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Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation.

Totsukawa G, Yamakita Y, Yamashiro S, Hosoya H, Hartshorne DJ, Matsumura F - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855, USA.

ABSTRACT
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.

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Myosin phosphatase activity of in vitro  phosphorylated myosin phosphatase. (a) Immunoblotting  analysis of in vitro phosphorylated myosin phosphatase.  Interphase myosin phosphatase was prepared by immunoprecipitation and phosphorylated in vitro with  Xenopus interphase (lane 1)  or mitotic extracts (lane 2).  After extensive washing,  phosphorylated samples were  analyzed by immunoblotting  with the mAb, pAb against  MYPT, or anti-PP1c antibody. (b) Myosin phosphatase activity of the in  vitro phosphorylated myosin  phosphatase. Left column,  myosin phosphatase phosphorylated with interphase  extracts; right column, myosin phosphatase phosphorylated with mitotic extracts.  The values shown are  means ± SEM from three independent experiments.
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Figure 5: Myosin phosphatase activity of in vitro phosphorylated myosin phosphatase. (a) Immunoblotting analysis of in vitro phosphorylated myosin phosphatase. Interphase myosin phosphatase was prepared by immunoprecipitation and phosphorylated in vitro with Xenopus interphase (lane 1) or mitotic extracts (lane 2). After extensive washing, phosphorylated samples were analyzed by immunoblotting with the mAb, pAb against MYPT, or anti-PP1c antibody. (b) Myosin phosphatase activity of the in vitro phosphorylated myosin phosphatase. Left column, myosin phosphatase phosphorylated with interphase extracts; right column, myosin phosphatase phosphorylated with mitotic extracts. The values shown are means ± SEM from three independent experiments.

Mentions: The enhanced myosin binding activity of mitotic MYPT suggested that myosin phosphatase activity may be increased during mitosis. To test this possibility, MYPT was immunoprecipitated using buffer II (to retain the catalytic subunit in complex with MYPT), phosphorylated with either Xenopus mitotic or interphase extracts, and used to assay phosphatase activities. Again, the MYPT phosphorylated with Xenopus mitotic extracts showed loss of reactivity to the mAb (lane 2 of the upper panel of Fig. 5 a), indicating that mitosis-specific phosphorylation of MYPT occurred. It was also confirmed, by immunoblotting with the pAb against MYPT and with the mAb to PP1c (middle and lower panel of Fig. 5 a), that essentially identical amounts of MYPT and the catalytic subunit were present in the immunoprecipitates treated with mitotic or interphase Xenopus extracts. As Fig. 5 b shows, the myosin phosphatase treated with mitotic extracts had approximately twice the activity than that treated with interphase extracts. Similar results were obtained with chick gizzard myosin phosphatase (data not shown). These results indicate that the enhanced myosin binding ability was accompanied by a higher phosphatase activity.


Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation.

Totsukawa G, Yamakita Y, Yamashiro S, Hosoya H, Hartshorne DJ, Matsumura F - J. Cell Biol. (1999)

Myosin phosphatase activity of in vitro  phosphorylated myosin phosphatase. (a) Immunoblotting  analysis of in vitro phosphorylated myosin phosphatase.  Interphase myosin phosphatase was prepared by immunoprecipitation and phosphorylated in vitro with  Xenopus interphase (lane 1)  or mitotic extracts (lane 2).  After extensive washing,  phosphorylated samples were  analyzed by immunoblotting  with the mAb, pAb against  MYPT, or anti-PP1c antibody. (b) Myosin phosphatase activity of the in  vitro phosphorylated myosin  phosphatase. Left column,  myosin phosphatase phosphorylated with interphase  extracts; right column, myosin phosphatase phosphorylated with mitotic extracts.  The values shown are  means ± SEM from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132942&req=5

Figure 5: Myosin phosphatase activity of in vitro phosphorylated myosin phosphatase. (a) Immunoblotting analysis of in vitro phosphorylated myosin phosphatase. Interphase myosin phosphatase was prepared by immunoprecipitation and phosphorylated in vitro with Xenopus interphase (lane 1) or mitotic extracts (lane 2). After extensive washing, phosphorylated samples were analyzed by immunoblotting with the mAb, pAb against MYPT, or anti-PP1c antibody. (b) Myosin phosphatase activity of the in vitro phosphorylated myosin phosphatase. Left column, myosin phosphatase phosphorylated with interphase extracts; right column, myosin phosphatase phosphorylated with mitotic extracts. The values shown are means ± SEM from three independent experiments.
Mentions: The enhanced myosin binding activity of mitotic MYPT suggested that myosin phosphatase activity may be increased during mitosis. To test this possibility, MYPT was immunoprecipitated using buffer II (to retain the catalytic subunit in complex with MYPT), phosphorylated with either Xenopus mitotic or interphase extracts, and used to assay phosphatase activities. Again, the MYPT phosphorylated with Xenopus mitotic extracts showed loss of reactivity to the mAb (lane 2 of the upper panel of Fig. 5 a), indicating that mitosis-specific phosphorylation of MYPT occurred. It was also confirmed, by immunoblotting with the pAb against MYPT and with the mAb to PP1c (middle and lower panel of Fig. 5 a), that essentially identical amounts of MYPT and the catalytic subunit were present in the immunoprecipitates treated with mitotic or interphase Xenopus extracts. As Fig. 5 b shows, the myosin phosphatase treated with mitotic extracts had approximately twice the activity than that treated with interphase extracts. Similar results were obtained with chick gizzard myosin phosphatase (data not shown). These results indicate that the enhanced myosin binding ability was accompanied by a higher phosphatase activity.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus