Spatiotemporal Regulation of Non-Canonical MAPK Signaling

Mitogen activated protein kinases (MAPKs) are conserved signaling enzymes that regulate an array of essential cellular processes in eukaryotic cells. Canonical MAPK signaling is defined by a three-tier kinase module in which a dual-specificity MAPK kinase phosphorylates the T-X-Y motif in the activation loop of the MAPK. MAPKs can also be activated by non-canonical signaling pathways, yet relatively little is known about these pathways and how they are controlled. Smk1 is a non-canonically activated MAPK in the yeast S. cerevisiae that is expressed specifically in meiotic cells and controls spore wall morphogenesis. The Smk1 pathway represents an excellent model to study mechanisms that control non-canonical MAPKs in the context of a developmental program. Prior studies demonstrated that the activation loop T in Smk1 (T207) is phosphorylated by the CDK activating kinase, Cak1, and the activation-loop Y (Y209) is autophosphorylated in an intramolecular reaction that requires a meiosis-specific protein, Ssp2. Data presented in this thesis show that Smk1 is almost immediately phosphorylated by Cak1 on T207 when it is produced during meiosis I, yet this early form of Smk1 is inactive and Smk1 is not activated until later in the program. Activation occurs specifically at membranes that surround incipient haploids termed prospore membranes (PSMs) in a switch-like fashion as cells exit meiosis II. This tight spatiotemporal control of Smk1 activation is achieved through Ssp2. The temporal control is achieved by a meiosis-specific translational regulatory pathway that represses SSP2 mRNA until anaphase II, while spatial control is achieved by an amino-terminal domain of Ssp2 that is both necessary and sufficient for PSM targeting. Kinase activation is achieved by Ssp2’s carboxy-terminal kinase activating domain (KAD) that activates catalysis by directly binding to Smk1. Ssp2 binding also triggers Smk1 autophosphorylation of Y209. Heterologous expression/reconstitution experiments demonstrate that phosphorylation of Smk1-T207 and Y209 increases Ssp2/Smk1 catalytic activity. These studies also identify Y-X-X-X-S/T-P as the unique phosphorylation consensus motif of Smk1. Together, these data define a novel mechanism of non-canonical MAPK activation and provide new insights into how MAPKs are spatiotemporally regulated in the context of a developmental program