Figure 1.

MAPK Slt2 phosphorylates TORC2 subunits Avo2 and Avo3. (A) The primary structure of each indicated TORC2 subunit is depicted schematically, with domains labeled as in Gaubitz et al. (2016). (Black asterisk) An -SP- or -TP- site; (red asterisk) an -SP- and -TP- detectably phosphorylated in vivo in various phosphoproteomic analyses, as cataloged in the Saccharomyces Genome Database (http://www.yeastgenome.org). (B) Wild-type cells (JTY5336) carrying both a CEN plasmid expressing Avo2-3xFlag (pKL1) from the AVO2 promoter and a multicopy (2 µm DNA) vector expressing either Pkc1* (pJT5660) or catalytically inactive Pkc1*KD (pJEN12) from the GAL1 promoter were cultured to mid-exponential phase in selective minimal medium containing 2% raffinose and 0.2% sucrose. Expression of Pkc1* or Pkc1*KD was induced by addition of galactose (2% final concentration). Cell samples were removed at the indicated times and lysed, the proteins in the resulting extracts were resolved by Phos-tag SDS-PAGE, and the indicated proteins were analyzed by immunoblotting with the appropriate antibodies, all as described in the Materials and Methods. Pgk1 was the loading control. (C) As in B except the cells carried plasmids expressing either Pkc1*-myc (pAEA376) or Pkc1*KD-myc (pJEN13), and the extracts were resolved by standard SDS-PAGE prior to immunoblotting. (D) Wild-type cells (JTY5336) or otherwise isogenic slt2Δ (YFR549) or hog1Δ (YFR538-A) derivatives carrying plasmids expressing either Avo2-3xFlag (pKL1) or Avo2^9A-3xFlag (pKL2), as indicated, as well as either empty vector (yEPlac112) or the same vector expressing Pkc1* from the GAL1 promoter (pJT5660), as shown, were grown to mid-exponential phase in selective minimal medium containing 2% raffinose and 0.2% sucrose. After addition of galactose (2% final concentration), the cells were cultured for 3 h, harvested, and lysed, and the indicated proteins were analyzed as in B. (E) Activated wild-type Slt2 (pKL63) and kinase-dead (KD) Slt2 (pKL64) were purified from Saccharomyces cerevisiae as described in the Materials and Methods and incubated with [γ-³²P]ATP and either GST-Avo2^WT (pKL16) or GST-Avo2^9A (pKL17) purified from Escherichia coli as described in the Materials and Methods. Reaction products were resolved by SDS-PAGE and analyzed by Coomassie blue staining (left) and autoradiography (right). (F) As in E, except the substrates were purified recombinant GST-Avo3(1-100) (pKL81) or GST-Avo3(1-100)^6A (pKL82). (G) A strain (YFR617) expressing Avo3-3xFlag and carrying a plasmid expressing either Pkc1*KD (pJEN12) or Pkc1* (pJT5660) from the GAL1 promoter was grown, induced with galactose for 2.5 h, lysed, and analyzed by Phos-tag SDS-PAGE as in B.