BRO1, a Novel Gene That Interacts with Components of the Pkc1p–Mitogen-Activated Protein Kinase Pathway in Saccharomyces cerevisiae

Abstract

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 Ca 2؉ 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 mitogenactivated 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.Cellular responses to a variety of environmental signals are mediated by modular protein kinase cascades whose ultimate targets are mitogen-activated protein (MAP) kinases (5). Roles for MAP kinases (also called ERKs) have been established in cells undergoing mitosis, meiosis, and differentiation (36). MAP kinase activation requires phosphorylation by a MAP kinase kinase, or MEK (for MAP kinase/ERK kinase). The MEK is, in turn, activated via phosphorylation by a member of the MEK kinase (MEKK) family, the upstream component of the MAP kinase functional unit. MAP kinase modules have been identified in a variety of eukaryotic species, ranging from mammals (46) to budding and fission yeasts (14).Maintenance of cell integrity in the budding yeast Saccharomyces cerevisiae requires a distinct MAP kinase pathway, which includes a MEKK, encoded by the BCK1/SLK1 gene (9, 23), two MEKs, products of the MKK1 and MKK2 genes (17), and a MAP kinase, encoded by the MPK1/SLT2 gene (22,30,47; reviewed in reference 12). Null mutants lacking any one of these functional units, the MAP kinase, the two MEKs, or the MEKK, display a cell lysis defect at elevated temperatures. Addition of osmotic stabilizing agents (e.g., 1 M sorbitol) to the growth medium suppresses this lysis defect.Genetic and biochemical data have demonstrated that signaling through the MAP kinase pathway that mediates maintenance of cell integrity is triggered by activation of yeast protein kinase C, product of the PKC1 gene (12, 25). Disruption of PKC1 results in a cell lysis defect at all temperatures as a result of defective cell wall construction (24, 37). Because loss of PKC1 results in a more severe phenotype than does loss of BCK1 or other downstream components of this cascade, one proposed model (23) suggests that the MAP kinase module constitutes one branch of a bifurcated pathway which is regulated by Pkc1p.Here we report the isolation of a yeast mutant, bro1, exhibiting a temperature-dependent, osmoremedial growth defect which is suppressed by Ca 2ϩ or by centromere-based plasmids cont...

Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix

“…35,36 Our results are consistent with and complement these results, suggesting a novel function for AIP1/Alix as an inhibitor of paraptosis.…”

“…Mutations in BRO1 result in a phenotype similar to that associated with mutations in BCK1, the latter of which is a MAPK that mediates cell integrity. 35 Given the findings described above suggesting that paraptosis is mediated by MAPK activation, we evaluated the effects of AIP1/Alix on both apoptotic and paraptotic cell death. Whereas AIP1/Alix inhibited paraptosis induced by both IGFIR-IC and IGFIR full length (Figure 6a-f) as determined by biochemical and morphological criteria, it did not inhibit apoptosis (Figure 6g).…”

Involvement of Nst1p/YNL091w and Msl1p, a U2B″ splicing factor, in Saccharomyces cerevisiae salt tolerance

“…Ethidium bromide-stained rRNA was used as loading control and Northern analysis with probes for the CTT1 and HAL2 transcripts as positive controls for the NaCl and 3-AT induction, respectively. The fold increase in the ratio of the different mRNA signals to rRNA fluorescence, relative to the value in control SCD medium, is given below the panels 198 A. Goossens, J. Forment and R. Serrano with defects in components mediating maintenance of cell integrity (Gentzsch and Tanner, 1996;Nickas and Yaffe, 1996;Bach et al, 2000) and caffeine has recently been demonstrated to activate the Slt2 MAP kinase pathway regulating cell wall biosynthesis, probably by acting on some intracellular target mediating alterations of the cell surface (Martin et al, 2000). On the other hand, msl1 mutants showed an increased sensitivity to the microtubule-destabilizing compound thiabendazole (Entian et al, 1999).…”

Characterization of the pH signal transduction pathway gene palA of Aspergillus nidulans and identification of possible homologs

“…3), PalA shows 29.4% identity over 850 residues with an 882-residue hypothetical protein from the nematode C. elegans (accession number Z29561), 23.1% identity over 769 residues with a 701-residue hypothetical protein from the fission yeast S. pombe (accession number Z54354) (not shown), and 20.8% identity over 852 residues with the 844-residue Bro1p protein (14) of the budding yeast S. cerevisiae. Although no function has yet been identified for the probable C. elegans PalA homolog (or the possible S. pombe homolog), Bro1p is likely to be in a pathway that interacts with (possibly sharing downstream targets with) the protein kinase C-mitogen-activated protein kinase pathway in S. cerevisiae (14). Like Bro1p (14), PalA contains putative SH3 domain-binding motifs (consensus PXXP [12,13,20]) with PVPP beginning at residue 317 and with PVKP and PQPP beginning at residues 770 and 775, respectively.…”

“…Although no function has yet been identified for the probable C. elegans PalA homolog (or the possible S. pombe homolog), Bro1p is likely to be in a pathway that interacts with (possibly sharing downstream targets with) the protein kinase C-mitogen-activated protein kinase pathway in S. cerevisiae (14). Like Bro1p (14), PalA contains putative SH3 domain-binding motifs (consensus PXXP [12,13,20]) with PVPP beginning at residue 317 and with PVKP and PQPP beginning at residues 770 and 775, respectively. A very recent database entry (accession number Z75183) for a hypothetical protein encoded by chromosome XV of S. cerevisiae has 30.2% identity over 732 residues in a Bestfit alignment with PalA.…”