Phosphorylation of the myosin heavy chain. Its effect on actin-activated Mg2+-stimulated ATPase in leukaemic myeloblasts.
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ABSTRACT:
SUBMITTER: Sagara J
PROVIDER: S-EPMC1152321 | biostudies-other | 1983 Sep
REPOSITORIES: biostudies-other
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Project description:Nonmuscle myosin-IIA (NMHC-IIA) heavy chain phosphorylation has gained recognition as an important feature of myosin-II regulation. In previous work, we showed that phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances EGF-stimulated lamellipod extension of breast tumor cells. However, the contribution of NMHC-IIA S1943 phosphorylation to the modulation of invasive cellular behavior and metastasis has not been examined. Stable expression of phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA in breast cancer cells revealed that S1943 phosphorylation enhances invadopodia function, and is critical for matrix degradation in vitro and experimental metastasis in vivo. These studies demonstrate a novel link between NMHC-IIA S1943 phosphorylation, the regulation of extracellular matrix degradation and tumor cell invasion and metastasis.
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Project description:The tail of the yeast myosin V encoded by Myo2p is known to bind several receptors for cargo delivery along polarized actin cables. However, it is not known how Myo2p activity is regulated or how it selects between cargoes. Here we show that Myo2p is reversibly phosphorylated in vivo. A short peptide at the N-terminal end of the cargo-binding domain contains three residues contributing to single or doubly phosphorylated species. We confirm that the tail consists of two proteolytically resistant subdomains and identify a functionally important region N-terminal to subdomain 1 that includes the phosphorylation sites. Mutagenesis of the phosphorylation sites to alanine abolished a mobility shift diagnostic of phosphorylation, whereas mutagenesis to glutamic acid produced the shift and the formation of an additional phosphorylated species. These substitutions did not affect overall cell growth. However, one of the sites is predicted to be a substrate of cAMP-dependent protein kinase (PKA), and yeast expressing Myo2p with alanine substitutions is resistant to otherwise lethal overexpression of PKA, whereas the glutamic acid mutant is supersensitive to overexpression of PKA. These results suggest that in yeast, Myo2p is subject to phosphoregulation involving a PKA-related signaling pathway.
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Project description:Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.
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Project description:Heavy chain phosphorylation plays a central role in regulating myosin II bipolar filament assembly in Dictyostelium, as well as in higher eukaryotic nonmuscle cells. Our previous work has demonstrated that the WD-repeat domain of Dictyostelium myosin II heavy chain kinase B (MHCK-B), unlike its counterpart in MHCK-A, is not absolutely required for targeting of the kinase to phosphorylate MHC. Thus, we tested the hypothesis that an asparagine-rich and structurally disordered region that is unique to MHCK-B can by itself function in substrate targeting.Biochemical assays comparing the activities of full-length MHCK-B, a truncation lacking only the WD-repeat domain (B-Delta-WD), and a truncation lacking both the N-rich region and the WD-repeat domain (B-Delta-N-WD) revealed that the N-rich region targets MHCK-B to phosphorylate MHC in a manner that leads to bipolar filament disassembly. This targeting is physiologically relevant since cellular over-expression of the B-Delta-WD truncation, but not the B-Delta-N-WD truncation, leads to dramatically reduced levels of myosin II filament assembly and associated defects in cytokinesis and multicellular development.The results presented here demonstrate that an intrinsically unstructured, and asparagine-rich, region of a MHCK-B can mediate specific targeting of the kinase to phosphorylate myosin II heavy chain. This targeting involves a direct binding interaction with myosin II filaments. In terms of regulating myosin bipolar filament assembly, our results suggest that factors affecting the activity of this unique region of MHCK-B could allow for regulation of MHCK-B in a manner that is distinct from the other MHCKs in Dictyostelium.
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Project description:The actin activated ATPase of myosin at low ionic strength shows a complex dependence on actin concentration, in contrast with the simple hyperbolic actin activation kinetics of heavy meromyosin and subfragment-1. To investigate how the aggregation of myosin influences the actomyosin ATPase kinetics, we have studied the actin-activated ATPase of mixed filaments in which the myosin molecules are separated from each other by copolymerization with myosin rod. Electron microscopy of copolymer filaments, alone and bound to actin, indicates that the myosin heads are distributed randomly along the co-polymer filaments. The actin-activated ATPase of myosin decreases with increasing rod, approaching a plateau of about 30% of the control at a rod/myosin molar ratio of 4:1. The decrease in ATPase persists even at Vmax, the extrapolated limit at infinite actin, indicating that it is not due merely to the loss of cooperative actin binding. Furthermore, the actin dependence of the ATPase still shows a biphasic character like that of control myosin, even at rod/myosin ratio of 12:1, so this complexity is not probably due solely to the structural proximity of myosin molecules, but may involve a non-equivalence of myosin heads or myosin molecules in the filament environment.
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Project description:Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian alpha-kinase TRPM7 inhibits myosin II-based contractility in a Ca(2+)- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains--the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the alpha-helical tail of the myosin IIA heavy chain.
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Project description:Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.
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Project description:Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a 'coiled-coil'-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent K(D) of approx. 0.5 muM and a stoichiometry of approx. 5:1 [actin/C(1-498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.
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Project description:BACKGROUND: Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event. RESULTS: A combined bioinformatics approach of motif prediction and evolutionary and structural analyses identified tyrosines163 and 1856 of the skeletal muscle heavy chain as the leading candidate for the sites of insulin-mediated tyrosine phosphorylation. CONCLUSION: Our work is suggestive that tyrosine phosphorylation of myosin heavy chain, whether in skeletal muscle or in platelets, is a significant event that may initiate cytoskeletal reorganization of muscle cells and platelets. Our studies provide a good starting point for further functional analysis of MHC phosphor-signalling events within different cells.
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