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Eukaryotic Cell, December 2002, p. 1032-1040, Vol. 1, No. 6
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.6.1032-1040.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation

Christian Young, James Mapes, Jennifer Hanneman, Sheikha Al-Zarban, and Irene Ota*

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215

Received 15 May 2002/ Accepted 10 September 2002

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°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 Vmax by 1.6-fold and increased Km by 2-fold. Thus Ptc2 requires an additional amino acid sequence beyond the catalytic domain defined for PTCs for full activity.

* Corresponding author. Mailing address: Myriad Proteomics, 2150 W. Dauntless Ave., Salt Lake City, UT 84116. Phone: (801) 303-1799. Fax: (801) 303-1701. E-mail: ota{at}myriad-proteomics.com.

Eukaryotic Cell, December 2002, p. 1032-1040, Vol. 1, No. 6
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.6.1032-1040.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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