FIG. 3.

Tor2 directly phosphorylates Ypk2 in vitro. A. Detection of immunoprecipitated HA-tagged Tor2 (^HATOR2). ^HATor2^{wild type} (WT; lanes 1 and 2) or ^HATor2^D2298E (kinase dead mutant [KD]; lanes 3 and 4) were immunoprecipitated in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of anti-HA antibody. Immunocomplexes were subjected to 7% SDS-PAGE gels, and ^HATor2 protein was detected by immunoblotting with anti-HA antibody. B. In vitro Tor2 kinase assay. Immunoprecipitated ^HATor2 (wild type [WT] or kinase dead [KD]) was incubated with [γ-³²P]ATP and 4E-BP (lanes 1 to 4 and lanes 9 to 12) or recombinant Ypk2 (lanes 5 to 8 and lanes 13 to 16) at 30°C for 60 min and stopped with sample buffer, and proteins were separated by SDS-PAGE. The results of Coomassie staining (left) and autoradiography (kinase assay) (right) are shown. * and **, migration of heavy and light chains of the immunoprecipitation antibody. C. Tor2 phosphorylates Ypk2 at the turn motif and the hydrophobic motifs. In vitro kinase assays were carried out using the wild type (WT; lanes 1, 2, and 4) and the turn- and hydrophobic-motif mutants (S641A T659A, S641A, and T659A; lanes 3, 5, and 6, respectively) of Ypk2.