A) In vitro CK2-dependent threonine phosphorylation of full-length and N-domain WT and T22A yHsp90.
B) Serine phosphorylation of p50-Cdc37-GFP chimera in yeast expressing either WT or ck2-ts mutant. Phosphorylation of S13 was detected by anti-phospho-Ser13 antibody and p50-Cdc37-GFP was visualized with anti-GFP antibody (See Figure S1).
C) Schematic diagram of temperature-dependent CK2 enzyme inactivation in ck2-ts yeast mutant.
D) CK2 phosphorylates yHsp90-T22 in vivo. N-domain WT and T22A yHsp90 threonine phosphorylation was detected in WT (CK2) and ck2-ts yeast at 25°C and 34°C.

A) ATPase activity of purified WT yHsp90, T22A and T22E mutants. Error bars represent standard deviation of three independent experiments. Kd of each protein for AMPPNP is shown in inset. The ATPase activity of WT yHsp90 (K_cat = 0.94 min⁻¹ +/− 0.03) is set to 100%.
B) Aha1 (20 μM) stimulates ATPase activity of purified WT, T22A and T22E yHsp90. Error bars represent standard deviation of three independent experiments. Data are expressed as fold increase in Hsp90 ATPase activity in the presence of Aha1 compared to activity of the same Hsp90 protein in the absence of Aha1.
C) AMPPNP (PNP)-stabilized N-domain dimerization of WT, T22A, and T22E yHsp90 proteins was determined after crosslinking and polyacrylamide gel electrophoresis. N-domain dimerized species run with an apparent molecular weight of approximately 190 kDa.

A) Yeast expressing WT, T22A, or T22E yHsp90 were transformed with v-SRC, and total cellular phosphotyrosine & v-Src expression were analyzed by immunoblotting; α-tubulin was used as loading control.
B) Growth of the same strains on glucose- or galactose-containing media.
C) Yeast expressing WT, T22A, or T22E yHsp90, and containing Ste11ΔN-His₆, were grown on glucose- or galactose-containing media. Ste11ΔN-His₆ expression was detected by immunoblotting; α-tubulin was used as loading control.
D) GR-lacZ activity was assessed in the same strains. Data are shown as percentage of WT activity, and are depicted as mean +/− standard deviation derived from four independent experiments. Lysates from yeast expressing His-tagged WT, T22A, or T22E yHsp90 were precipitated by Ni-NTA, and associating GR was detected by immunoblotting.

A) NIH 3T3 cells stably expressing v-Src were transfected with empty vector pcDNA3 (c), FLAG-tagged WT hHsp90α (wt), hHsp90α-T36A (T36A), and hHsp90α-T36E (T36E) mutants. After v-Src IP, FLAG-hHsp90α interaction was detected by immunoblotting.
B) Empty vector pcDNA3 (c), FLAG-tagged WT hHsp90α (wt), hHsp90α-T36A (T36A), and hHsp90α-T36E (T36E) mutants were expressed in COS7 cells. After 24 hrs, interaction of endogenous Raf-1, ErbB2 and CDK4 with FLAG IPs were monitored by immunoblotting.
C) COS7 cells were transfected with GR and indicated FLAG-hHsp90α constructs. After 24 hrs, lysates were immunoprecipitated with FLAG antibody-conjugated agarose; co-precipitating GR was detected by immunoblotting.
D) HEK293 cells were transfected with CFTR and indicated FLAG-hHsp90α constructs. After 24 hrs CFTR and FLAG-hHsp90α were detected by immunoblotting. α-tubulin was used as loading control.

(A) Lysates from yeast expressing WT, T22A, or T22E yHsp90 were precipitated by Ni-NTA, and associating Cdc37^p50 and;
(B) yAha1 were detected by immunoblotting.
(C) COS7 cells were transfected with indicated FLAG-hHsp90α constructs. After FLAG-hHsp90α IP, associated p50^Cdc37 and;
(D) hAha1 were detected by immunoblotting.
(E) The yeast strains used above were transformed with either yAha1-FLAG or empty plasmid. Interaction of T22A and T22E yHsp90 with Cdc37^p50 and yAha1-FLAG was examined by immunoblotting.
(F) COS7 cells were co-transfected as in (C), and also with either an empty plasmid or with hAha1 plasmid. FLAG-hHsp90α was immunoprecipitated and associated p50^Cdc37 and hAha1 was detected by immunoblotting.

A) Yeast expressing WT, T22A, or T22E yHsp90 were transformed with v-SRC and with either yAha1-FLAG or empty plasmid as shown. Total cellular phosphotyrosine and v-Src expression were analyzed by immunoblotting; α-tubulin was used as loading control.
B) Yeast cells expressing WT, T22A, or T22E yHsp90, and also Ste11ΔN-His₆, were transformed with either yAha1-FLAG or empty plasmid as shown. Ste11ΔN-His₆ expression was detected by immunoblotting; α-tubulin was used as loading control.
C) YIL117c-LacZ activity was measured in yeast expressing WT yHsp90-His₆, yHsp90-T22A, or yHsp90-T22E, with (+) or without (−) yAha1-FLAG. Cells were grown to mid-log and then stressed with 8 mM caffeine for 4 h. Data are depicted as mean +/− standard deviation derived from three independent experiments. yAha1-FLAG was visualized by immunoblotting and α-tubulin was used as loading control.
D) GR activity was measured in yeast cells expressing WT, T22A, or T22E yHsp90. Cells were transformed with either yAha1-FLAG or empty plasmid as shown. Data are displayed as % of WT activity. Bars represent mean +/− standard deviation derived from four independent experiments. Lysates from yeast expressing WT, T22A, or T22E yHsp90 were precipitated by Ni-NTA, and associating GR was detected by immunoblotting. yAha1 was detected by anti-FLAG antibody and α-tubulin was used as loading control.
E) HEK293 cells were transfected with wild type CFTR, the indicated FLAG-hHsp90α constructs, and either an empty plasmid or hAha1 plasmid. After 24 hrs, CFTR, FLAG-hHsp90α and hAha1 were detected by immunoblotting. α-tubulin was used as loading control.