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. Author manuscript; available in PMC: 2016 Mar 4.
Published in final edited form as: Cell Rep. 2015 Jul 30;12(6):1006–1018. doi: 10.1016/j.celrep.2015.07.004

Figure 3. Tyrosine Phosphorylation of hAha1 Is Essential for Binding to Hsp90.

Figure 3

(A) HEK293 cells were transiently transfected with empty plasmid (C), WT hAha1-FLAG (WT), non-phosphorylatable Y223F, or phosphomimetic Y223E mutants. The hAha1-FLAG protein was immunoprecipitated (IP) and co-immunoprecipitating (co-IP) of hHsp90 (including α and β isoforms) was examined by immunoblotting.

(B) HEK293 cells transiently expressing hAha1-FLAG (WT) or the non-phosphorylatable Y223F mutant were treated with 20 μM DPH for 6 hr prior to lysis. The hAha1-FLAG proteins were immunoprecipitated (IP) and co-immunoprecipitating (co-IP) hHsp90 was detected by immunoblotting.

(C) The c-Abl+/+ and c-Abl deficient c-Abl−/− MEF cells were transiently transfected with hAha1-FLAG (WT) or the non-phosphorylatable Y223F mutant. The immunoprecipitation of hAha1-FLAG proteins and co-immunoprecipitating (co-IP) of hHsp90 were detected by immunoblotting.

(D) Co-immunoprecipitating (co-IP) of PP5 and Hsp70 in (A) were examined by immunoblotting.

(E) In vitro ATPase activity of hHsp90α-HA isolated from PC3 cells. The reaction was also performed in the presence of 10 μM ganetespib. All the data represent mean ± SD (**p < 0.005).

(F) The ATPase activity was stimulated by hAha1-FLAG (WT) or the phospho mutants Y223E and Y223F isolated from HEK293 cells. All the data represent mean ± SD (**p < 0.005).

(G) Indicated hAha1-FLAG constructs were transfected in HEK293 cells. The hAha1 was immunoprecipitated (IP) with anti-FLAG agarose; co-immunoprecipitating (co-IP) c-Abl and phospho-Tyr89, Raf-1, c-Src, Wee1, Ulk1, and Cdk4 proteins were detected by immunoblotting.

(H) HEK293 cells were co-transfected with CFTR and hAha1-FLAG (WT) and Y223F and Y223E mutants. After 24 hr, CFTR, hAha1-FLAG, and hHsp90 were detected by immunobloting.

(I) HEK293 cells transiently transfected with hAha1-FLAG, Y223F, and Y223E for 24 hr and then treated with 10 μM dexamethasone for 1 hr (time 0). The cells were then washed and incubated in dexamethasone free media. The GR activity was monitored by western blot analysis using anti-phospho-Ser211-GR and total GR antibodies.