FIGURE 3:

Hsp70 promotes ubiquitylation of kinases for their degradation. (A) Ubiquitylation of Ste11ΔN^K444R was assayed in SSA1 and ssa1-45 cells at permissive (30°C) and nonpermissive (37°C) temperatures. MG-132 (100 μM) was added for 2 h to block the proteasome. Ste11ΔN^K444R was immunoprecipitated from these cells with anti-His antibody. Ubiquitylated Ste11ΔN^K444R was visualized by Western blotting followed by immunostaining with antiubiquitin antibody. Five microliters of immunoprecipitated Ste11ΔN^K444R from each sample was loaded onto an SDS–PAGE, and the kinase level was identified by Western blotting probed with anti-His antibody. Vector control (NT) was used to visualize any nonspecific ubiquitination. Ubiquitinated Ste11ΔN^K444R was quantified and represented in the graph. (n = 3, bar = ±SE). (B) Ubiquitination of Ste11ΔN^K444R kinase was checked after WT yeast cells were treated with the proline analogue AZC (50 mM) alone or in combination with myricetin (100 μM) or PES (150 μM) for 2 h. In this case, Myc-tag ubiquitin was expressed from the CUP promoter after induction with 100 μM of copper. The Ste11ΔN^K444R was immunoprecipitated, and polyubiquitylation of kinase was identified by the anti-myc antibody. AZC-induced Ste11ΔN^K444R ubiquitylation was checked in the ubiquitin ligases of ubr1Δsan1Δ knockout yeast cells. GA (50 μM)-induced Ste11ΔN^K444R ubiquitylation in the presence of MG132 (100 μM) was used as a control. The polyubiquitylation of Ste11ΔN^K444R was quantified and represented in the associated graph (n = 3, bar = ± SE). * indicates the significance of p ≤ 0.05. (C) Western blotting with anti-myc antibody showed the same level of myc-ub expression in the yeast cell lysates used for Ste11ΔN^K444R ubiquination in B. Vector control (NT) was used for nonspecific interaction with the antibody. Pgk1 served as a loading control.