Non-Canonical MAP Kinase Signaling and Meiotic Development in Saccharomyces cerevisiae

Mitogen-activated protein kinases (MAPKs) regulate multiple cellular processes, including cellular survival, proliferation, and differentiation in a multitude of cell types. Smk1, a meiosis-specific MAPK, is required for spore morphogenesis in the yeast Saccharomyces cerevisiae. In the absence of Smk1, cells complete the meiotic divisions but exhibit multiple aberrant spore structures, suggesting that Smk1 regulates several steps during spore differentiation. Like other MAPKs, Smk1 is activated by dual phosphorylation of a conserved TX-Y activation motif, yet Smk1 activaiton occurs through a novel pathway. Several positive regulators of Smk1 have been identified, including the cyclin-dependent protein kinase activating kinase (CAK), Cak1; the meiosis-specific subunit of the anaphase-promoting E3 ubiquitin ligase complex/cyclosome (APC/C), Ama1; and Ssp2, a meiosis-specific protein of unknown function. Data presented in this thesis demonstrate that Cak1 directly phosphorylates Smk1 on T207. Phosphorylation of Y209 occurs through an intramolecular, autocatalytic reaction that requires the presence of Ssp2, the 5’UTR of the SMK1 message, and the N-terminus of the Smk1 protein. In a proposed model for Smk1 activation, Ssp2 promotes the formation of a transitory intermediate form of Smk1 that possesses tyrosine kinase activity as the protein emerges from the ribosome or during protein folding. SMK1 and SSP2 are both expressed as middle meiotic genes, yet Ssp2 is translated after Smk1. Therefore, the initial Smk1 produced undergoes partial activation through phosphorylation of T207 by Cak1. Complete activation of Smk1 only occurs after Ssp2 appears. This establishes distinct pools of differentially active forms of Smk1. Indeed, completion of progressively later steps in spore morphogenesis has been shown to require increasing thresholds of Smk1 kinase activity. Ama1 is a meiosis-specific subunit of the APC/C. The requirement of Ama1 for Smk1 activation suggests an Smk1 inhibitor that is removed through ubiquitin-mediated proteasomal degradation. Mutations that suppress the sporulation defect of an smk1 hypomorph are predicted to occur in negative regulators of Smk1. Several recessive extragenic s uppressors of the smk1-4 (ess) temperature-sensitive sporulation defect were isolated. Cloning by complementation with a genomic library and whole genome sequencing were employed in an attempt to identify the proposed inhibitor of Smk1 mutated in the ess background