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J. Biol. Chem., Vol. 282, Issue 43, 31174-31185, October 26, 2007

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Retrophosphorylation of Mkk1 and Mkk2 MAPKKs by the Slt2 MAPK in the Yeast Cell Integrity Pathway^*

From the Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain

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 Ser⁵⁰ 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.

Received for publication, July 30, 2007

^* This work was supported in part by Grant BIO2004-02019 from the Comisión Interministerial de Ciencia y Tecnología (CYCyT). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a predoctoral Formación de Profesorado Universitario fellowship from the Spanish Ministerio de Educación y Ciencia.

² To whom correspondence should be addressed: Dept. de Microbiología II, Facultad de Farmacia UCM, Pza de Ramón y Cajal s/n., 28040 Madrid, Spain. Tel.: 34-91-394-1888; Fax: 34-91-394-1745; E-mail: vicjcid{at}farm.ucm.es.

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