Wilkinson, Marc G.; (1998) Structural and functional analysis of the fission yeast Sty1 stress-activated MAP kinase pathway. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The fission yeast Sty1 MAP kinase displays strong sequence homology with the budding yeast Hogl and mammalian stress-activated MAP kinases (SAPKs). Loss of Sty1 function results in several phenotypes: cells are not only sensitive to environmental stress but are also sterile and delayed in the timing of mitotic initiation. In this thesis I demonstrate that Sty1 and the Wis1 MAP2K form the core of a previously unidentified MAP kinase pathway which is activated in response to both nitrogen starvation and multiple environmental stresses. I have identified a number of target genes important for the response to stress which are induced rapidly in a Sty1-dependent manner. One such gene encodes the Pyp2 tyrosine-specific phosphatase which binds to and tyrosine dephosphosphorylates Sty1 in response to prolonged stress, demonstrating a negative feedback loop. Significantly, expression of all stress-induced genes identified is dependent on the BZip transcription factor Atf1, a homolog of human ATF-2. ATF-2 binds to and is phosphorylated by the stress-activated SAPK1 enzyme. Similarly, Atf1 binds to Sty1 and is inducibly phosphorylated by Sty1 in response to stress. These data demonstrate structural and functional conservation between human and yeast SAPK pathways. I have also characterised the role of the Sty1 MAPK pathway in sexual differentiation. Cells lacking Sty1, wis1 or atf1 are sterile, at least partly due to an inability to activate Sty1, arrest in G1 or express ste11, a transcription factor essential for conjugation and meiosis. Atf1 is not the only substrate of Sty1 since atfl cells are still sensitive both to certain stresses and to overexpression of wis1. I sought alternative Sty1 targets using the yeast two-hybrid system and isolated a novel protein, Sin1. Sin1 associates with Sty1 in vitro and displays sequence homology to a human cDNA which when overexpressed suppresses the defect of a budding yeast strain carrying an activated RAS allele. Taken together, the results presented in this thesis identify multiple roles for the Sty1 MAPK pathway in the fission yeast life cycle and demonstrate remarkable conservation, both in structure and function between human and fission yeast SAPK pathways.

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