Solution structure of the inhibitory phosphorylation domain of myosin phosphatase targeting subunit 1.
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ABSTRACT:
SUBMITTER: Mori S
PROVIDER: S-EPMC2796628 | biostudies-literature | 2009 Nov
REPOSITORIES: biostudies-literature
Publications
Solution structure of the inhibitory phosphorylation domain of myosin phosphatase targeting subunit 1.
Mori Shunsuke S Iwaoka Ryou R Eto Masumi M Ohki Shin-ya SY
Proteins 20091101 3
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Project description:In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.
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Project description:Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction, and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2), and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, whereas the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M(21)) that increased the sensitivity to Ca(2+) in muscle contraction. In this study, we investigated the role of hHS-M(21) in the regulation of MLCP phosphorylation. Two isoforms of hHS-M(21), hHS-M(21)A and hHS-M(21)B, preferentially bound the C-terminal one-third region of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser-852, impaired the binding of MYPT1 and hHS-M(21). The hHS-M(21) increased the phosphorylation level of MYPT1 at Thr-696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M(21) bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M(21) is a heart-specific effector of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr-696 by ROCK.
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Project description:The myosin light chain phosphatase (MLCP) is a cytoskeleton-associated protein phosphatase-1 (PP1) holoenzyme and a RhoA/ROCK effector, regulating cytoskeletal reorganization. ROCK-induced phosphorylation of the MLCP regulatory subunit (MYPT1) at two sites, Thr696 and Thr853, suppresses the activity, although little is known about the difference in the role. Here, we developed a new method for the preparation of the recombinant human MLCP complex and determined the molecular and cellular basis of inhibitory phosphorylation. The recombinant MLCP partially purified from mammalian cell lysates retained characteristics of the native enzyme, such that it was fully active without Mn(2+) and sensitive to PP1 inhibitor compounds. Selective thio-phosphorylation of MYPT1 at Thr696 with ROCK inhibited the MLCP activity 30%, whereas the Thr853 thio-phosphorylation did not alter the phosphatase activity. Interference with the docking of phospho-Thr696 at the active site weakened the inhibition, suggesting selective autoinhibition induced by phospho-Thr696. Both Thr696 and Thr853 sites underwent autodephosphorylation. Compared with that of Thr853, phosphorylation of Thr696 was more stable, and it facilitated Thr853 phosphorylation. Endogenous MYPT1 at Thr696 was spontaneously phosphorylated in quiescent human leiomyosarcoma cells. Serum stimulation of the cells resulted in dissociation of MYPT1 from myosin and PP1C in parallel with an increase in the level of Thr853 phosphorylation. The C-terminal domain of human MYPT1(495-1030) was responsible for the binding to the N-terminal portion of myosin light meromyosin. The spontaneous phosphorylation at Thr696 may adjust the basal activity of cellular MLCP and affect the temporal phosphorylation at Thr853 that is synchronized with myosin targeting.
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Project description:Myosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatory light chain phosphorylation with small molecule inhibitors alters the shape, adhesion, and migration of many types of smooth muscle and cancer cells. Dephosphorylation is mediated by myosin phosphatase (MP), a complex that consists of a catalytic subunit (protein phosphatase 1c, PP1c), a large subunit (myosin phosphatase targeting subunit, MYPT), and a small subunit of unknown function. MYPT functions by targeting PP1c onto its substrate, phosphorylated myosin II. Using RNA interference, we show here that stability of PP1c beta and MYPT1 is interdependent; knocking down one of the subunits decreases the expression level of the other. Associated changes in cell shape also occur, characterized by flattening and spreading accompanied by increased cortical actin, and cell numbers decrease secondary to apoptosis. Of the three highly conserved isoforms of PP1c, we show that MYPT1 binding is restricted to PP1c beta, and, using chimeric analysis and site-directed mutations, that the central region of PP1c beta confers the isoform-specific binding. This finding was unexpected because the MP crystal structure has been solved and was reported to identify the variable, C-terminal domain of PP1c beta as being the region key for isoform-specific interaction with MYPT1. These findings suggest a potential screening strategy for cardiovascular and cancer therapeutic agents based on destabilizing MP complex formation and function.
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Project description:Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.
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Project description:Myosin phosphatase is an evolutionarily conserved regulator of actomyosin contractility, comprised of a regulatory subunit (Mypt1), and a catalytic subunit (PP1). Zebrafish has become an ideal model organism for the study of the genetic and cell physiological role of the myosin phosphatase in morphogenesis and embryonic development. We identified and characterized a novel splice variant of Mypt1 (ppp1r12a-tv202) from zebrafish, which is widely expressed during early embryonic development. Importantly, mutant alleles and antisense morpholinos that have been used to demonstrate the important role of Mypt1 in early development, not only disrupt the longer splice variants, but also tv202. The protein product of ppp1r12a-tv202 (Mypt1-202) contains the PP1-binding N-terminus, but lacks the regulatory C-terminus, which contains two highly conserved inhibitory phosphorylation sites. We observed that the protein product of tv202 assembled a constitutively active myosin phosphatase uninhibited by kinases such as Zipk. Thus, we propose that Mypt1-202 plays an important role in maintaining baseline Mlc2 dephosphorylation and actomyosin relaxation during early zebrafish development.
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Project description:BackgroundThe myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase.Methodology/principal findingsWe observed that in zebrafish two paralogous genes encoding PP1β, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function.Conclusions/significanceOur work demonstrates that zebrafish have two genes encoding PP1β, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required.
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Project description:Aging causes major alterations of all components of the neurovascular unit and compromises brain blood supply. Here, we tested how aging affects vascular reactivity in basilar arteries from young (<10 weeks; y-BA), old (>22 months; o-BA) and old (>22 months) heterozygous MYPT1-T-696A/+ knock-in mice. In isometrically mounted o-BA, media thickness was increased by ∼10% while the passive length tension relations were not altered. Endothelial denudation or pan-NOS inhibition (100 µmol/L L-NAME) increased the basal tone by 11% in y-BA and 23% in o-BA, while inhibition of nNOS (1 µmol/L L-NPA) induced ∼10% increase in both ages. eNOS expression was ∼2-fold higher in o-BA. In o-BA, U46619-induced force was augmented (pEC50 ∼6.9 vs. pEC50 ∼6.5) while responsiveness to DEA-NONOate, electrical field stimulation or nicotine was decreased. Basal phosphorylation of MLC20-S19 and MYPT1-T-853 was higher in o-BA and was reversed by apocynin. Furthermore, permeabilized o-BA showed enhanced Ca2+-sensitivity. Old T-696A/+ BA displayed a reduced phosphorylation of MYPT1-T696 and MLC20, a lower basal tone in response to L-NAME and a reduced eNOS expression. The results indicate that the vascular hypercontractility found in o-BA is mediated by inhibition of MLCP and is partially compensated by an upregulation of endothelial NO release.
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