An Analog-sensitive Version of the Protein Kinase Slt2 Allows Identification of Novel Targets of the Yeast Cell Wall Integrity Pathway

Esmeralda Alonso-Rodríguez,Pablo Fernández-Piñar,Almudena Sacristán-Reviriego,María Molina,Humberto Martín

doi:10.1074/jbc.m115.683680

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.

Highlights

2 To whom correspondence should be addressed: Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain Tel.: 34-91394188; Fax: 34913941745; E-mail: molmifa@ucm.es. 3 The abbreviations used are: MAP, mitogen-activated protein; CWI, cell wall integrity; ATP␥S, adenosine 5Ј-[␥-thio]triphosphate; as, analog-sensitive; kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK) that are sequentially phosphorylated at their activation loop upon stimulation [1]
This is a very powerful technique that brings about the identification of the phosphorylation sites within the CR, Congo red; 2,3-DMB-PP1, 4-Amino-1-tert-butyl-3-(2,3-dimethylbenzyl)pyrazolo[3,4-d]pyrimidine; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; Carboxypeptidase Y (CPY), carboxypeptidase Y; 4E-BP, translation initiation factor eIF4E-binding protein; GGA, Golgi-associated, ␥-adaptin ear-containing, ARF-binding protein; RCAN, regulator of the Ca2ϩ and calmodulin-dependent serine-threonine phosphatase calcineurin; AP, alkaline phosphatase
By using a quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based phosphoproteomic approach, we previously identified a number of putative Slt2 targets that displayed enhanced phosphorylation at (S/T)P sites upon CWI pathway stimulation triggered by Pkc1 hyperactivation [18]

Summary

Experimental Procedures

Microorganisms and Culture Conditions—For the cloning and amplification of plasmid DNA, the Escherichia coli strain DH5␣ (supE44, ⌬lacU169, hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was used. Non-phosphorylatable Myc-tagged versions of Msg were expressed in S. cerevisiae from Ycplac22-based plasmids [29] To this end, the custom-made plasmids pUC57-msg5N9A and pUC57-msg5C7A were obtained from GenScript bearing the MSG5 N-terminal and the C-terminal coding region, respectively, with mutations to change every serine or threonine followed by proline to alanine. To express GST-fused proteins in E. coli, the DNA fragments encoding the corresponding proteins were amplified using PCR with the primers described in Table 1 using wild-type BY4741 genomic DNA as a template and cloned into pGEMT. Expression of Recombinant GST Fusion Proteins in E. coli— E. coli Rosetta cells transformed with the corresponding pGEXKG-based plasmids were cultured in LB with ampicillin to an A600 ϭ 0.5, and isopropyl-1-thio-␤-D-galactopyranoside was added to a final concentration of 0.5 mM for 3 h.

No addition

Results

Discussion