Myosin binding subunit
Two genes have been identified that are associated with the hypodermal cell shape changes that occur during elongation of the C. elegans embryo. The first gene, let-502, encodes a protein with high similarity to Rho-binding Ser/Thr kinases and to human myotonic dystrophy kinase (DM-kinase). Strong mutations in let-502 block embryonic elongation, and let-502 reporter constructs are expressed in hypodermal cells at the elongation stage of development. The second gene, mel-11, was identified by mutations that act as extragenic suppressors of let-502. mel-11 encodes a protein similar to the 110- to 133-kD regulatory subunits of vertebrate smooth muscle myosin-associated phosphatase (PP-1M). It is suggested that the LET-502 kinase and the MEL-11 phosphatase subunit act in a pathway linking a signal generated by the small GTP-binding protein Rho to a myosin-based hypodermal contractile system that drives embryonic elongation. LET-502 may directly regulate the activity of the MEL-11 containing phosphatase complex and the similarity between LET-502 and DM-kinase suggests a similar function for DM-kinase (Wissmann, 1997).
C. 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). 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).
let-502 rho-binding kinase and mel-11 myosin phosphatase regulate C. elegans embryonic morphogenesis. Genetic analysis establishes the following modes of let-502 action: (1) loss of only maternal let-502 results in abnormal early cleavages; (2) loss of both zygotic and maternal let-502 causes elongation defects, and (3) loss of only zygotic let-502 results in sterility. The morphogenetic function of let-502 and mel-11 is apparently redundant with another pathway since elimination of these two genes results in progeny that undergo near-normal elongation. Triple mutant analysis indicates that unc-73 (Rho/Rac guanine exchange factor) and mlc-4 (myosin light chain) act in parallel to or downstream of let-502/mel-11. In contrast mig-2 (Rho/Rac), daf-2 (insulin receptor), and age-1 (PI3 kinase) act within the let-502/mel-11 pathway. Mutations in the sex-determination gene fem-2, which encodes a PP2c phosphatase (unrelated to the MEL-11 phosphatase), enhance mutations of let-502 and suppress those of mel-11. fem-2's elongation function appears to be independent of its role in sexual identity since the sex-determination genes fem-1, fem-3, tra-1, and tra-3 have no effect on mel-11 or let-502. By itself, fem-2 affects morphogenesis with low penetrance. fem-2 blocks the near-normal elongation of let-502; mel-11 indicating that fem-2 acts in a parallel elongation pathway. The action of two redundant pathways likely ensures accurate elongation of the C. elegans embryo (Piekny, 2000).
The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component is the catalytic subunit, while the 130-kDa and 20-kDa components form a regulatory complex that enhances catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppresses its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits (Alessi, 1992).
Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacts with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylates MBS and consequently inactivates myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increases phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase (Kimura, 1996).
Myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin phosphatase, plays a pivotal role in the regulation of myosin phosphatase activity. A novel isoform of MYPT1, termed MYPT2, has been cloned from a human brain cDNA library screened with a cDNA fragment of rat MYPT1. Overlapping clones indicate an open reading frame of 3763 nucleotides and a predicted polypeptide of mass 110,398. Ankyrin repeats and leucine zipper motifs were identified for the sequences 57-316 and 956-982, respectively. Overall, the deduced amino acid sequence of MYPT2 is 61% identical to MYPT1. MYPT2 gene is transcribed abundantly in heart and skeletal muscle, while Western blots using an antibody specific for MYPT2 show exclusive expression of MYPT2 in heart and brain. A recombinant of the N-terminal two-thirds of MYPT2 binds to the catalytic subunit of type 1 phosphatase (delta isoform) and increases activity toward phosphorylated myosin light chain. In situ hybridization localizes the human MYPT2 gene on chromosome 1q32.1, compared to the chromosomal location 12q15-q21-2 for MYPT1. It is suggested that the products of the two gene families of myosin phosphatase target subunit may be localized differently among various tissues (Fujioka, 1998).
Rho-associated kinase phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. The sites of phosphorylation of MBS by Rho-kinase have been identified as Thr-697 and Ser-854; antibody has been prepared that specifically recognizes MBS phosphorylated at Ser-854. It has been found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induces the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration are induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibit the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhances the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 are localized in the leading edge and posterior region. Phosphorylated MBS is localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupts stress fibers and weakens the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulate at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 is increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates, including MLC and ERM family proteins in vivo in a cooperative manner (Kawano, 1999).
The molecular mechanism of myosin-bound protein phosphatase (MBP) regulation by insulin has been examined and the role of MBP in insulin-mediated vasorelaxation has been evaluated. Insulin rapidly stimulates MBP in confluent primary vascular smooth muscle cell (VSMC) cultures. In contrast, VSMCs isolated from diabetic and hypertensive rats exhibit impaired MBP activation by insulin. Insulin-mediated MBP activation is accompanied by a rapid time-dependent reduction in the phosphorylation state of the myosin-bound regulatory subunit (MBS) of MBP. The decrease observed in MBS phosphorylation is due to insulin-induced inhibition of Rho kinase activity. Insulin also prevents a thrombin-mediated increase in Rho kinase activation and abolishes the thrombin-induced increase in MBS phosphorylation and MBP inactivation. These data are consistent with the notion that insulin inactivates Rho kinase and decreases MBS phosphorylation to activate MBP in VSMCs. Furthermore, treatment with synthetic inhibitors of phosphatidylinositol-3 kinase (PI3-kinase), nitric oxide synthase (NOS), and cyclic guanosine monophosphate (cGMP) all block insulin's effect on MBP activation. It is concluded that insulin stimulates MBP via its regulatory subunit, MBS, partly by inactivating Rho kinase and stimulating NO/cGMP signaling via PI3-kinase as part of a complex signaling network that controls 20-kDa myosin light chain (MLC20) phosphorylation and VSMC contraction (Begum, 2000).
Dephosphorylation of the two key regulatory factors of myosin light chain phosphatase (MLCP), CPI17 and MBS (myosin binding subunit) of MLCP was studied. While Thr38 phosphorylated CPI17 is quite susceptible to protein phosphatases, phosphorylated MBS is highly resistant to dephosphorylation. Type 2A, 2B and 2C protein phosphatases (PP2A, PP2B and PP2C), but not type 1 (PP1), dephosphorylated CPI17. The majority of the CPI17 phosphatase activity in smooth muscle is attributed to PP2A and PP2C. Phospholipids inhibit dephosphorylation of MBS and arachidonic acid (AA) inhibits PP2A activity against both MBS and CPI17, raising the possibility that AA favors the preservation of active MLCP. Consistently, while the phosphorylation of CPI17 is promptly decreased when the agonist is removed, the phosphorylation of MBS is unchanged in intact smooth muscle fiber. The results suggest that MBS phosphorylation mediated regulation of MLCP is not suitable for regulating rapid change in myosin phosphorylation. In contrast, phosphorylated CPI17 is readily dephosphorylated and thus likely to play a role in regulating fast phosphorylation-dephosphorylation cycle in cells (Takizawa, 2002).
Raf-1 serine/threonine protein kinase plays an important role in cell survival, proliferation, and migration; however, the specific targets of Raf-1 in diverse cellular processes are not clearly defined. Myosin phosphatase activity is critical to the regulation of cytoskeletal reorganization, cytokinesis, and cell motility. Raf-1 associates with myosin phosphatase, and the regulatory myosin-binding subunit (MBS) of myosin phosphatase is phosphorylated by Raf-1. Treatment of cells with phorbol 12-myristate 13-acetate stimulates Raf-1 protein kinase. To determine the effect of enzymatic activation of Raf-1 on MBS phosphorylation, COS-1 cells were transiently transfected with FLAG-tagged full-length Raf-1. A significantly higher phosphorylation of purified glutathione S-transferase-tagged truncated MBS protein (amino acids 654-880) occurs in the presence of FLAG-Raf-1 immunoprecipitated from phorbol 12-myristate 13-acetate-treated cells compared with untreated cells (approximately 3.0-fold). Using a sequential kinase-phosphatase assay and phosphorylated myosin light chain as substrate in the phosphatase reaction, it has been shown that Raf-1-associated protein phosphatase-specific activity is inhibited (relative phosphatase activity without and with adenosine 5'-O-(3-thiotriphosphate): 100% and approximately 30%, respectively). Ionizing radiation activates Raf-1. Exposure of cells to ionizing radiation results in the increased association of Raf-1 with MBS (3-6-fold versus unirradiated control) and inhibition of Raf-1-associated protein phosphatase-specific activity (relative phosphatase activity without and with ionizing radiation: 100% and approximately 54%, respectively). These studies identify MBS as a new substrate of Raf-1 and implicate a role for Raf-1 in the regulation of pathways involving myosin phosphatase activity (Broustas, 2002).
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