Tor2 Directly Phosphorylates the AGC Kinase Ypk2 To Regulate Actin Polarization

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Abstract
The target of rapamycin (TOR) protein kinases, Tor1 and Tor2, form two distinct complexes (TOR complex 1 and 2) in the yeast Saccharomyces cerevisiae. TOR complex 2 (TORC2) contains Tor2 but not Tor1 and controls polarity of the actin cytoskeleton via the Rho1/Pkc1/MAPK cell integrity cascade. Substrates of TORC2 and how TORC2 regulates the cell integrity pathway are not well understood. Screening for multicopy suppressors of tor2, we obtained a plasmid expressing an N-terminally truncated Ypk2 protein kinase. This truncation appears to partially disrupt an autoinhibitory domain in Ypk2, and a point mutation in this region (Ypk2D239A) conferred upon full-length Ypk2 the ability to rescue growth of cells compromised in TORC2, but not TORC1, function. YPK2D239A also suppressed the lethality of tor2Δ cells, suggesting that Ypks play an essential role in TORC2 signaling. Ypk2 is phosphorylated directly by Tor2 in vitro, and Ypk2 activity is largely reduced in tor2Δ cells. In contrast, Ypk2D239A has increased and TOR2-independent activity in vivo. Thus, we propose that Ypk protein kinases are direct and essential targets of TORC2, coupling TORC2 to the cell integrity cascade.