Protein-protein interactions in the yeastPKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with theSaccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in thePKC1-mediated signalling pathway. Subsequently, Pkc1p and the otherPKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of thePKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.     

Protein-protein interactions in the yeastPKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with theSaccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in thePKC1-mediated signalling pathway. Subsequently, Pkc1p and the otherPKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of thePKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.     

Differential roles of p39Mos-Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes

Fully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathways. The latter cascade is activated upon p39(Mos) accumulation. Using U0126, a MEK1 inhibitor, and p39(Mos) antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39(Mos) accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42(Mpk1) but not p90(Rsk). Second, inhibition of either p39(Mos) accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39(Mos) or p90(Rsk) alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathway is required for bipolar organization of the meiotic spindle at the cortex.     

The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint

The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (GAP) previously shown to act on Rho1p, and we now show that it also acts on Cdc42p, the GTPase primarily responsible for establishment of cell polarity in yeast. Whereas the morphogenesis role of Bem2p required GAP activity, the checkpoint role of Bem2p did not. Instead, this function required an N-terminal Bem2p domain. Thus, this single protein has a GAP-dependent role in promoting cell polarity and a GAP-independent role in responding to defects in cell polarity by enacting the checkpoint. Surprisingly, Swe1p accumulation occurred normally in bem2 cells, but they were nevertheless unable to promote Cdc28p phosphorylation. Therefore, Bem2p defines a novel pathway in the morphogenesis checkpoint.     

Control of Swe1p degradation by the morphogenesis checkpoint.

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.     

Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint

Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a cyclin-dependent kinase (CDK) mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive-feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive-feedback loops active at the G2/M transition.     

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.     

Afr1p has a role in regulating the localization of Mpk1p at the shmoo tip in Saccharomyces cerevisiae

Afr1p functions to promote adaptation to pheromone-induced growth arrest and morphogenesis. We show here that Afr1p regulates polarized localization of the Mpk1p MAP kinase in shmooing cells. Deletion of AFR1 results in mislocalization of Mpk1p although the scaffold protein Spa2p localizes normally at shmoo tip, and overexpression of Spa2 cannot rescue this defect, indicating Afr1p in required for Spa2p to recruit Mpk1 to the site of polarized growth during mating. Overexpression of SPA2 partially suppresses the morphogenetic defect of afr1Delta cells upon alpha-factor induction, suggesting the two proteins function in the same genetic pathway with Spa2p acts downstream of Afr1p.     

A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea

Many fungal pathogens invade plants by means of specialized infection structures called appressoria. In the rice (Oryza sativa) blast fungus Magnaporthe grisea, the pathogenicity mitogen-activated protein (MAP) kinase1 (PMK1) kinase is essential for appressorium formation and invasive growth. In this study, we functionally characterized the MST7 and MST11 genes of M. grisea that are homologous with the yeast MAP kinase kinase STE7 and MAP kinase kinase kinase STE11. Similar to the pmk1 mutant, the mst7 and mst11 deletion mutants were nonpathogenic and failed to form appressoria. When a dominant MST7 allele with S212D and T216E mutations was introduced into the mst7 or mst11 mutant, appressorium formation was restored in the resulting transformants. PMK1 phosphorylation also was detected in the vegetative hyphae and appressoria of transformants expressing the MST7(S212D T216E) allele. However, appressoria formed by these transformants failed to penetrate and infect rice leaves, indicating that constitutively active MST7 only partially rescued the defects of the mst7 and mst11 mutants. The intracellular cAMP level was reduced in transformants expressing the MST7(S212D T216E) allele. We also generated MST11 mutant alleles with the sterile alpha motif (SAM) and Ras-association (RA) domains deleted. Phenotype characterizations of the resulting transformants indicate that the SAM domain but not the RA domain is essential for the function of MST11. These data indicate that MST11, MST7, and PMK1 function as a MAP kinase cascade regulating infection-related morphogenesis in M. grisea. Although no direct interaction was detected between PMK1 and MST7 or MST11 in yeast two-hybrid assays, a homolog of yeast STE50 in M. grisea directly interacted with both MST7 and MST11 and may function as the adaptor protein for the MST11-MST7-PMK1 cascade.     

A novel role for integrin-linked kinase in epithelial sheet morphogenesis

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.     

Questioning the role of checkpoint kinase 2 in the p53 DNA damage response

Cdc25C and p53 have been reported to be physiological targets of checkpoint kinase 2 (Chk2). Surprisingly, although Chk2 purified from DNA damage sustaining cells has dramatically increased ability to phosphorylate Cdc25C when compared with untreated cells, its ability to phosphorylate p53 is weak before treatment, and there is no increase in its activity toward p53 after DNA damage by gamma irradiation or the radiomimetic agent neocarzinostatin. Furthermore, introduction of Chk2 short interfering RNA into three different human tumor cell lines leads to marked reduction of Chk2 protein, but p53 is still stabilized and active after DNA damage. The results with Chk1 short interfering RNA indicate as well that Chk1 does not play a role in human p53 stabilization after DNA damage. Thus, Chk1 and Chk2 are unlikely to be regulators of p53 in at least some human tumor cells. We discuss our results in the context of previous findings demonstrating a requirement for Chk2 in p53 stabilization and activity.     

