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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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Identification of one of the mitosis-specific phosphorylation sites of MYPT. (a) Schematic presentation of the truncated  mutants of chick MYPT. The reactivities to the mAb of truncated  mutants are shown. MYPT truncated mutants were immunoblotted with the mAb against MYPT. The protein expression in bacteria was confirmed with an antipolyhistidine antibody. The  epitope of the mAb should be located between 421 and 432. (b)  The epitope region of MYPT to the mAb. The sequence shown is  Glu 421 to Arg 432 of chick MYPT (which corresponds to Glu  426 to Arg 437 of rat MYPT), and contains two serine residues of  Ser 427 and Ser 430. To mimic phosphorylation, Ser 427 and Ser  430 were mutated to Asp (S427D) and Glu (S430E), respectively,  and the mutants were expressed in bacteria. (c) Immunoblot  analysis of the phosphorylation-mimicking mutants of MYPT.  The S427D and S430E mutants were separated by SDS-PAGE  followed by immunoblotting analysis using the antipolyhistidine  antibody or the mAb against MYPT. Lane 1, wild-type  MYPT304-511; lane 2, S427D mutant; lane 3, S430E mutant. (d)  Phosphopeptide mapping analysis of a synthetic peptide (NH2-ISPKEEERKDESPASWRLGLRKC-COOH) containing Ser  430. P, peptide map of the synthetic peptide phosphorylated by  Xenopus mitotic extracts; X, map of rat MYPT phosphorylated  by Xenopus mitotic extracts; Mix, a map of a mixture of the synthetic peptide and MYPT. The two spots in P (indicated by arrows) match M2 and M3 of mitotic spots.
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Figure 3: Identification of one of the mitosis-specific phosphorylation sites of MYPT. (a) Schematic presentation of the truncated mutants of chick MYPT. The reactivities to the mAb of truncated mutants are shown. MYPT truncated mutants were immunoblotted with the mAb against MYPT. The protein expression in bacteria was confirmed with an antipolyhistidine antibody. The epitope of the mAb should be located between 421 and 432. (b) The epitope region of MYPT to the mAb. The sequence shown is Glu 421 to Arg 432 of chick MYPT (which corresponds to Glu 426 to Arg 437 of rat MYPT), and contains two serine residues of Ser 427 and Ser 430. To mimic phosphorylation, Ser 427 and Ser 430 were mutated to Asp (S427D) and Glu (S430E), respectively, and the mutants were expressed in bacteria. (c) Immunoblot analysis of the phosphorylation-mimicking mutants of MYPT. The S427D and S430E mutants were separated by SDS-PAGE followed by immunoblotting analysis using the antipolyhistidine antibody or the mAb against MYPT. Lane 1, wild-type MYPT304-511; lane 2, S427D mutant; lane 3, S430E mutant. (d) Phosphopeptide mapping analysis of a synthetic peptide (NH2-ISPKEEERKDESPASWRLGLRKC-COOH) containing Ser 430. P, peptide map of the synthetic peptide phosphorylated by Xenopus mitotic extracts; X, map of rat MYPT phosphorylated by Xenopus mitotic extracts; Mix, a map of a mixture of the synthetic peptide and MYPT. The two spots in P (indicated by arrows) match M2 and M3 of mitotic spots.

Mentions: The loss of reactivity to the mAb indicated that the epitope of the mAb may contain a site of mitosis-specific phosphorylation. It was known that the epitope to the mAb was between residues 371 and 511 (Hartshorne, D.J., unpublished results) and to define more precisely the epitope a series of truncation mutants was analyzed. In Fig. 3 a it is shown that fragment 421–511 has a positive reaction with the mAb while 432–511 was negative, indicating that the epitope is localized between residues 421 and 432. There are two serines (no threonine) in this sequence, Ser 427 and Ser 430 (Fig. 3 b), and these two residues were mutated to Asp and Glu, respectively. The resultant point mutants were expressed in bacteria, and the reactivities of these two mutants to the mAb were examined by immunoblotting. It was found that mutation of Ser 430 to Glu (S430E) resulted in complete loss of reactivity (lane 3 of Fig. 3 c). In contrast, the mutant replacing Ser 427 with Asp (S427D) still showed a strong reactivity to the mAb, though the reactivity was weaker than the control (lane 2 of Fig. 3 c). A reasonable conclusion from these results is that Ser 430 of MYPT is a site phosphorylated during mitosis, although it is possible that simultaneous phosphorylation at Ser 427 may also occur.


