Figure 3.

TORC2 phosphorylates the novel C-terminal sites. (A) WT (BY4741) or otherwise isogenic tor2^ts cells expressing Ypk1^5A-myc (pFR246) were grown at 26° to midexponential phase and then either kept at 26° or shifted to 37° for 2 hr, harvested, lysed, and samples of the resulting extracts resolved by Phos-tag SDS-PAGE and analyzed by immunoblotting with anti-myc mAb 9E10. (B) TOR1-1 avo3^∆CT cells (left) expressing Ypk1^5A-myc (pFR246) were grown to midexponential phase, treated for 20 min with either vehicle alone (Tween 20:ethanol, 10:90) or 200 nM rapamycin in the same solvent as indicated, collected, and analyzed as in (A). WT cells (BY4741) expressing Ypk1^5A-myc (pFR246) (right) were treated in the same manner in a separate experiment. (C) Strain yAM135-A (ypk1^as ypk2∆) expressing Ypk1^5A-myc (pFR246) was grown to midexponential phase and then treated with either vehicle alone (DMSO) or 10 µM 3-MB-PP1 for 1 hr, harvested, and then analyzed as in (A). (D) WT (Mock IP) (yKL4) and Avo3-3C-3XFLAG (TORC2 IP) (yNM695) strains were grown in YPD to midexponential phase, harvested, lysed, and TORC2 immunoprecipitated from the resulting extracts using anti-FLAG antibody-coupled agarose resin. Immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting with anti-Avo3 and anti-Tor2 antibodies. (E) Mock and TORC2 preparations, as in (D), were incubated with [γ-³²P]ATP, either alone or in the presence of purified analog-sensitive Ypk1^as, in either the absence or presence of the TORC2 inhibitor NVP-BEZ235, as indicated. Reaction products were resolved by SDS-PAGE and analyzed by Coomassie staining and, after drying the gel, by autoradiography.