Pil1p and Lsp1p negatively regulate the 3-phosphoinositide-dependent protein kinase-like kinase Pkh1p and downstream signaling pathways Pkc1p and Ypk1p

The Saccharomyces cerevisiae homologs, Pkh1/2p, of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1) regulate the Pkc1-MAP kinase cascade and the partially parallel Ypk1/2p pathway(s) that control growth and cell integrity. Mammalian PDK1 is regulated by 3-phosphoinositides, whereas Pkh1/2p are regulated by sphingolipid long-chain bases (LCBs). Recently Pkh1/2p were found to complex with two related proteins, Pil1p (Ygr086) and Lsp1p (Ypl004). Because these two proteins are not related to any known protein we sought to characterize their functions. We show that Pkh1p phosphorylates both proteins in vitro in a reaction that is only weakly regulated by LCBs. In contrast, LCBs inhibit phosphorylation of Pil1p by Pkh2p, whereas LCBs stimulate phosphorylation of Lsp1p by Pkh2p. We find that Pil1p and Lsp1p down-regulate resistance to heat stress and, specifically, that they down-regulate the activity of the Pkc1p-MAP and Ypk1p pathways during heat stress. Pil1p and Lsp1p are thus the first proteins identified as regulators of Pkh1/2p. An unexpected finding was that the level of Ypk1p is greatly reduced in pkc1Delta cells, indicating that Pkc1p controls the level of Ypk1p. Homologs of Pil1p and Lsp1p are widespread in nature, and our results suggest that they may be negative regulators of PDK-like protein kinases and their downstream cellular pathways that control cell growth and survival.     

Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade

Production of the essential phospholipid PI4P at the Golgi by the Pik1 kinase is required for protein secretion, while a distinct pool of PI4P generated by the Stt4 kinase is critical for normal actin cytoskeleton organization. We identify a transmembrane protein that stabilizes Stt4 at the plasma membrane where it directs synthesis of PI4P, which is required for activation of the Rho1/Pkc1-mediated MAP kinase cascade. Inactivation of Stt4 or the PI4P 5-kinase Mss4 results in mislocalization of the Rho-GTPase GEF Rom2. Rom2 binds PI4,5P(2) through its PH domain and represents the first identified effector in the Stt4-Mss4 pathway. Based on these results, we propose that Stt4-Mss4 generates PI4,5P(2) at the plasma membrane, required to recruit/activate effector proteins such as Rom2.     

A monitor for bud emergence in the yeast morphogenesis checkpoint

Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation.     

Pkc1p modifies CPY* degradation in the ERAD pathway

The process of endoplasmic reticulum-associated degradation (ERAD) involved in the degradation of misfolded N-linked glycoproteins utilizes Cdc48p which extracts misfolded glycoproteins from the lumen to the cytosol to present them for deglycosylation and degradation. Pkc1p has been identified as a component of the ERAD pathway, because deletion of the pkc1 gene impairs ERAD and causes accumulation of CPY* in the lumen of the ER, most probably because of the mislocalization of Cdc48p. In addition, we show that Cdc48p interacts in the cytosol with the deglycosylation enzyme, PNGase, only when Cdc48p is associated with a misfolded glycoprotein.     

PDK1 homologs activate the Pkc1-mitogen-activated protein kinase pathway in yeast

PDK1 (phosphoinositide-dependent kinase 1) is a mammalian growth factor-regulated serine/threonine kinase. Using a genetic selection based on a mutant form of the yeast MAP kinase kinase Ste7, we isolated a gene, PKH2, encoding a structurally and functionally conserved yeast homolog of PDK1. Yeast cells lacking both PKH2 and PKH1, encoding another PDK1 homolog, were nonviable, indicating that Pkh1 and Pkh2 share an essential function. A temperature-sensitive mutant, pkh1(D398G) pkh2, was phenotypically similar to mutants defective in the Pkc1-mitogen-activated protein kinase (MAPK) pathway. Genetic epistasis analyses, the phosphorylation of Pkc1 by Pkh2 in vitro, and reduced Pkc1 activity in the pkh1(D398G) pkh2 mutant indicate that Pkh functions upstream of Pkc1. The Pkh2 phosphorylation site in Pkc1 (Thr-983) is part of a conserved PDK1 target motif and essential for Pkc1 function. Thus, the yeast PDK1 homologs activate Pkc1 and the Pkc1-effector MAPK pathway.