Ptc1, a Type 2C Ser/Thr Phosphatase, Inactivates the HOG Pathway by Dephosphorylating the Mitogen-Activated Protein Kinase Hog1

Mitogen-activated protein kinase (MAPK) pathways comprise three sequentially acting kinases, MEKK (or Raf), MEK, and MAPK, known as the MAPK module 16, 22, 48. MEKKs can be activated by interaction with upstream components and phosphorylation on Ser, Thr, and Tyr residues. Activated MEKKs then activate MEK by phosphorylating Ser/Thr residues. Activated MEK activates MAPK by phosphorylating a Thr and a Tyr residue in the phosphorylation lip. Phosphorylation of both residues is required for full MAPK activation. There are three groups of MAPKs, classified by the signals that activate them and their phosphorylation lip sequence. One group, including vertebrate ERK1 and ERK2, contains the phosphorylation lip sequence TEY and is activated by growth factors, mitogens, and cytokines (22, 48). A second group, vertebrate c-Jun N-terminal kinase (JNK)–stress-activated protein kinase, contains the phosphorylation lip sequence TPY and is activated by UV light, osmotic stress, tumor necrosis factor, and interleukin-1 (22, 48). A third group, including Saccharomyces cerevisiae Hog1, Schizosaccharomyces pombe Spc1, and vertebrate p38, contains the phosphorylation lip sequence TGY and is activated by osmotic stress and other environmental stresses (16, 22, 48). At least six MAPK cascades operate in S. cerevisiae to regulate physiologically distinct responses (16). The HOG pathway allows yeast to grow in high-osmolarity environments by inducing the expression of osmoprotectants. The upstream portion of this pathway has two branches (Fig. ​(Fig.1).1). One branch is the two-component signaling system comprising Sln1, Ypd1, and Ssk1 (25, 33, 37). Genetic and biochemical data support the following model for activation of this pathway. Sln1 is a plasma membrane-bound His/Asp kinase that is phosphorylated in the absence of stress. Osmotic stress causes its dephosphorylation, leading to dephosphorylation of Ypd1, a His kinase, and Ssk1, an Asp kinase (37). Unphosphorylated Ssk1 activates the MEKKs, Ssk2 and Ssk22, by binding to their N-terminal inhibitory domains (23). For Ssk2, this has been shown to result in autophosphorylation of a Thr residue and kinase activation (34). Activated Ssk2 and Ssk22 then activate the MEK, Pbs2, by phosphorylating a Ser and a Thr residue in the T loop. Activated Pbs2 activates the MAPK, Hog1, by phosphorylation of a Thr and Tyr residue in the phosphorylation lip (4, 42). A second branch of the HOG pathway is activated by the osmosensor Sho1 (23), which signals to Ste20, Ste50, Ste11, and then Pbs2 (32, 35, 36, 38, 50). FIG. 1 Protein phosphatase inactivation of the HOG pathway. The HOG pathway is regulated by two membrane-bound osmosensors, Sln1 and Sho1. Both Sln1 and Sho1 activate the MAPK cascade (boxed). The two-component regulators, Sln1, Ypd1, and Ssk1, negatively regulate ... The identity of physiologically relevant protein phosphatases that inactivate MAPK pathways is less well established. Protein phosphatases can inactivate MEKK, MEK, and MAPK, since they require phosphorylation on Ser, Thr, and Tyr residues for activity (21, 22). Vertebrate MEKK and MEK are inactivated by the type 2A Ser/Thr phosphatase (PP2A) in vitro and may be inactivated by PP2A in vivo (1, 2, 17, 45). Stress-activated vertebrate MEKs, MKK6 and SEK1 (MKK4/JNKK1), are also inactivated by the type 2C Ser/Thr-specific phosphatase (PP2C) in vivo and in vitro (46). Since MAPKs require phosphorylation of a Thr and Tyr residue in the phosphorylation lip to be active, they can be inactivated by Ser/Thr phosphatases, protein tyrosine phosphatases (PTPs) specific for phosphotyrosine (pY), and dual-specificity phosphatases, capable of dephosphorylating both the phosphorylation lip phosphothreonine (pT) and pY residues (21, 22). Among the Ser/Thr phosphatases, PP2A has been shown to inactivate MAPK (1, 15, 45), and PP2C has been found to inactivate the stress-activated MAPK, p38 (46). In S. pombe, there are conflicting reports regarding the activity of PP2C on the stress-activated MAPK, Spc1/Sty1. Two PP2C phosphatases, Ptc1 and Ptc3, were shown to be important for inactivating Spc1/Sty1 activated by heat stress (31), while Ptc1 was shown to be unimportant for inactivating Spc1/Sty1 activated by osmotic stress (13). In S. cerevisiae, MAPKs have been shown to be inactivated by a dual-specificity phosphatase, PTPs, and potentially PP2Cs. The dual-specificity phosphatase Msg5 inactivates Fus3 (11) but has not been shown to inactivate other MAPKs. The PTPs Ptp2 and Ptp3 inactivate Hog1, Fus3, and Mpk1 with different specificities (Fig. ​(Fig.1)1) (19, 27, 51, 52). The identity of Ser/Thr phosphatases that inactivate S. cerevisiae MAPKs has not been established. Two PP2Cs, Ptc1 and Ptc3, have been implicated as negative regulators of the HOG pathway by genetic means, but their substrates have not been identified. Initially, Maeda et al. identified PTC1 as a gene whose mutation produced a synthetic growth defect with deletion of PTP2 (24). We showed that this growth defect was suppressed by deletion of HOG1 (19), further suggesting that Ptc1 negatively regulates the HOG pathway. Previous studies also showed that overexpression of PTC1 or PTC3 suppressed lethality conferred by deletion of SLN1, which is known to hyperactivate the HOG pathway (25). In this work, we show that PTC2, encoding a type 2C Ser/Thr phosphatase closely related to Ptc3, also inactivates this pathway, and that Ptc1 inactivates the HOG pathway by dephosphorylating the MAPK, Hog1. Unlike vertebrate PP2C, Ptc1 does not have a strong effect on the MEK, Pbs2.