UC Berkeley

Protein kinases are enzymes that transfer the γ-phosphate of ATP to acceptor residues, most often serine, threonine or tyrosine, in proteins. Phosphorylation by protein kinases are integral to the signaling processes that control virtually all aspects of cellular physiology and phosphorylation is the most abundant post-translational modification in signal transduction networks. Covalent attachment of a phosphate group to a protein can cause a conformational change, introduce a new epitope that enhances or prevents interaction with another protein, increase or decrease the rate of turnover, and/or affect subcellular localization. In these ways, phosphorylation-dependent events can influence cell metabolism, morphology, and differentiation.

Target of rapamycin (TOR) complex 2 (TORC2) is an evolutionarily conserved multi-subunit protein kinase and indispensable regulator of plasma membrane homeostasis. Genetic studies in yeast have demonstrated that the sole essential downstream target and effector of TORC2 is the protein kinase Ypk1 (and its paralog Ypk2), whose mammalian ortholog is SGK1. In humans, mutations in the TORC2 signaling network have been implicated in inflammatory disorders, metabolic diseases, and multiple cancers.

In this doctoral dissertation, I describe the studies I undertook to explore novel aspects of TORC2-Ypk1 signaling. Because a Rab5-specific guanine nucleotide exchange factor (GEF), Muk1, was identified in a prior global screen for candidate Ypk1 targets, I investigated the role of Rab5-type GTPases in the TORC2 signaling network. I confirmed first that Muk1 is a substrate of Ypk1 and demonstrated that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Second, and strikingly, I found that yeast lacking its two Rab5 GEFs (Muk1 and Vps9), or its three Rab5 paralogs (Vps21/Ypt51, Ypt52 and Ypt53), or overexpressing Msb3 (a Rab5-directed GTPase-activating protein), all exhibited pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Finally, I showed that Vps21 co-immunoprecipitates with TORC2, and furthermore, that purified GTP-bound Vps21 is able to stimulate in vitro the activity of TORC2 immuno-enriched from Rab5-deficient cells. My findings suggest that TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling