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Vol. 18, Issue 11, 4405-4419, November 2007

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Stress-activated Protein Kinase-mediated Down-Regulation of the Cell Integrity Pathway Mitogen-activated Protein Kinase Pmk1p by Protein Phosphatases

Yeast Physiology Group, Department of Genetics and Microbiology, Facultad de Biología, University of Murcia, 30071 Murcia, Spain

Submitted May 23, 2007; Revised July 11, 2007; Accepted August 15, 2007
Monitoring Editor: Fred Chang

Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it becomes activated under multiple stresses, including hyper- or hypotonic conditions, glucose deprivation, cell wall-damaging compounds, and oxidative stress. The only protein phosphatase known to dephosphorylate and inactivate Pmk1p is Pmp1p. We show here that the stress-activated protein kinase (SAPK) pathway and its main effector, Sty1p MAPK, are essential for proper deactivation of Pmk1p under hypertonic stress in a process regulated by Atf1p transcription factor. We demonstrate that tyrosine phosphatases Pyp1p and Pyp2p, and serine/threonine phosphatase Ptc1p, that negatively regulate Sty1p activity and whose expression is dependent on Sty1p-Atf1p function, are involved in Pmk1p dephosphorylation under osmostress. Pyp1p and Ptc1p, in addition to Pmp1p, also control the basal level of MAPK Pmk1p activity in growing cells and associate with, and dephosphorylate Pmk1p both in vitro and in vivo. Our results with Ptc1p provide the first biochemical evidence for a PP2C-type phosphatase acting on more than one MAPK in yeast cells. Importantly, the SAPK-dependent down-regulation of Pmk1p through Pyp1p, Pyp2p, and Ptc1p was not complete, and Pyp1p and Ptc1p phosphatases are able to negatively regulate MAPK Pmk1p activity by an alternative regulatory mechanism. Our data also indicate that Pmk1p phosphorylation oscillates as a function of the cell cycle, peaking at cell separation during cytokinesis, and that Pmp1p phosphatase plays a main role in regulating this process.

Address correspondence to: Mariano Gacto (maga{at}um.es).

Abbreviations used: GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; HA6H, epitope comprising hemagglutinin antigen plus six histidine residues; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; SAPK, stress-activated protein kinase.