The Identification of Novel Targets of the Fission Yeast Sty1 MAP Kinase

Abstract

In order to survive exposure to adverse environmental conditions, cells must initiate a stress response. In the fission yeast Schizosaccharomyces pombe, the stress response is mediated by the mitogen activated protein kinase (MAPK) Sty1. MAPK signalling pathways are highly conserved between yeast and mammalian cells, and the Sty1 pathway provides a useful model for the study of the MAPK pathways. Sty1 mediates the stress response through the phosphorylation of downstream target proteins that are involved in the regulation of a range of different cellular processes. A pool of Sty1 has recently been found to present within the mitochondria of S. pombe cells, suggesting that Sty1 may mediate part of the stress response through the phosphorylation of mitochondrial proteins. In order to fully understand the role of Sty1 within the mitochondria, it is necessary to identify mitochondrial targets of Sty1 phosphorylation. It is also essential that Sty1 activity is tightly regulated, and this is achieved by the action of both activating kinases, and negatively regulating phosphatases. Following exposure to oxidative stress, mitochondrial Sty1 activity is attenuated by the PP2C phosphatase Ptc4. However, it is not yet known whether additional phosphatases are involved in the regulation of mitochondrial Sty1 activity.In this study two further phosphatases, Azr1, a PP2C phosphatase, and Pyp1, a known regulator of basal Sty1 activity, were identified as potential mitochondrial proteins. However, Azr1 was not found to be involved in the regulation of mitochondrial Sty1 activity, and the potential role of Pyp1 within the mitochondria requires further investigation.To examine the role of Sty1 within the mitochondria, Deltasty1 cells were assayed for mitochondrial defects. This revealed that Sty1 appears to be involved in the regulation of respiration. To identify how Sty1 regulates mitochondrial function, two separate approaches were utilised to identify novel Sty1 target proteins. The first approach involved the use of the peptidyl-prolyl cis/trans isomerase, Pin1. The Pin1 binding assay was previously developed within our laboratory, and I used the Pin1 binding assay to identify proteins phosphorylated at the MAPK consensus site, serine/threonine-proline, in mitochondrial lysates. The second approach used was SILAC, which was performed using whole cell lysates. None of the proteins that were validated as putative Sty1 targets were known mitochondrial proteins, although one protein, Efc25, contains a potential mitochondrial targeting sequence. Interestingly, Wis1, the MAPK kinase upstream of Sty1, was found to be phosphorylated in a Sty1-dependent manner. Mutation of the potential Sty1 phosphorylation sites in Wis1 leads to reduced Sty1 phosphorylation following oxidative stress exposure, suggesting that Sty1 phosphorylates Wis1 to increase Wis1 activity, thus forming a positive feedback loop to further modulate Sty1 activation.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nicholas Jones (Supervisor) & Caroline Wilkinson (Supervisor)