1. Volume 41, Issue 6, Pages 672-681 (March 2011)
Threonine 22 Phosphorylation Attenuates Hsp90 Interaction with Cochaperones and Affects Its Chaperone Activity 
Mehdi Mollapour, Shinji Tsutsumi, Andrew W. Truman, Wanping Xu, Cara K. Vaughan, Kristin Beebe, Anna Konstantinova, Srinivas Vourganti, Barry Panaretou, Peter W. Piper, Jane B. Trepel, Chrisostomos Prodromou, Laurence H. Pearl, Len Neckers 
Molecular Cell 
Volume 41, Issue 6, Pages (March 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 CK2 Phosphorylates yHsp90-T22 In Vitro and In Vivo
(A) In vitro CK2-dependent threonine phosphorylation of full-length and N-domain wild-type (wt) and T22A yHsp90.
(B) Serine phosphorylation of p50-Cdc37-GFP chimera in yeast expressing either wild-type or ck2-ts mutants. Phosphorylation of S13 was detected by antiphospho-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 wild-type and T22A yHsp90 threonine phosphorylation was detected in wild-type (CK2) and ck2-ts yeast at 25°C and 34°C.
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3. Figure 2 Chaperone Function of T22 Phospho-Mutants
(A) ATPase activity of purified wild-type yHsp90, T22A, and T22E mutants. Error bars represent standard deviation of three independent experiments. The KD of each protein for AMPPNP is shown in the inset. The ATPase activity of wild-type yHsp90 (Kcat = 0.94/min ± 0.03) is set to 100%.
(B) Aha1 (20 μM) stimulates ATPase activity of purified wild-type, 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 wild-type, T22A, and T22E yHsp90 proteins was determined after crosslinking and polyacrylamide gel electrophoresis. N-domain dimerized species run with an apparent molecular weight of ∼190 kDa.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Chaperone Function of T22 Phospho-Mutants in Yeast
(A) Yeast expressing wild-type, T22A, or T22E yHsp90 were transformed with v-SRC, and total cellular phosphotyrosine and 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 wild-type, 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 wild-type activity and are depicted as mean ± standard deviation derived from four independent experiments. Lysates from yeast expressing His-tagged wild-type, T22A, or T22E yHsp90 were precipitated by Ni-NTA and associating GR was detected by immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Chaperone Function of T36 Phospho-Mutants in Mammalian Cells
(A) NIH 3T3 cells stably expressing v-Src were transfected with empty vector pcDNA3 (c), FLAG-tagged wild-type hHsp90α (wt), hHsp90α-T36A (T36A), and hHsp90α-T36E (T36E) mutants. After v-Src immunoprecipitation (IP) FLAG-hHsp90α interaction was detected by immunoblotting.
(B) Empty vector pcDNA3 (c), FLAG-tagged wild-type hHsp90α (wt), hHsp90α-T36A (T36A), and hHsp90α-T36E (T36E) mutants were expressed in COS7 cells. After 24 hr, interaction of endogenous Raf-1, ErbB2, and CDK4 with FLAG IPs was monitored by immunoblotting.
(C) COS7 cells were transfected with GR and indicated FLAG-hHsp90α constructs. After 24 hr, lysates were immunoprecipitated with FLAG antibody-conjugated agarose; coprecipitating GR was detected by immunoblotting.
(D) HEK293 cells were transfected with CFTR and indicated FLAG-hHsp90α constructs. After 24 hr CFTR and FLAG-hHsp90α were detected by immunoblotting; α-tubulin was used as loading control.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Reduced Interaction of Aha1 and Cdc37p50 Cochaperones with T22 and T36 Phospho-Mutants
(A) Lysates from yeast expressing wild-type, T22A, or T22E yHsp90 were precipitated by Ni-NTA and associating Cdc37p50.
(B) yAha1 was detected by immunoblotting.
(C) COS7 cells were transfected with indicated FLAG-hHsp90α constructs. After FLAG-hHsp90α IP, associated p50Cdc37 was detected by immunoblotting.
(D) hAha1 was 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 Cdc37p50 and yAha1-FLAG was examined by immunoblotting.
(F) COS7 cells were cotransfected as in (C) and with either an empty plasmid or with hAha1 plasmid. FLAG-hHsp90α was immunoprecipitated and associated p50Cdc37 and hAha1 were detected by immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Aha1 Overexpression Compensates for Defective Chaperone Function of yHsp90-T22 Phospho-Mutants
(A) Yeast expressing wild-type, 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 wild-type, T22A, or T22E yHsp90 and 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 wild-type yHsp90-His6, yHsp90-T22A, or yHsp90-T22E with (+) or without (–) yAha1-FLAG. Cells were grown to mid-log phase and then stressed with 8 mM caffeine for 4 hr. Data are depicted as mean ± standard deviation derived from three independent experiments. yAha1-FLAG was visualized by immunoblotting; α-tubulin was used as loading control.
(D) GR activity was measured in yeast cells expressing wild-type, T22A, or T22E yHsp90. Cells were transformed with either yAha1-FLAG or empty plasmid as shown. Data are displayed as percent of wild-type activity. Bars represent mean ±standard deviation derived from four independent experiments. Lysates from yeast expressing wild-type, T22A, or T22E yHsp90 were precipitated by Ni-NTA and associating GR was detected by immunoblotting. yAha1 was detected by anti-FLAG antibody; 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 hr, CFTR, FLAG-hHsp90α, and hAha1 were detected by immunoblotting; α-tubulin was used as loading control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions