Zeo1, a negative regulator of the cell integrity MAPK pathway, a substrate for the Yck1,2 casein kinase 1 protein kinases

Abstract

Yeast casein kinase 1 (CK1) proteins Yck1 and 2 are redundant, essential plasma membrane-associated protein kinases in budding yeast. Yck1,2 are required for endocytic trafficking of receptors and permeases, cellular morphogenesis and cytokinesis. Although much is known about Yck1,2 function, it has been difficult to identify substrates using genetic approaches. We used a comparative phosphoproteomic approach to identify Yck1,2 substrates. Protein extracts from wild-type cells and cells with low Yck1,2 activity (yckts cells), were digested and enriched for phosphopeptides. Phosphopeptides were identified by mass spectrometry, and their abundances were compared. One phosphopeptide found to be substantially more abundant in wild-type protein extracts than in yckts extracts is a peptide from Zeo1 that is phosphorylated within a weak CK1 consensus site. Zeo1 was identified as a negative regulator of the cell integrity Pkc1-Slt2 MAP kinase pathway. Zeo1 interacts with Mid2, an upstream regulator of Rho1, and deletion of ZEO1 causes constitutive activation of the Slt2 MAPK. We found that the yckts mutant also shows constitutive activation of Slt2, and yckts genetically interacts with rom2Δ and sac7Δ similar to those for zeo1Δ. We are now testing for in vitro phosphorylation of Zeo1 by Yck2, and are confirming preliminary observations that mutation of the Zeo1 phosphorylation site affects Zeo1 activity in vivo.