Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation

It has been demonstrated previously that during mitosis the sites of myosin phosphorylation are switched between the inhibitory sites, Ser 1/2, and the activation sites, Ser 19/Thr 18 (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129- 137; Satterwhite, L.L., M.J. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), suggesting a regulatory role of myosin phosphorylation in cell division. To explore the function of myosin phosphatase in cell division, the possibility that myosin phosphatase activity may be altered during cell division was examined. We have found that the myosin phosphatase targeting subunit (MYPT) undergoes mitosis-specific phosphorylation and that the phosphorylation is reversed during cytokinesis. MYPT phosphorylated either in vivo or in vitro in the mitosis-specific way showed higher binding to myosin II (two- to threefold) compared to MYPT from cells in interphase. Furthermore, the activity of myosin phosphatase was increased more than twice and it is suggested this reflected the increased affinity of myosin binding. These results indicate the presence of a unique positive regulatory mechanism for myosin phosphatase in cell division. The activation of myosin phosphatase during mitosis would enhance dephosphorylation of the myosin regulatory light chain, thereby leading to the disassembly of stress fibers during prophase. The mitosis-specific effect of phosphorylation is lost on exit from mitosis, and the resultant increase in myosin phosphorylation may act as a signal to activate cytokinesis.     

Isolation and characterization of microsatellite loci in the two‐spotted spider mite,Tetranychus urticae (Acari: Tetranychidae)

Sixteen microsatellite loci are described for the two‐spotted spider mite Tetranychus urticae, which is an agricultural pest. The microsatellite loci were obtained through the construction of an enriched library; these loci exhibited polymorphisms (2–5 alleles per locus) and high levels of observed (0.033–0.667, average 0.415) and expected (0.033–0.602, average 0.336) heterozygosities. The isolated microsatellite markers are expected to be useful for the construction of a linkage map of this species.     

Phosphorylation of caldesmon

The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin-dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulin-dependent protein kinase II.     

Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.     

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)