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

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Abstract

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.

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References

  • Angel P, Imagawa R, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M (1987) Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729–739

    Google Scholar 

  • Baeuerle PA, Baltimore D (1988) Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor. Cell 53:211–217

    Google Scholar 

  • Berridge MJ (1987) Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56:159–193

    Google Scholar 

  • Berridge MJ, Irvine RF (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312:215–321

    Google Scholar 

  • Breeden L, Nasmyth K (1993) Regulation of the yeastHO gene. Cold Spring Harbor Symp Quant Biol 50:643–650

    Google Scholar 

  • Chiu R, Imagawa M, Imbra J, Bockover JR, Karin M (1987) Multiple cis- and trans-acting elements mediate the transcriptional response to phorbol esters. Nature 329:648–651

    Google Scholar 

  • Cobb MH, Boulton TG, Robbins DJ (1991) Extracellular signal-regulated kinases: ERKs in progress. Cell Regulation 2:965–978

    Google Scholar 

  • Colamonici OR, Trepel JB, Vidal CA, Neckers LM (1986) Phorbol ester inducesc-sis gene transcription in stem cell line K-562. Mol Cell Biol 6:1847–1850

    Google Scholar 

  • Coughlin SR, Lee WMF, Williams PW, Giels GM, Williams LT (1985)c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF. Cell 43:243–251

    Google Scholar 

  • Dekker LV, Parker PJ (1994) Protein kinase C — a question of specificity. Trends Biochem Sci 19:73–77

    Google Scholar 

  • Durfee T, Becherer K, Chen P-L, Yeh S-H, Yang Y, Kilburn AE, Lee W-H, Elledge SJ (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev 7:555–569

    Google Scholar 

  • Elledge SJ, Mulligan JT, Ramer SW, Spottswood M, Davis RW (1991) λYES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast andEscherichia coli mutations. Proc Natl Acad Sci USA 88:1731–1735

    Google Scholar 

  • Errede B, Levin D (1993) A conserved kinase cascade for MAP kinase activation in yeast. Curr Opin Cell Biol 5:254–260

    Google Scholar 

  • Farley J, Auerbach S (1986) Protein kinase C activation induces conductance changes inHermissenda photoreceptors like those seen in associative learning. Nature 319:220–223

    Google Scholar 

  • Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246

    Google Scholar 

  • Greenberg ME, Ziff EB (1984) Stimulation of 3T3 cells induces transcription of thec-fos proto-oncogene. Nature 311:433–438

    Google Scholar 

  • Hokin LE (1985) Receptors and phosphoinositide-generated second messengers. Annu Rev Biochem 54:205–235

    Google Scholar 

  • Holt KH, Olson AL, Moye-Rowley WS, Pessin JE (1994) Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits. Mol Cell Biol 14:42–49

    Google Scholar 

  • Imagawa M, Chiu R, Karin M (1987) Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51:251–260

    Google Scholar 

  • Imbra RJ, Karin M (1986) Phorbol ester induces the transcriptional stimulatory activity of the SV40 enhancer. Nature 323:555–558

    Google Scholar 

  • Irie K, Takase M, Lee KS, Levin DE, Araki H, Matsumoto K, Oshima Y (1993)MKK1 andMKK2, which encodeSaccharomyces cerevisiae mitogen-activated protein kinase homologs, function in the pathway mediated by protien kinase C. Mol Cell Biol 13:3076–3083

    Google Scholar 

  • Jarvis EE, Hagen DC, Sprague GF Jr (1988) Identification of a DNA segment that is necessary and sufficient for α-specific gene control inSaccharomyces cerevisiae. Implications for regulationa-specific and α-specific genes. Mol Cell Biol 8:309–320

    Google Scholar 

  • Kelly K, Cochran BH, Stiles CD, Leder P (1983) Cell-specific regulation of thec-myc gene by lylmphocyte mitogens and platelet-derived growth factor. Cell 35:603–610

    Google Scholar 

  • Kikkawa U, Kishimoto A, Nishizuka Y (1989) The protein kinase C family: heterogeneity and its implications. Annu Rev Biochem 58:31–44

    Google Scholar 

  • Kishimoto A, Takai Y, Mori T, Kikkawa U, Nishizuka Y (1980) Activation of calcium and phospholipid dependent protein kinase by diacylglycerol: its possible relation to phosphatidyl inositol turnover. J Biol Chem 255:2273–2276

    Google Scholar 

  • Kruijer W, Cooper JA, Hunter T, Verma IM (1984) Platelet-derived growth factor induces rapid but transient expression of thec-fos gene and protein. Nature 312:711–716

    Google Scholar 

  • Lee KS, Levin DE (1992) Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for aSaccharomyces cerevisiae protein kinase C homolog. Mol Cell Biol 12:172–182

    Google Scholar 

  • Lee KS, Irie K, Gotoh Y, Watanabe Y, Araki H, Nishida E, Matsumoto K, Levin DE (1993) A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C. Mol Cell Biol 13:3067–3075

    Google Scholar 

  • Lee W, Haslinger A, Karin M, Tjian R (1987) Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40. Nature 325:368–372

    Google Scholar 

  • Levin DE, Errede B (1995) The proliferation of MAP kinase signaling pathways in yeast. Curr Opin Cell Biol 7:197–202

    Google Scholar 

  • Levin DE, Fields FO, Kunisawa R, Bishop JM, Thorner J (1990) A candidate protein kinase C gene,PKC1, is required for theS. cerevisiae cell cycle. Cell 62:213–224

    Google Scholar 

  • Levin DE, Blowers B, Chen C-Y, Kamada Y, Watanabe M (1994) Dissecting the protein kinase C/MAP kinase signalling pathway ofSaccharomyces cerevisiae. Cell Mol Biol Res 40:229–239

    Google Scholar 

  • Madison DV, Malenka RC, Nicoll RA (1986) Phorobol esters block a voltage-sensitive chloride current in hippocampal pyramidal cells. Nature 321:695–697

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Miller J (1972) Experiments in Molecular Genetics

  • Nishizuka Y (1986) Studies and perspectives of protein kinase C. Science 233:305–312

    Google Scholar 

  • Nishizuka Y (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334:661–665

    Google Scholar 

  • Nonaka H, Tanaka K, Hirano H, Fujiwara T, Kohno H, Umikawa M, Mino A, Takai Y (1995) A downstream target of Rhol small GTP-binding protein is Pkc1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade inSaccharomyces cerevisiae. EMBO J 14:5931–5938

    Google Scholar 

  • Ohmura E, Friesen HG (1985) 12-O-Tetradecanoyl phorbol-13-acetate stimulates rat growth hormone (GH) release by different pathways from that of human pancreatic GH-releasing factor. Endocrinology 116:728–733

    Google Scholar 

  • Ohno S, Akita Y, Konno S, Imajoh S, Suzuki K (1988) A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family. Cell 53:731–741

    Google Scholar 

  • Ono Y, Fuji K, Ogita U, Kikkawa K, Igarashi K, Nishizuka Y (1989) Protein kinase C ζ subspecies from rat brain: its structure, expression, and properties. Proc Natl Acad Sci USA 86:3099–3103

    Google Scholar 

  • Paravicini G, Cooper M, Friedli L, Smith DJ, Carpenter J-L, Klig LS, Payton MA (1992) The osmotic integrity of the yeast cell requires a functionalPKC1 gene product. Mol Cell Biol 12:4896–4905

    Google Scholar 

  • Pelech SL, Sanghera JS (1988) Mitogen-activated protein kinases: versatile transducers for cell signaling. Trends Biochem Sci 17:233–238

    Google Scholar 

  • Persons DA, Wilkison WO, Bell RM, Finn OJ (1988) Altered growth regulation and enhanced tumorigenicity of NIH3T3 fibroblasts transfected with protein kinase C-1 cDNA. Cell 52:447–458

    Google Scholar 

  • Printen JA, Sprague GF (1994) Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics 138:609–619

    Google Scholar 

  • Rose MD, Winston F, Heiter P (1990) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Plainview, New York

    Google Scholar 

  • Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Meth Enzymol 194:281–301

    Google Scholar 

  • Rozengurt E, Rodriguez-Pena A, Coombs M, Sinnet-Smith J (1984) Diacyglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells: role of Ca2+-sensitive phospholipid-dependent protein kinase. Proc Natl Acad Sci USA 81:5748–5752

    Google Scholar 

  • Ryba NJP, Findlay JBC, Reid JD (1993) The molecular cloning of the squid (Loligo forbesi) visual Gqα subunit and its expression inSaccharomyces cerevisiae. Biochem J 292:333–341

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single-stranded nucleic acids as a carrier. Curr Genet 16:339–346

    Google Scholar 

  • Soler M, Plovins A, Martin H, Molina M, Nombela C (1995) Characterization of domains in the yeast MAP kinase Slt2 (Mpk1) required for functional activity and in vivo interaction with protein kinases Mkk1 and Mkk2. Mol Microbiol 17:833–842

    Google Scholar 

  • Stabel S, Parker P (1991) Protein kinase C. Pharmacol Ther 51:71–95

    Google Scholar 

  • Stolz A, Linder P (1990) TheADE2 gene fromSaccharomyces cerevisiae: sequence and new vectors. Gene 95:91–98

    Google Scholar 

  • Thomas G (1992) MAP kinase by any other name smells just as sweet. Cell 68:3–6

    Google Scholar 

  • Torres L, Martin H, Garcia-Saez MI, Arroyo J, Molina M, Sanchez M, Nombela C (1991) A protein kinase gene complements the lytic phenotype ofS. cerevisiae lyt2 mutants. Mol Microbiol 5:2845–2854

    Google Scholar 

  • Weinstein IB, Borner CM, Krauss RS, O'Driscoll K, Choi PM, Moritomi M, Hoshina S, Hsieh LL, Tchou-Wong KM, Guadagno SN, Ueffing M, Guillem J (1991) Pleiotropic effects of protein kinase C and the concept of carcinogenesis as a progressive disorder in signal transduction. In: Brugge J, Curran T, Harlow E, McCormick F (eds). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 113–123

    Google Scholar 

  • Wickerham LJ (1946) A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeasts. J Bacteriol 52:293–301

    Google Scholar 

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  1. GLAXO Institute for Molecular Biology, Chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland

    G. Paravicini & L. Friedli

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  1. G. Paravicini
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  2. L. Friedli
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Communicated by D. Y. Thomas

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Paravicini, G., Friedli, L. Protein-protein interactions in the yeastPKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade. Molec. Gen. Genet. 251, 682–691 (1996). https://doi.org/10.1007/BF02174117

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  • DOI: https://doi.org/10.1007/BF02174117

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