title: An Analog-sensitive Version of the Protein Kinase Slt2 Allows Identification of Novel Targets of the Yeast Cell Wall Integrity Pathway.
abstract: The yeast cell wall integrity MAPK Slt2 mediates the transcriptional response to cell wall alterations through phosphorylation of transcription factors Rlm1 and SBF. However, the variety of cellular functions regulated by Slt2 suggests the existence of a significant number of still unknown substrates for this kinase. To identify novel Slt2 targets, we generated and characterized an analog-sensitive mutant of Slt2 (Slt2-as) that can be specifically inhibited by bulky kinase inhibitor analogs. We demonstrated that Slt2-as is able to use adenosine 5'-[γ-thio]triphosphate analogs to thiophosphorylate its substrates in yeast cell extracts as well as when produced as recombinant proteins in Escherichia coli. Taking advantage of this chemical-genetic approach, we found that Slt2 phosphorylates the MAPK phosphatase Msg5 both in the N-terminal regulatory and C-terminal catalytic domains. Moreover, we identified the calcineurin regulator Rcn2, the 4E-BP (translation initiation factor eIF4E-binding protein) translation repressor protein Caf20, and the Golgi-associated adaptor Gga1 as novel targets for Slt2. The Slt2 phosphorylation sites on Rcn2 and Caf20 were determined. We also demonstrated that, in the absence of SLT2, the GGA1 paralog GGA2 is essential for cells to survive under cell wall stress and for proper protein sorting through the carboxypeptidase Y pathway. Therefore, Slt2-as provides a powerful tool that can expand our knowledge of the outputs of the cell wall integrity MAPK pathway.
authors.affiliation: From the Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), 28040 Madrid, Spain.
authors.forename: Esmeralda
authors.initials: E
authors.lastname: Alonso-Rodríguez
authors.affiliation: From the Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), 28040 Madrid, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Fernández-Piñar
authors.affiliation: From the Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), 28040 Madrid, Spain.
authors.forename: Almudena
authors.initials: A
authors.lastname: Sacristán-Reviriego
authors.affiliation: From the Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), 28040 Madrid, Spain. Electronic address: molmifa@ucm.es.
authors.forename: María
authors.initials: M
authors.lastname: Molina
authors.affiliation: From the Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), 28040 Madrid, Spain.
authors.forename: Humberto
authors.initials: H
authors.lastname: Martín
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C555411:Caf20 protein, S cerevisiae; C497361:Gga2 protein, S cerevisiae; C541019:Rcn2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae; C085791:MSG5 protein, S cerevisiae; D017027:Protein Tyrosine Phosphatases
keywords: ATPγS analog; Slt2; cell wall; cell wall integrity; mitogen-activated protein kinase (MAPK); phosphorylation; signaling; yeast