Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation

• Published in: Molecular biology of the cell Vol. 27; no. 17; pp. 2771 - 2783
• Main Authors: Goshen-Lago, Tal, Goldberg-Carp, Anat, Melamed, Dganit, Darlyuk-Saadon, Ilona, Bai, Chen, Ahn, Natalie G, Admon, Arie, Engelberg, David
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01.09.2016
• Subjects: 3T3 Cells; Amino Acid Motifs; Amino Acid Sequence; Animals; Cell Culture Techniques; Cell Wall - metabolism; Extracellular Signal-Regulated MAP Kinases - metabolism; HEK293 Cells; Humans; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases - metabolism; Mitogen-Activated Protein Kinases - genetics; Mitogen-Activated Protein Kinases - metabolism; Mutation; Phosphorylation; Protein-Serine-Threonine Kinases - metabolism; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Signal Transduction - physiology
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E16-03-0167

MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect.