Abstract
In Saccharomyces cerevisiae, a variety of stresses and aggressions to the cell wall stimulate the activation of the cell wall integrity MAPK pathway, which triggers the expression of a series of genes important for the maintenance of cell wall homeostasis. This MAPK module lies downstream of the Rho1 small GTPase and protein kinase C Pkc1 and consists of MAPKKK Bck1, MAPKKs Mkk1 and Mkk2, and the Slt2 MAPK. In agreement with previous reports suggesting that Mkk1 and Mkk2 were functionally redundant, we show here that both Mkk1 and Mkk2 alone or even chimerical proteins constructed by interchanging their catalytic and regulatory domains are able to efficiently maintain signal transduction through the pathway. Both Mkk1 and Mkk2 are phosphorylated in vivo concomitant to activation of the cell integrity pathway. Interestingly, hyperphosphorylation of the MEKs required not only the upstream components of the pathway, but also a catalytically competent Slt2 MAPK downstream. Active Slt2 purified from yeast extracts was able to phosphorylate Mkk1 and Mkk2 in vitro. We have mapped Ser(50) as a direct phosphorylation target for Slt2 in Mkk2. However, substitution of all (Ser/Thr)-Pro canonical MAPK target sites with alanine did not totally abrogate Slt2-dependent Mkk2 phosphorylation. Mutation or deletion of a conserved MAPK-docking site at the N-terminal extension of Mkk2 precluded its interaction with Slt2 and negatively affected retrophosphorylation. Our data show that the cell wall integrity MAPKKs are targets for their downstream MAPK, suggesting the existence of complex feedback regulatory mechanisms at this level.
Highlights
Through this pathway by the sequential phosphorylation of a module composed of three protein kinases
Slt2 Retrophosphorylates Mkk1 and Mkk2 MAPKKs tion is controversial, as some researchers did not find any variation on the catalytic activity of MEK1 in respect to its unphosphorylated form [18, 19], whereas others suggested a negative role for this retrophosphorylation mechanism [20]
Mkk1 and Mkk2, the MAPKKs that operate in the cell wall integrity (CWI) MAPK pathway in S. cerevisiae were reported to be functionally redundant when first uncovered [40]
Summary
YPD (1% yeast extract, 2% peptone, and 2% glucose) broth or agar was the general non-selective medium used for yeast cell growth. Synthetic minimal medium (SD) contained 0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulfate, and 2% glucose and lacking appropriate amino acids and nucleic acid bases to maintain selection for plasmids. Plasmids pRS306-Mkk1-6myc and pRS306-Mkk2-6myc were generated by amplification by PCR from the Mkk and Mkk coding sequence using primers MKK1Up and MKK1Lo for MKK1 and MKK2Up and MKK2Lo for MKK2, and cloned into the BamHI site in a yeast vector, pRS306-myc, which had been previously generated by inserting a myc epitope into the polylinker in the integrative URA3 vector, pRS306 [35]. The plasmid carrying the pRS306-MKK1(1– 222)-MKK2(216 –506)-6myc chimera was constructed by overlapping PCR using primers MKK1Up and NB for amplifying the region corresponding to the N-terminal extension of OCTOBER 26, 2007 VOLUME 282 NUMBER 43
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