We show that S. cerevisiae has two PDK1 homologs, Pkh1 and Pkh2. Single pkh1Δ and pkh2Δ mutants are viable, but the pkh1Δ pkh2Δ double mutant is nonviable, indicating that Pkh1 and Pkh2 share a role that is essential for cell growth. Expression of mammalian PDK1 suppresses the lethality of pkh1Δ pkh2Δ cells, demonstrating that Pkh1 and Pkh2 are functionally similar to PDK1. As many signal transduction pathways and mechanisms that regulate cell growth and proliferation are conserved between mammalian and yeast cells, it is not unexpected that PDK1 homologs are present in yeast. PDK1 activates PKB, PKCs and p70S6K by phosphorylating the Thr residue in a conserved sequence motif located within the activation loops of their catalytic domains (Fig. D). This conserved sequence motif is also found in the activation loop of the S. cerevisiae Pkc1 protein, raising the possibility that Pkc1 is a physiological substrate for Pkh1 and Pkh2.
In mammalian cells, PDK1 has been shown to phosphorylate PKC isoforms (24
). However, the biological role of this phosphorylation in mammalian cells is not understood. Here we provide evidence indicating that Pkh1 and Pkh2 function in the Pkc1-MAPK pathway. First, temperature-sensitive pkh1ts pkh2
Δ mutants display phenotypes similar to those of mutants defective in the Pkc1-MAPK pathway, notably osmoremedial cell lysis and loss of actin cytoskeleton polarity. Second, pkh1ts pkh2
Δ mutants are defective in activation of the transcription factor Rlm1, whose activity is dependent on the Pkc1-MAPK pathway. Third, an activating mutation in PKC1
, or MKK1
partially suppresses the growth defect of a pkh1ts pkh2
Δ mutant. Fourth, Pkc1 activity is decreased in a pkh1ts pkh2
Δ mutant. Finally, the Pkh2 protein phosphorylates Pkc1 in vitro at Thr-983; this residue is part of the conserved PDK1 target motif in the Pkc1 activation loop and is essential for Pkc1 function. Thus, Pkh1 and Pkh2 are in the Pkc1-MAPK pathway as activators of Pkc1.
Many protein kinases require phosphorylation within their activation loops to be fully activated. Phosphorylation within the activation loop is also important for protein kinases stringently regulated by allosteric effectors. This is exemplified by PKC, where activation of the Ca2+
/diacylglycerol-dependent isotypes PKCα and PKCβ absolutely requires phosphorylation of respective activation loops (8
). PDK1 regulates multiple protein kinases, including PKB, p70S6K
, and PKC isoforms (2
). The specificity of PDK1 action on its downstream protein kinase targets could be determined by target-specific regulators. In S. cerevisiae
, the GTP-bound form of Rho1 functions as an activator of Pkc1 (19
), raising the possibility that this Rho1-dependent activation of Pkc1 is controlled through Pkh1/Pkh2-dependent phosphorylation of the Pkc1 activation loop.
Several observations suggest that Pkc1 is not the only target of the Pkh kinases. pkh1ts pkh2
Δ mutants resemble pkc1
mutants in that they also exhibit defects in cell integrity resulting from aberrant cell wall construction. However, whereas cell lysis caused by loss of PKC1
function is suppressed by osmotic stabilizing agents (26
), the growth defect in pkh1Δ pkh2
Δ mutants was not. Based on the observation that mammalian PDK1 activates PKB (2
), it is possible that the yeast PKB-like protein kinases, Ypk1 and Ypk2, which play an essential role in yeast cell growth (9
), are also targets of Pkh. Consistent with this possibility, the sequence surrounding the site in PKB phosphorylated by PDK1 is also conserved in Ypk1 and Ypk2 (Fig. D). Furthermore, Casamayor et al. have recently shown that Pkh1 activates Ypk1 in vitro by phosphorylating the Thr-504 residue that corresponds to the site of PDK1 phosphorylation (7
). Thus, Pkh1 and Pkh2 are likely to play a role in activating at least two types of protein kinases that are essential for cell growth, Pkc1 and Ypk1/Ypk2.
In this study, we isolated six different ts pkh1 mutants and divided them into two classes. The growth defect of the first class, typified by pkh1D398G, was rescued by osmotic stabilization, whereas the growth defect of the second class (unpublished data) was not. This suggests that the second class of mutants may be defective in activation of Ypk1/Ypk2. We thus attempted to rescue this class of mutants by overexpression of Ypk1 or Ypk2, but growth was not restored even in the presence of osmotic stabilizing agents (unpublished data). We suspect that activation of Ypk1 and Ypk2 may absolutely require phosphorylation by Pkh1 and Pkh2. If true, this hypothesis suggests a genetic approach to identify constitutive mutations in YPK1 or YPK2, i.e., by isolating mutations that suppress the growth defect of a pkh1ts pkh2Δ strain. This work may elucidate the different modes by which mammalian PDK1 and PKB are regulated.
Overexpression of the PKH2
gene, but not the PKH1
gene, was shown to activate Ste7S368P
, which suggests that Pkh2 basal kinase activity is higher than that of Pkh1. Consistent with this possibility, we could detect Pkh2 but not Pkh1 kinase activity when Pkc1 was used as a substrate (unpublished data). However, the pkh2
Δ single mutant grows normally, indicating that endogenous Pkh1 activity is sufficient for growth. It is therefore likely that the activity of Pkh1 is tightly regulated. The fact that PDK1 contains a PH domain and can bind lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate or phosphatidylinositol 3,4-bisphosphate (39
) may suggest that 3-phosphorylated lipids can activate PDK1 in some way. In contrast, Pkh1 and Pkh2 lack any obvious PH domain. It will be interesting to identify upstream activators of Pkh1. These analyses should further our understanding of the signal(s) that activates the Pkh-Pkc1 pathway.
The STE7S368P mutant, which activates mating response genes in the absence of pheromone, can be upregulated by overexpression of PKH2, whereas the wild-type STE7 cannot. This suggests that Pkh2 phosphorylates and activates the Ste7S368P variant but not wild-type Ste7. The Ste7 protein contains a threonine residue in its activation loop, Thr-363, which is known to be the site of Ste11 phosphorylation. This residue is also analogous to a conserved Thr residue in the phosphorylation site of PDK1 (Fig. D). The residue in Ste7 that is mutated in Ste7S368P, i.e., Ser-368, lies within the activation loop between subdomains VII and VIII in proximity to the Thr-363 residue. Interestingly, all PDK1 target protein kinases except p70S6K have a proline residue at the site corresponding to Ser-368 of Ste7, which may be important for their interaction with PDK1, Pkh1, and/or Pkh2. Thus, the serine-to-proline change in Ste7S368P may convert Ste7 to a substrate of Pkh2, perhaps by altering the conformation of the activation loop in a way that makes the Thr-363 residue accessible to Pkh2.
We also identified a novel protein kinase, Pkh3, that functions as a multicopy suppressor of a pkh1ts pkh2Δ mutant and which exhibits homology to PDK1, Pkh1, and Pkh2. Growth of the pkh3Δ single mutant is normal and indistinguishable from that of wild-type cells. Also, pkh1Δ pkh3Δ and pkh2Δ pkh3Δ double mutants display no apparent phenotype. These results suggest that Pkh3 is able to phosphorylate the same essential target substrates as Pkh1 and Pkh2 when overexpressed but that Pkh1 and Pkh2 play a more important role in regulating cell growth under physiological conditions.