Characterization of domains in the yeast MAP kinase Slt2 (Mpk1) required for functional activity and in vivo interaction with protein kinases Mkk1 and Mkk2.
MKK1/MKK2 and SLT2 (MPK1) are three Saccharomyces cerevisiae genes, coding for protein kinases, that have been postulated to act sequentially as part of the Pkc1p signalling pathway, a phosphorylation cascade essential for cell integrity. By using the 'two-hybrid system' and co-purification experiments on glutathione-agarose beads, we have shown that Slt2p interacts in vivo and in vitro with both Mkk1p and Mkk2p, thus confirming a previous suggestion based on epistasis experiments of the corresponding genes. Plasmid constructs of the SLT2 gene, deleted in the whole C-terminal non-kinase region or part of it, and therefore containing all of the conserved kinase subdomains, were still functional in complementation of the slt2 lytic phenotype and in vivo interaction with Mkk1p and Mkk2p. In contrast, the Slt2p C-terminal domain (162 residues) that carries a glutamine-rich fragment followed by a 16 polyglutamine tract, was shown to be dispensable for complementation and in vivo association with Mkk1p and Mkk2p. We have also demonstrated that the N-terminal putative regulatory domain of these two MAP kinase activators is the main region involved in the interaction with Slt2p.
Soler
M
M
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Plovins
A
A
Martín
H
H
Molina
M
M
Nombela
C
C
D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D015533:Transcriptional Activation
D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases