Role of Ptc2 type 2C Ser/Thr phosphatase in yeast high-osmolarity glycerol pathway inactivation.
Three type 2C Ser/Thr phosphatases (PTCs) are negative regulators of the yeast Saccharomyces cerevisiae high-osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway. Ptc2 and Ptc3 are 75% identical to each other and differ from Ptc1 in having a noncatalytic domain. Previously, we showed that Ptc1 inactivates the pathway by dephosphorylating the Hog1 MAPK; Ptc1 maintains low basal Hog1 activity and dephosphorylates Hog1 during adaptation. Here, we examined the function of Ptc2 and Ptc3. First, deletion of PTC2 and/or PTC3 together with PTP2, encoding the protein tyrosine phosphatase that inactivates Hog1, produced a strong growth defect at 37 degrees C that was dependent on HOG1, providing further evidence that PTC2 and PTC3 are negative regulators. Second, overexpression of PTC2 inhibited Hog1 activation but did not affect Hog1-Tyr phosphorylation, suggesting that Ptc2 inactivates the pathway by dephosphorylating the Hog1 activation loop phosphothreonine (pThr) residue. Indeed, in vitro studies confirmed that Ptc2 was specific for Hog1-pThr. Third, deletion of both PTC2 and PTC3 led to greater Hog1 activation upon osmotic stress than was observed in wild-type strains, although no obvious change in Hog1 inactivation during adaptation was seen. These results indicate that Ptc2 and Ptc3 differ from Ptc1 in that they limit maximal Hog1 activity. The function of the Ptc2 noncatalytic domain was also examined. Deletion of this domain decreased V(max) by 1.6-fold and increased K(m) by 2-fold. Thus Ptc2 requires an additional amino acid sequence beyond the catalytic domain defined for PTCs for full activity.
Young
Christian
C
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Mapes
James
J
Hanneman
Jennifer
J
Al-Zarban
Sheikha
S
Ota
Irene
I
D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D009995:Osmosis; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D000071636:Protein Phosphatase 2C; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D010769:Phosphothreonine; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; C000607464:PTC3 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; D005990:Glycerol