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)

Identification of one of the mitosis-specific phosphorylation sites of MYPT. (a) Schematic presentation of the truncated  mutants of chick MYPT. The reactivities to the mAb of truncated  mutants are shown. MYPT truncated mutants were immunoblotted with the mAb against MYPT. The protein expression in bacteria was confirmed with an antipolyhistidine antibody. The  epitope of the mAb should be located between 421 and 432. (b)  The epitope region of MYPT to the mAb. The sequence shown is  Glu 421 to Arg 432 of chick MYPT (which corresponds to Glu  426 to Arg 437 of rat MYPT), and contains two serine residues of  Ser 427 and Ser 430. To mimic phosphorylation, Ser 427 and Ser  430 were mutated to Asp (S427D) and Glu (S430E), respectively,  and the mutants were expressed in bacteria. (c) Immunoblot  analysis of the phosphorylation-mimicking mutants of MYPT.  The S427D and S430E mutants were separated by SDS-PAGE  followed by immunoblotting analysis using the antipolyhistidine  antibody or the mAb against MYPT. Lane 1, wild-type  MYPT304-511; lane 2, S427D mutant; lane 3, S430E mutant. (d)  Phosphopeptide mapping analysis of a synthetic peptide (NH2-ISPKEEERKDESPASWRLGLRKC-COOH) containing Ser  430. P, peptide map of the synthetic peptide phosphorylated by  Xenopus mitotic extracts; X, map of rat MYPT phosphorylated  by Xenopus mitotic extracts; Mix, a map of a mixture of the synthetic peptide and MYPT. The two spots in P (indicated by arrows) match M2 and M3 of mitotic spots.
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Figure 3: Identification of one of the mitosis-specific phosphorylation sites of MYPT. (a) Schematic presentation of the truncated mutants of chick MYPT. The reactivities to the mAb of truncated mutants are shown. MYPT truncated mutants were immunoblotted with the mAb against MYPT. The protein expression in bacteria was confirmed with an antipolyhistidine antibody. The epitope of the mAb should be located between 421 and 432. (b) The epitope region of MYPT to the mAb. The sequence shown is Glu 421 to Arg 432 of chick MYPT (which corresponds to Glu 426 to Arg 437 of rat MYPT), and contains two serine residues of Ser 427 and Ser 430. To mimic phosphorylation, Ser 427 and Ser 430 were mutated to Asp (S427D) and Glu (S430E), respectively, and the mutants were expressed in bacteria. (c) Immunoblot analysis of the phosphorylation-mimicking mutants of MYPT. The S427D and S430E mutants were separated by SDS-PAGE followed by immunoblotting analysis using the antipolyhistidine antibody or the mAb against MYPT. Lane 1, wild-type MYPT304-511; lane 2, S427D mutant; lane 3, S430E mutant. (d) Phosphopeptide mapping analysis of a synthetic peptide (NH2-ISPKEEERKDESPASWRLGLRKC-COOH) containing Ser 430. P, peptide map of the synthetic peptide phosphorylated by Xenopus mitotic extracts; X, map of rat MYPT phosphorylated by Xenopus mitotic extracts; Mix, a map of a mixture of the synthetic peptide and MYPT. The two spots in P (indicated by arrows) match M2 and M3 of mitotic spots.
Mentions: The loss of reactivity to the mAb indicated that the epitope of the mAb may contain a site of mitosis-specific phosphorylation. It was known that the epitope to the mAb was between residues 371 and 511 (Hartshorne, D.J., unpublished results) and to define more precisely the epitope a series of truncation mutants was analyzed. In Fig. 3 a it is shown that fragment 421–511 has a positive reaction with the mAb while 432–511 was negative, indicating that the epitope is localized between residues 421 and 432. There are two serines (no threonine) in this sequence, Ser 427 and Ser 430 (Fig. 3 b), and these two residues were mutated to Asp and Glu, respectively. The resultant point mutants were expressed in bacteria, and the reactivities of these two mutants to the mAb were examined by immunoblotting. It was found that mutation of Ser 430 to Glu (S430E) resulted in complete loss of reactivity (lane 3 of Fig. 3 c). In contrast, the mutant replacing Ser 427 with Asp (S427D) still showed a strong reactivity to the mAb, though the reactivity was weaker than the control (lane 2 of Fig. 3 c). A reasonable conclusion from these results is that Ser 430 of MYPT is a site phosphorylated during mitosis, although it is possible that simultaneous phosphorylation at Ser 427 may also occur.

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