Regulation of Pkc1 Hyper-Phosphorylation by Genotoxic Stress.

Li Liu,John C Samuelson,Jiri Veis,Catherine E Costello,Wolfgang Reiter,David E Levin,Gustav Ammerer,Edwin Motari

doi:10.3390/jof7100874

Abstract

The cell wall integrity (CWI) signaling pathway is best known for its roles in cell wall biogenesis. However, it is also thought to participate in the response to genotoxic stress. The stress-activated protein kinase Mpk1 (Slt2, is activated by DNA damaging agents through an intracellular mechanism that does not involve the activation of upstream components of the CWI pathway. Additional observations suggest that protein kinase C (Pkc1), the top kinase in the CWI signaling cascade, also has a role in the response to genotoxic stress that is independent of its recognized function in the activation of Mpk1. Pkc1 undergoes hyper-phosphorylation specifically in response to genotoxic stress; we have found that this requires the DNA damage checkpoint kinases Mec1 (Mitosis Entry Checkpoint) and Tel1 (TELomere maintenance), but not their effector kinases. We demonstrate that the casein kinase 1 (CK1) ortholog, Hrr25 (HO and Radiation Repair), previously implicated in the DNA damage transcriptional response, associates with Pkc1 under conditions of genotoxic stress. We also found that the induced association of Hrr25 with Pkc1 requires Mec1 and Tel1, and that Hrr25 catalytic activity is required for Pkc1-hyperphosphorylation, thereby delineating a pathway from the checkpoint kinases to Pkc1. We used SILAC mass spectrometry to identify three residues within Pkc1 the phosphorylation of which was stimulated by genotoxic stress. We mutated these residues as well as a collection of 13 phosphorylation sites within the regulatory domain of Pkc1 that fit the consensus for CK1 sites. Mutation of the 13 Pkc1 phosphorylation sites blocked hyper-phosphorylation and diminished RNR3 (RiboNucleotide Reductase) basal expression and induction by genotoxic stress, suggesting that Pkc1 plays a role in the DNA damage transcriptional response.

Highlights

We found that the protein kinase C 1 (Pkc1) band-shift was induced in response to DNA damage by alkylating agent methylmethane sulfonate (MMS), by dNTP depletion by hydroxyurea (HU) treatment, or by UV irradiation, but not in response to cell wall stress treatments calcofluor white (CFW) or elevated growth temperature (Figure 1a)
Lambda protein phosphatase (Figure 1b). We found that it is dependent on the partially redundant DNA damage checkpoint kinases Mec1 and Tel1 (Figure 1c)
This is in contrast to the findings of Soriano-Carot, et al [30], who found that Tel1 was uniquely responsible for the Pkc1 band-shift

Summary

Introduction

The cell wall integrity (CWI) signaling pathway of the budding yeast Saccharomyces cerevisiae has been well characterized with regard to its regulation by cell wall stress [1,2,3,4]. This pathway regulates biosynthesis of cell wall polymers, organization of the actin cytoskeleton, exocytosis, and the protein kinase C 1 (Pkc1)-mediated stress-activated protein kinase (SAPK) cascade through activation of the small GTPase, Rho. Loss-of-function mutants in the SAPK cascade display cell lysis defects that are suppressed by external osmotic support [2], highlighting the central role of this signaling pathway in the maintenance of cell wall integrity

Methods

Results

Conclusion