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 Mpk 1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1Δmkk2Δ and mkk1Δmkk2Δpbs2Δste7Δ 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 mkk1Δmkk2Δ 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.
Bibliographical noteFunding Information:
We thank Michal Bell for critically reading and editing the manuscript. This study was supported by the Israel Science Foundation (Center of Excellence Grants 180/09 and 1772/13 and personal grant 593/15), the Bi-National US-Israel Science Foundation (Grant 2009116), the Israel Cancer Research Fund, and the Singapore National Research Foundation under its HUJ-NUS partnership program at the Campus for Research Excellence and Technology Enterprise (CREATE).
© 2016 Goshen-Lago, Goldberg-Carp, et al.