Differential regulation of the cell wall integrity mitogen-activated protein kinase pathway in budding yeast by the protein tyrosine phosphatases Ptp2 and Ptp3.

Mitogen-activated protein kinase (MAPK) pathways regulate growth, development, and the response to stress in eukaryotes (2, 11, 28, 30, 36, 77). These pathways regulate mitosis and differentiation in vertebrates, vulval development in Caenorhabditis elegans, and photoreceptor development in Drosophila melanogaster (42). In the yeast Saccharomyces cerevisiae, six different MAPK pathways regulate mating, osmosensing, pseudohyphal growth, invasive growth, spore formation, and cell wall biosynthesis (28, 44). The proper regulation of MAPK pathways is important since mutations that alter their activity cause defects in growth and development. Mutations that hyperactivate MAPK modules lead to cell transformation in vertebrates, developmental defects in C. elegans and Drosophila, and growth defects in S. cerevisiae (12, 30, 33, 45, 68, 71, 73, 77). Common to all MAPK pathways are MAPK modules comprising three sequentially acting kinases termed MEK kinase (MEKK), MEK, and MAPK (11). MEKK activates MEK, which in turn activates MAPK by phosphorylating a conserved threonine and tyrosine residue in the phosphorylation lip sequence (11, 42, 74). Although the activation of MAPK pathways has been intensively investigated, the inactivation of these pathways is less well understood. Most is known about the inactivation of MAPKs. Since MAPKs require phosphorylation of both a Thr and a Tyr for full activity (11, 42), it is possible to inactivate them by dephosphorylating either phosphothreonine, phosphotyrosine (pY), or both residues. Dual-specificity phosphatases, capable of dephosphorylating both phosphothreonine and pY residues, inactivate MAPKs in vertebrates, Drosophila, and S. cerevisiae (17, 25, 41, 42, 47, 70). In vertebrates, there are at least nine dual-specificity phosphatases that inactivate the three MAPK family members, ERK, JNK/SAPK, and p38, with variable specificity (42). In Drosophila, a dual-specificity phosphatase, puckered, has been identified as a negative regulator of JNK in a developmental pathway (47). In the S. cerevisiae genome, six genes encode proteins similar to dual-specificity phosphatases. One of these, Msg5, has been shown to inactivate the MAPK, Fus3, in the pheromone response pathway (17, 76). In yeasts, MAPKs have also been shown to be inactivated by a novel mechanism involving protein tyrosine phosphatases (PTPs) specific for pY (33, 50, 66, 73). The S. cerevisiae MAPKs, Hog1, in the osmotic stress-activated high-osmolarity glycerol (HOG) pathway, and Fus3, in the pheromone response pathway, are inactivated by two protein tyrosine phosphatases, Ptp2 and Ptp3 (33, 73, 76). These two PTPs contain a catalytic domain of ∼400 residues 57% similar to each other and significantly similar to vertebrate PTPs (5, 7, 9). Analysis of human PTP1B showed that the Cys residue in the conserved sequence, (I/V)HCXAGXXR(S/T)G, acts as a nucleophile in pY hydrolysis (26). Mutation of the corresponding residue in Ptp2 and Ptp3 inactivates them in vivo and in vitro (33, 73, 76). In Schizosaccharomyces pombe, two protein tyrosine phosphatases, Pyp1 and Pyp2, have been shown to inactivate a MAPK, Spc1/Sty1, in a stress response pathway analogous to the HOG pathway (14, 50, 66). Protein phosphatases that regulate MAPKs show variable levels of specificity for their substrates. For example, the vertebrate dual-specificity phosphatase, MKP-3/PYST1 (23, 53, 56), is highly specific for ERK, while other dual-specificity phosphatases, MKP-1, MKP-2, and MKP-4, inactivate multiple MAPKs (3, 10, 42, 54, 70). Part of this specificity is due to noncatalytic domains that recognize their substrates through tight binding interactions (55). PTPs also show different specificities for MAPKs. For example, the S. cerevisiae Ptp2 is a more effective negative regulator of Hog1 than Ptp3 (33, 73). We have shown that Ptp2 binds Hog1 more effectively than Ptp3 (33), and this likely contributes to its greater ability to inactivate Hog1. In contrast, Ptp3 is a more effective negative regulator of Fus3 than Ptp2 (76), but the reason for this is unknown. To further explore the functions of S. cerevisiae Ptp2 and Ptp3 in MAPK signaling and their different specificities for MAPKs, we examined whether these phosphatases could inactivate Mpk1, a MAPK in the cell wall integrity pathway (37). This pathway activates the biosynthesis of the yeast cell wall during vegetative growth, during mating, and in response to stresses such as heat and hypo-osmotic shock (6, 13, 22, 34, 40, 43, 46, 49, 52, 75). The activation of this pathway has been well characterized, but its inactivation is poorly understood. The transmembrane proteins Hcs77, Mid2, and Mtl1 signal to Rho1, protein kinase C, and a MAPK module comprising a MEKK called Bck1, redundant MEKs called Mkk1 and Mkk2, and a single MAPK, Mpk1 (22, 32, 35, 37, 38, 40, 58, 61). MSG5 was isolated as a multicopy suppressor of growth defects due to the hyperactive MEK mutant, MKK1-386; however, it has not been established whether it is a physiologically relevant regulator of Mpk1 (71). In this work, we show that both Ptp2 and Ptp3 inactivate Mpk1; however, Ptp2 is a more potent negative regulator than Ptp3. The difference in their activities is likely explained by at least three factors. First, Ptp2 binds Mpk1 and dephosphorylates Mpk1-pY more effectively than Ptp3. Second, these PTPs show differences in subcellular localization; Ptp2 is predominantly nuclear, while Ptp3 is cytoplasmic. The nuclear localization of Ptp2 may account for its greater effectiveness, since activation of MAPKs often results in their translocation to the nucleus. Last, heat shock increased the level of PTP2 transcript in a Mpk1-dependent manner, suggesting that Ptp2 acts in a negative feedback loop to inactivate Mpk1. Thus, Ptp2 and Ptp3 are global regulators of MAPK signaling, inactivating the HOG, pheromone response, and cell wall integrity pathways but show differences in specificity toward the MAPKs in these pathways.