MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1.     

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.     

BRO1, a novel gene that interacts with components of the Pkc1p-mitogen-activated protein kinase pathway in Saccharomyces cerevisiae.

Yeast cells with mutations in BRO1 display phenotypes similar to those caused by deletion of BCK1, a gene encoding a MEK kinase that functions in a mitogen-activated protein kinase pathway mediating maintenance of cell integrity. bro1 cells exhibit a temperature-sensitive growth defect that is suppressed by the addition of osmotic stabilizers or Ca2+ to the growth medium or by additional copies of the BCK1 gene. At permissive temperatures, bro1 mutants are sensitive to caffeine and respond abnormally to nutrient limitation. A null mutation in BRO1 is synthetically lethal with null mutations in BCK1, MPK1, which encodes a mitogen-activated protein kinase that functions downstream of Bck1p, or PKC1, a gene encoding a protein kinase C homolog that activates Bck1p. Analysis of the isolated BRO1 gene revealed that it encodes a novel, 97-kDa polypeptide which contains a putative SH3 domain-binding motif and is homologous to a protein of unknown function in Caenorhabditis elegans.     

Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae

Loss of yeast protein kinase C function results in three distinct phenotypes: staurosporine sensitivity, cell lysis and blockage of cell cycle progression at the G2/M boundary. Genetic analysis of the PKC1/STT1 protein kinase C gene and its interactions with STT4, encoding an upstream phosphatidylinositol 4-kinase, and BCK1, encoding a downstream protein kinase, reveal that they form part of a single pathway. However, the BCK1-20 mutation (a gain-of-function mutation of BCK1) or overexpression of PKC1 cannot suppress all of the phenotypes caused by the loss of STT4 function, strongly suggesting the existence of a branch point between STT4 and PKC1. We also describe the MSS4 gene, a multicopy suppressor of the temperature-sensitive stt4-1 mutation. MSS4 is predicted to encode a hydrophilic protein of 779 amino acid residues and is essential for cell growth. Based on genetic and biochemical data, we suggest that MSS4 acts downstream of STT4, but in a pathway that does not involve PKC1. GenBank accession number: The accession number for the MSS4 sequence reported in this paper is D13716.     

Genetic interactions between GLC7, PPZ1 and PPZ2 in saccharomyces cerevisiae

GLC7 encodes an essential serine/threonine protein type I phosphatase in Saccharomyces cerevisiae. Three other phosphatases (Ppz1p, Ppz2p, and Sal6p) share >59% identity in their catalytic region with Glc7p. ppz1 ppz2 null mutants have no apparent growth defect on rich media. However, null alleles of PPZ1 and PPZ2, in combination with mutant alleles of GLC7, confer a range of growth defects varying from slow growth to lethality. These results indicate that Glc7p, Ppz1p, and Ppz2p may have overlapping functions. To determine if this overlap extends to interaction with targeting subunits, Glc7p-binding proteins were tested for interaction in the two-hybrid system with the functional catalytic domain of Ppz1p. Ppz1p interacts strongly with a number of Glc7p regulatory subunits, including Glc8p, a protein that shares homology with mammalian PP1 inhibitor I2. Genetic data suggest that Glc8p positively affects both Glc7p and Ppz1p functions. Together our data suggest that Ppz1p and Ppz2p may have overlapping functions with Glc7p and that all three phosphatases may act through common regulatory proteins.     

Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta- glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.     

Mutation of the gene encoding protein kinase C 1 stimulates mitotic recombination in Saccharomyces cerevisiae.

We have isolated a recessive allele of the yeast protein kinase C gene (PKC1) which promotes an elevated rate of mitotic recombination and confers a temperature-sensitive growth defect. The rate of recombination was increased between genes in direct repeat and at a series of heteroalleles and was dependent upon the RAD52 gene product. The mutant pkc1 allele was sequenced and found to encode a single amino acid change within the catalytic domain. Osmotic stabilizing agents rescued the temperature-sensitive growth defect but not the hyperrecombination phenotype, indicating that the two traits are separable. This separability suggests that the PKC1 gene product (Pkc1p) regulates DNA metabolism by an alternate pathway to that used in the regulation of cell lysis. The regulation of recombination is a previously unidentified role for Pkc1p.     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.     

The osmotic integrity of the yeast cell requires a functional PKC1 gene product.

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.     

The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway.

We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

Functional Interaction of Isr1, a Predicted Protein Kinase,with the Pkc1 Pathway in Saccharomyces cerevisiae

Staurosporine is a potent inhibitor of protein kinase C. To identify the genes that functionally interact with the Pkc1 pathway of the yeast Saccharomyces cerevisiae, we screened for the genes that cause induced staurosporine sensitivity when overexpressed from a galactose-inducible promoter. The novel gene ISR1 encodes a predicted protein kinase with the highest sequence similarity to mammalian Raf in the kinase domain. Drug sensitivity induced by ISR1 overexpression is specific to staurosporine. Although ISR1 disruption causes no obvious phenotype, it does exacerbate the phenotypes of a temperature-sensitive allele (stt1-1) of PKC1, but not of the mpk1 and bck1 mutants of the Mpk1 MAP kinase pathway. These results suggest that Isr1 functions in an event important for growth in a manner redundant with a Mpk1-independent branch of the Pkc1 signalling pathways.