Figure 2
Phosphorylation of uS7 by Ypk1. (A) uS7 phosphorylation in vitro by Ypk1. Logarithmically growing yeast cells were harvested and Ypk1 was immunoprecipitated by anti-Ypk1 and Sepharose-conjugated protein G. GST-conjugated uS7 was expressed in E. coli and purified by glutathione-Sepharose. Ypk1 containing resin was suspended with uS7 and incubated with [γ-32P]-ATP as described in experimental procedures. Post-incubation samples were separated on SDS-PAGE gel and radioactivity was measured by exposing to an image plate for BAS-2500. Mean relative kinase activity was plotted from triplicate experiments with SD error. Statistical significance was assessed by Student’s t-test. The triple asterisk denotes that p is less than 0.001. To examine the background level of assay, lysate prepared from ypk1∆ strain with empty vector was used. (B) Schematic presentation of uS7 phosphorylation site candidates. Open reading frame of uS7 is depicted as a rectangle. Potential serine or threonine residues identified by NetPhosYeast 2.0 software are expressed as vertical lines. (C) Alanine mutation of uS7. A series of alanine mutations were introduced to phosphorylation candidate sites. Mutant uS7s were expressed from plasmid vector pRS415 with its own promoter whereas endogenous uS7 was turned off utilizing Tet-OFF with doxycycline. Serial dilutions of indicated yeast cells were spotted onto SD plate and incubated at 30 °C with or without 10 μg/mL doxycycline (Dox). (D) Involvement of Pkc1 but Ypk2 in uS7 phosphorylation. In vitro phosphorylation assay was carried out as Fig. 2A except Ypk2 and Pkc1 was also utilized as a kinase source and S223A (uS7-S223A) mutant was also utilized as substrate. Mean relative kinase activity was plotted from triplicate experiments with SD error. Statistical significance was assessed by Student’s t-test. The single asterisk denotes that p is less than 0.05.
