were constructed bearing various combinations of a predicted coiled coil region, extensions preceding this coiled coil region, and the leucine-zipper motif. The results show, firstly, that while the leucine-zipper motif at the N terminus of PKG forms a homodimeric coiled coil, the one at the C terminus of MYPT1 is monomeric and non-helical. Secondly, the leucine-zipper motif of PKG binds to that of MYPT1 to form a heterodimer. Thirdly, when the leucine-zipper motif of MYPT1 is absent, the PKG leucine-zipper motif binds to the coiled coil region and upstream segments of MYPT1 via formation of a heterotetramer. These results provide rationalization of some of the findings by others using alternative binding analyses (Lee, 2007).

The repressive activity of histone deacetylase 7 (HDAC7), a class IIa HDAC expressed in CD4+CD8+ double-positive thymocytes, is regulated by its nucleocytoplasmic shuttling. In resting thymocytes, HDAC7 is nuclear and functions as a transcriptional repressor. After T-cell receptor (TCR) activation, the serine/threonine kinase PKD1 phosphorylates HDAC7, resulting in its nuclear export and the derepression of its target genes. This study identifies protein phosphatase 1beta (PP1beta) and myosin phosphatase targeting subunit 1 (MYPT1), two components of the myosin phosphatase complex, as HDAC7-associated proteins in thymocytes. Myosin phosphatase dephosphorylates HDAC7 and promotes its nuclear localization, leading to the repression of the HDAC7 target, Nur77, and the inhibition of apoptosis in CD4+CD8+ thymocytes (Parra, 2007).

Myosin II phosphorylation-dependent cell motile events are regulated by myosin light chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin binding subunit of MLCP, plays a critical role in MLCP regulation. This study reports a new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3beta to MYPT1 diminishes the direct binding between MYPT1 and myosin II, and 14-3-3beta overexpression abolishes MYPT1 localization at stress fiber. Furthermore, 14-3-3beta inhibits MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3beta overexpression increased myosin II phosphorylation in cells. MYPT1 phosphorylation at Ser472 is critical for the binding to 14-3-3. EGF-stimulation increases both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreases EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase-dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity (Koga, 2007).

Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin

Anillin is a scaffolding protein that organizes and stabilizes actomyosin contractile rings and was previously thought to function primarily in cytokinesis. Using Xenopus laevis embryos as a model system to examine Anillin's role in the intact vertebrate epithelium, this study found that a population of Anillin surprisingly localizes to epithelial cell-cell junctions throughout the cell cycle, whereas it was previously thought to be nuclear during interphase. Furthermore, Anillin was shown to play a critical role in regulating cell-cell junction integrity. Both tight junctions and adherens junctions are disrupted when Anillin is knocked down, leading to altered cell shape and increased intercellular spaces. Anillin interacts with Rho, F-actin, and myosin II, all of which regulate cell-cell junction structure and function. When Anillin is knocked down, active Rho (Rho-guanosine triphosphate [GTP]), F-actin, and myosin II are misregulated at junctions. Indeed, increased dynamic 'flares' of Rho-GTP are observed at cell-cell junctions, whereas overall junctional F-actin and myosin II accumulation is reduced when Anillin is depleted. It is proposed that Anillin is required for proper Rho-GTP distribution at cell-cell junctions and for maintenance of a robust apical actomyosin belt, which is required for cell-cell junction integrity. These results reveal a novel role for Anillin in regulating epithelial cell-cell junctions (Reyes, 2014).

Myosin biology and biophysics

zipper Evolutionary Homologs continued: part 3/3

zipper: | Biological Overview | Evolutionary Homologs part 1/3 | Regulation | Developmental Biology | Effects of Mutation | References

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