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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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Mitosis-specific phosphorylation of MYPT. (a) Mitotic  MYPT loses a reactivity to the mAb specific to MYPT. Total cell  lysates of interphase (lanes 1 and 3) or mitotic cells (lanes 2 and  4) were immunoblotted with rabbit pAb (lanes 1 and 2) or mouse  mAb (lanes 3 and 4). The arrow indicates the position of MYPT  (130 kD). Molecular mass markers are indicated in kD at the left  of the figure. (b) Treatment with PP1c restores the reactivity of  mitotic MYPT to the mAb. MYPT was immunoprecipitated from  interphase (lanes 1 and 3) or mitotic cells (lanes 2 and 4). Half of  the immunoprecipitate was treated with PP1c (lanes 3 and 4).  Both treated or untreated samples were immunoblotted with the  mAb or pAb. (c) Time course of mitosis-specific modification of  MYPT during cell division. Total cell lysates were prepared from  interphase cells (lane I), mitotic cells (lane M), and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and blotted with either the mAb or the pAb. Cyto-kinesis occurred 40–60 min after release of mitotic arrest.
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Figure 1: Mitosis-specific phosphorylation of MYPT. (a) Mitotic MYPT loses a reactivity to the mAb specific to MYPT. Total cell lysates of interphase (lanes 1 and 3) or mitotic cells (lanes 2 and 4) were immunoblotted with rabbit pAb (lanes 1 and 2) or mouse mAb (lanes 3 and 4). The arrow indicates the position of MYPT (130 kD). Molecular mass markers are indicated in kD at the left of the figure. (b) Treatment with PP1c restores the reactivity of mitotic MYPT to the mAb. MYPT was immunoprecipitated from interphase (lanes 1 and 3) or mitotic cells (lanes 2 and 4). Half of the immunoprecipitate was treated with PP1c (lanes 3 and 4). Both treated or untreated samples were immunoblotted with the mAb or pAb. (c) Time course of mitosis-specific modification of MYPT during cell division. Total cell lysates were prepared from interphase cells (lane I), mitotic cells (lane M), and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and blotted with either the mAb or the pAb. Cyto-kinesis occurred 40–60 min after release of mitotic arrest.

Mentions: Initially a mitosis-specific modification of MYPT was observed by immunoblotting the mitotic cell lysates with the mAb specific for MYPT. As Fig. 1 a shows, the mAb did not detect a band in the total cell lysates of mitotic cells (lane 4) but the same mAb reacted strongly with MYPT from interphase cells (lane 3). The lack of reactivity in mitotic cell lysates was not due to degradation of MYPT during mitosis because the pAb, Ab1–38, reacted equally well with MYPT from either interphase (lane 1) or mitotic (lane 2) cells. In addition, in cells rounded by trypsin treatment the reactivity of MYPT with the mAb was retained (data not shown). Thus it is suggested that the loss of recognition of the mAb for MYPT was due to a modification of MYPT incurred 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)

Mitosis-specific phosphorylation of MYPT. (a) Mitotic  MYPT loses a reactivity to the mAb specific to MYPT. Total cell  lysates of interphase (lanes 1 and 3) or mitotic cells (lanes 2 and  4) were immunoblotted with rabbit pAb (lanes 1 and 2) or mouse  mAb (lanes 3 and 4). The arrow indicates the position of MYPT  (130 kD). Molecular mass markers are indicated in kD at the left  of the figure. (b) Treatment with PP1c restores the reactivity of  mitotic MYPT to the mAb. MYPT was immunoprecipitated from  interphase (lanes 1 and 3) or mitotic cells (lanes 2 and 4). Half of  the immunoprecipitate was treated with PP1c (lanes 3 and 4).  Both treated or untreated samples were immunoblotted with the  mAb or pAb. (c) Time course of mitosis-specific modification of  MYPT during cell division. Total cell lysates were prepared from  interphase cells (lane I), mitotic cells (lane M), and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and blotted with either the mAb or the pAb. Cyto-kinesis occurred 40–60 min after release of mitotic arrest.
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Related In: Results  -  Collection

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Figure 1: Mitosis-specific phosphorylation of MYPT. (a) Mitotic MYPT loses a reactivity to the mAb specific to MYPT. Total cell lysates of interphase (lanes 1 and 3) or mitotic cells (lanes 2 and 4) were immunoblotted with rabbit pAb (lanes 1 and 2) or mouse mAb (lanes 3 and 4). The arrow indicates the position of MYPT (130 kD). Molecular mass markers are indicated in kD at the left of the figure. (b) Treatment with PP1c restores the reactivity of mitotic MYPT to the mAb. MYPT was immunoprecipitated from interphase (lanes 1 and 3) or mitotic cells (lanes 2 and 4). Half of the immunoprecipitate was treated with PP1c (lanes 3 and 4). Both treated or untreated samples were immunoblotted with the mAb or pAb. (c) Time course of mitosis-specific modification of MYPT during cell division. Total cell lysates were prepared from interphase cells (lane I), mitotic cells (lane M), and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and blotted with either the mAb or the pAb. Cyto-kinesis occurred 40–60 min after release of mitotic arrest.
Mentions: Initially a mitosis-specific modification of MYPT was observed by immunoblotting the mitotic cell lysates with the mAb specific for MYPT. As Fig. 1 a shows, the mAb did not detect a band in the total cell lysates of mitotic cells (lane 4) but the same mAb reacted strongly with MYPT from interphase cells (lane 3). The lack of reactivity in mitotic cell lysates was not due to degradation of MYPT during mitosis because the pAb, Ab1–38, reacted equally well with MYPT from either interphase (lane 1) or mitotic (lane 2) cells. In addition, in cells rounded by trypsin treatment the reactivity of MYPT with the mAb was retained (data not shown). Thus it is suggested that the loss of recognition of the mAb for MYPT was due to a modification of MYPT incurred 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.

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