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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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Increased myosin  binding ability of mitotic  MYPT. (a) Mitotic or interphase MYPT was prepared  by immunoaffinity purification and myosin binding activity was determined by  cosedimentation assay with  phosphorylated myosin as  described in Materials and  Methods. The figure shows  myosin binding of MYPT  prepared from interphase  cells (I) or mitotic cells (M).  The values shown are the  means ± SEM from three independent experiments. (b)  Myosin binding activity of in  vitro phosphorylated MYPT.  MYPT was phosphorylated  in vitro with Xenopus mitotic  or interphase extracts and  myosin binding ability was  examined as described in  Materials and Methods.  MYPT phosphorylated with  mitotic extracts (▪); MYPT  phosphorylated with interphase extracts (○).
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Figure 4: Increased myosin binding ability of mitotic MYPT. (a) Mitotic or interphase MYPT was prepared by immunoaffinity purification and myosin binding activity was determined by cosedimentation assay with phosphorylated myosin as described in Materials and Methods. The figure shows myosin binding of MYPT prepared from interphase cells (I) or mitotic cells (M). The values shown are the means ± SEM from three independent experiments. (b) Myosin binding activity of in vitro phosphorylated MYPT. MYPT was phosphorylated in vitro with Xenopus mitotic or interphase extracts and myosin binding ability was examined as described in Materials and Methods. MYPT phosphorylated with mitotic extracts (▪); MYPT phosphorylated with interphase extracts (○).

Mentions: To explore the functional significance of mitosis-specific phosphorylation of MYPT, the myosin binding activities of MYPT from mitotic or interphase cells were compared. Mitotic and interphase MYPT were immuno-affinity purified, and their myosin binding abilities were examined using phosphorylated myosin in the presence of Mg-ATP, as described in Materials and Methods. As Fig. 4 a shows, the amount of mitotic MYPT bound to phosphorylated myosin was about threefold higher than MYPT from interphase cells.


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)

Increased myosin  binding ability of mitotic  MYPT. (a) Mitotic or interphase MYPT was prepared  by immunoaffinity purification and myosin binding activity was determined by  cosedimentation assay with  phosphorylated myosin as  described in Materials and  Methods. The figure shows  myosin binding of MYPT  prepared from interphase  cells (I) or mitotic cells (M).  The values shown are the  means ± SEM from three independent experiments. (b)  Myosin binding activity of in  vitro phosphorylated MYPT.  MYPT was phosphorylated  in vitro with Xenopus mitotic  or interphase extracts and  myosin binding ability was  examined as described in  Materials and Methods.  MYPT phosphorylated with  mitotic extracts (▪); MYPT  phosphorylated with interphase extracts (○).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132942&req=5

Figure 4: Increased myosin binding ability of mitotic MYPT. (a) Mitotic or interphase MYPT was prepared by immunoaffinity purification and myosin binding activity was determined by cosedimentation assay with phosphorylated myosin as described in Materials and Methods. The figure shows myosin binding of MYPT prepared from interphase cells (I) or mitotic cells (M). The values shown are the means ± SEM from three independent experiments. (b) Myosin binding activity of in vitro phosphorylated MYPT. MYPT was phosphorylated in vitro with Xenopus mitotic or interphase extracts and myosin binding ability was examined as described in Materials and Methods. MYPT phosphorylated with mitotic extracts (▪); MYPT phosphorylated with interphase extracts (○).
Mentions: To explore the functional significance of mitosis-specific phosphorylation of MYPT, the myosin binding activities of MYPT from mitotic or interphase cells were compared. Mitotic and interphase MYPT were immuno-affinity purified, and their myosin binding abilities were examined using phosphorylated myosin in the presence of Mg-ATP, as described in Materials and Methods. As Fig. 4 a shows, the amount of mitotic MYPT bound to phosphorylated myosin was about threefold higher than MYPT from interphase cells.

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