Skh1, the MEK component of the mkh1 signaling pathway in Schizosaccharomyces pombe.
We previously reported the identification of Mkh1, a MEK kinase in Schizosaccharomyces pombe that is required for cell wall integrity, and we presented genetic evidence that Pmk1/Spm1, a MAP kinase, functions downstream from Mkh1 in the same pathway. Here, we report the identification of Skh1, a MEK (MAP kinase kinase) in S. pombe. The sequence of Skh1 is nearly identical to that of the recently reported Pek1 sequence. We present biochemical and genetic evidence that Skh1 is the MEK component of the Mkh1-Spm1 MAP kinase cascade. Our yeast two-hybrid results indicate that Mkh1, Skh1, and Spm1 physically interact to form a ternary complex. Deletion of mkh1, skh1 or spm1 results in identical phenotypes, including sensitivity to (beta)-glucanase treatment, growth inhibition on media containing KCl, and filamentous growth on medium containing caffeine. Double mutant strains exhibit phenotypes that are identical to the single mutant strains. Furthermore, expression of an activated HA-Skh1(DD )protein suppressed these defects in mkh1(delta) cells, and overexpression of Spm1 suppressed these defects in skh1(delta) cells. We also show that HA-Spm1 is hyper-phosphorylated on tyrosine residues in cells co-expressing the activated HA-Skh1(DD) protein. Furthermore, we found the phosphorylated/activated form of GFP-HA-Spm1 at detectable levels in wild-type cells, but not at appreciable levels in mkh1(delta) or skh1(delta) cells expressing this fusion protein. Together, our results indicate that Mkh1, Skh1 and Spm1 constitute a MAPK cascade in fission yeast.
Loewith
R
R
Department of Biochemistry and Molecular Biology, University of Calgary Health Sciences Centre, Calgary, Alberta, T2N4N1, Canada.
Hubberstey
A
A
Young
D
D
D000595:Amino Acid Sequence; D002110:Caffeine / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D004843:Epistasis, Genetic; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D043326:Glucan 1,3-beta-Glucosidase; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D013172:Spores, Fungal / Q000235:genetics / Q000502:physiology; D013489:Suppression, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques; D001617:beta-Glucosidase / Q000378:metabolism / Q000494:pharmacology
D005656:Fungal Proteins; C085569:GAL83 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D019000:Phosphotyrosine; D002110:Caffeine; D011189:Potassium Chloride; D020930:MAP Kinase Kinase Kinases; C106762:Mkh1 protein, S pombe; D020929:Mitogen-Activated Protein Kinase Kinases; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase