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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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Phosphopeptide analysis of mitotic and interphase  MYPT and in vitro reconstitution of mitosis-specific phosphorylation of MYPT. (a) In vivo phosphorylation of MYPT from interphase or mitotic cells. MYPT was isolated by immunoprecipitation from interphase (lane 1) or mitotic cells (lane 2) that had  been labeled with 32P-orthophosphate. 32P-labeled MYPT was  separated by SDS-PAGE followed by autoradiography. (b) Two-  dimensional tryptic phosphopeptide mapping analysis of in vivo  phosphorylated MYPT. I, MYPT isolated from interphase; M,  MYPT from mitotic cells; Mix, a mixture of mitotic and interphase MYPT; M1–4, phosphopeptide spots specifically observed  in mitotic map; M5, a spot whose intensity is increased in mitotic  map. Phosphopeptide spots specifically observed in interphase  map and spots commonly observed in both interphase and mitotic MYPT are labeled with I and C, respectively. Arrow with e,  electrophoretic dimension; arrow with c, chromatographic dimension; ori, the origin. (c) In vitro reconstitution of mitotic  phosphorylation. Interphase MYPT was prepared by immunoprecipitation and phosphorylated with Xenopus mitotic or interphase extracts. Both samples were separated by SDS-PAGE followed by immunoblotting using the mAb or pAb. Lane 1, a  control without addition of Xenopus extracts; lane 2, MYPT  treated with mitotic extracts; lane 3, MYPT treated with interphase extracts. (d) Phosphopeptide mapping analysis of in vitro  phosphorylated MYPT. MYPT was phosphorylated in vitro using  Xenopus mitotic extracts and analyzed by phosphopeptide mapping (X). For comparison, a mixture of in vivo and in vitro phosphorylated MYPT (Mix) is shown. The mitosis-specific spots are  indicated by M.
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Figure 2: Phosphopeptide analysis of mitotic and interphase MYPT and in vitro reconstitution of mitosis-specific phosphorylation of MYPT. (a) In vivo phosphorylation of MYPT from interphase or mitotic cells. MYPT was isolated by immunoprecipitation from interphase (lane 1) or mitotic cells (lane 2) that had been labeled with 32P-orthophosphate. 32P-labeled MYPT was separated by SDS-PAGE followed by autoradiography. (b) Two- dimensional tryptic phosphopeptide mapping analysis of in vivo phosphorylated MYPT. I, MYPT isolated from interphase; M, MYPT from mitotic cells; Mix, a mixture of mitotic and interphase MYPT; M1–4, phosphopeptide spots specifically observed in mitotic map; M5, a spot whose intensity is increased in mitotic map. Phosphopeptide spots specifically observed in interphase map and spots commonly observed in both interphase and mitotic MYPT are labeled with I and C, respectively. Arrow with e, electrophoretic dimension; arrow with c, chromatographic dimension; ori, the origin. (c) In vitro reconstitution of mitotic phosphorylation. Interphase MYPT was prepared by immunoprecipitation and phosphorylated with Xenopus mitotic or interphase extracts. Both samples were separated by SDS-PAGE followed by immunoblotting using the mAb or pAb. Lane 1, a control without addition of Xenopus extracts; lane 2, MYPT treated with mitotic extracts; lane 3, MYPT treated with interphase extracts. (d) Phosphopeptide mapping analysis of in vitro phosphorylated MYPT. MYPT was phosphorylated in vitro using Xenopus mitotic extracts and analyzed by phosphopeptide mapping (X). For comparison, a mixture of in vivo and in vitro phosphorylated MYPT (Mix) is shown. The mitosis-specific spots are indicated by M.

Mentions: To examine whether net phosphate incorporation into MYPT was increased during mitosis, MYPT was immunoprecipitated from interphase and mitotic cells after in vivo labeling with 32P-orthophosphate. As Fig. 2 a shows, the level of phosphorylation of mitotic MYPT (lane 2) is similar to that of interphase MYPT (lane 1). One explanation for the lack of reactivity to the mAb could be that different sites were phosphorylated in the two stages, rather than a net increase in phosphorylation during mitosis.


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)

Phosphopeptide analysis of mitotic and interphase  MYPT and in vitro reconstitution of mitosis-specific phosphorylation of MYPT. (a) In vivo phosphorylation of MYPT from interphase or mitotic cells. MYPT was isolated by immunoprecipitation from interphase (lane 1) or mitotic cells (lane 2) that had  been labeled with 32P-orthophosphate. 32P-labeled MYPT was  separated by SDS-PAGE followed by autoradiography. (b) Two-  dimensional tryptic phosphopeptide mapping analysis of in vivo  phosphorylated MYPT. I, MYPT isolated from interphase; M,  MYPT from mitotic cells; Mix, a mixture of mitotic and interphase MYPT; M1–4, phosphopeptide spots specifically observed  in mitotic map; M5, a spot whose intensity is increased in mitotic  map. Phosphopeptide spots specifically observed in interphase  map and spots commonly observed in both interphase and mitotic MYPT are labeled with I and C, respectively. Arrow with e,  electrophoretic dimension; arrow with c, chromatographic dimension; ori, the origin. (c) In vitro reconstitution of mitotic  phosphorylation. Interphase MYPT was prepared by immunoprecipitation and phosphorylated with Xenopus mitotic or interphase extracts. Both samples were separated by SDS-PAGE followed by immunoblotting using the mAb or pAb. Lane 1, a  control without addition of Xenopus extracts; lane 2, MYPT  treated with mitotic extracts; lane 3, MYPT treated with interphase extracts. (d) Phosphopeptide mapping analysis of in vitro  phosphorylated MYPT. MYPT was phosphorylated in vitro using  Xenopus mitotic extracts and analyzed by phosphopeptide mapping (X). For comparison, a mixture of in vivo and in vitro phosphorylated MYPT (Mix) is shown. The mitosis-specific spots are  indicated by M.
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Figure 2: Phosphopeptide analysis of mitotic and interphase MYPT and in vitro reconstitution of mitosis-specific phosphorylation of MYPT. (a) In vivo phosphorylation of MYPT from interphase or mitotic cells. MYPT was isolated by immunoprecipitation from interphase (lane 1) or mitotic cells (lane 2) that had been labeled with 32P-orthophosphate. 32P-labeled MYPT was separated by SDS-PAGE followed by autoradiography. (b) Two- dimensional tryptic phosphopeptide mapping analysis of in vivo phosphorylated MYPT. I, MYPT isolated from interphase; M, MYPT from mitotic cells; Mix, a mixture of mitotic and interphase MYPT; M1–4, phosphopeptide spots specifically observed in mitotic map; M5, a spot whose intensity is increased in mitotic map. Phosphopeptide spots specifically observed in interphase map and spots commonly observed in both interphase and mitotic MYPT are labeled with I and C, respectively. Arrow with e, electrophoretic dimension; arrow with c, chromatographic dimension; ori, the origin. (c) In vitro reconstitution of mitotic phosphorylation. Interphase MYPT was prepared by immunoprecipitation and phosphorylated with Xenopus mitotic or interphase extracts. Both samples were separated by SDS-PAGE followed by immunoblotting using the mAb or pAb. Lane 1, a control without addition of Xenopus extracts; lane 2, MYPT treated with mitotic extracts; lane 3, MYPT treated with interphase extracts. (d) Phosphopeptide mapping analysis of in vitro phosphorylated MYPT. MYPT was phosphorylated in vitro using Xenopus mitotic extracts and analyzed by phosphopeptide mapping (X). For comparison, a mixture of in vivo and in vitro phosphorylated MYPT (Mix) is shown. The mitosis-specific spots are indicated by M.
Mentions: To examine whether net phosphate incorporation into MYPT was increased during mitosis, MYPT was immunoprecipitated from interphase and mitotic cells after in vivo labeling with 32P-orthophosphate. As Fig. 2 a shows, the level of phosphorylation of mitotic MYPT (lane 2) is similar to that of interphase MYPT (lane 1). One explanation for the lack of reactivity to the mAb could be that different sites were phosphorylated in the two stages, rather than a net increase in phosphorylation during mitosis.

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