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• W1708752746 abstract "•c-Abl phosphorylates the co-chaperone Aha1•c-Abl phosphorylation of Aha1 tyrosine 223 promotes association with Hsp90•Tyrosine phosphorylation of Aha1 leads to increased Hsp90 ATPase activity•Pharmacological inhibition of c-Abl sensitizes cancer cells to Hsp90 inhibitors The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo. The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo. The essential eukaryotic molecular chaperone Heat Shock Protein 90 (Hsp90) is involved in folding and stability of target proteins, also referred to as “clients” (Röhl et al., 2013Röhl A. Rohrberg J. Buchner J. The chaperone Hsp90: changing partners for demanding clients.Trends Biochem. Sci. 2013; 38: 253-262Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Taipale et al., 2010Taipale M. Jarosz D.F. Lindquist S. HSP90 at the hub of protein homeostasis: emerging mechanistic insights.Nat. Rev. Mol. Cell Biol. 2010; 11: 515-528Crossref PubMed Scopus (1292) Google Scholar). Hsp90 has approximately 200 clients (listed at http://www.picard.ch/downloads/Hsp90interactors.pdf). They are broadly classified as kinase clients, such as ErbB2, c-Met, and CDK4 and non-kinase clients including heat shock factor, steroid hormone receptors, and cystic fibrosis transmembrane conductance regulator (CFTR). The majority of the kinase clients are involved in oncogenesis, therefore Hsp90 is recognized as a facilitator of “oncogene addiction” (Neckers and Workman, 2012Neckers L. Workman P. Hsp90 molecular chaperone inhibitors: are we there yet?.Clin. Cancer Res. 2012; 18: 64-76Crossref PubMed Scopus (747) Google Scholar). The Hsp90 structure consists of homodimer molecules with N-, middle, and C-domains. ATP binding to the N-domain and its subsequent hydrolysis are linked to Hsp90 chaperone function (Obermann et al., 1998Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (492) Google Scholar, Panaretou et al., 1998Panaretou B. Prodromou C. Roe S.M. O’Brien R. Ladbury J.E. Piper P.W. Pearl L.H. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (625) Google Scholar). Nucleotide binding and Hsp90 ATPase activity confer different conformational states that allow clients to bind and dissociate from Hsp90 (Hessling et al., 2009Hessling M. Richter K. Buchner J. Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90.Nat. Struct. Mol. Biol. 2009; 16: 287-293Crossref PubMed Scopus (254) Google Scholar, Mickler et al., 2009Mickler M. Hessling M. Ratzke C. Buchner J. Hugel T. The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis.Nat. Struct. Mol. Biol. 2009; 16: 281-286Crossref PubMed Scopus (194) Google Scholar). The chaperone activity of Hsp90 is tightly regulated by co-chaperones and posttranslational modifications (PTMs) (Cox and Johnson, 2011Cox M.B. Johnson J.L. The role of p23, Hop, immunophilins, and other co-chaperones in regulating Hsp90 function.Methods Mol. Biol. 2011; 787: 45-66Crossref PubMed Scopus (24) Google Scholar, Walton-Diaz et al., 2013Walton-Diaz A. Khan S. Bourboulia D. Trepel J.B. Neckers L. Mollapour M. Contributions of co-chaperones and post-translational modifications towards Hsp90 drug sensitivity.Future Med. Chem. 2013; 5: 1059-1071Crossref PubMed Scopus (49) Google Scholar). Co-chaperones are groups of proteins that interact with distinct conformations of Hsp90, regulating chaperone function by either accelerating or decelerating the ATPase activity or simply acting as scaffolds between Hsp90 and its clients. Our work and studies by other groups have shown that PTMs of Hsp90 can impact its interaction with co-chaperones. The evolutionarily conserved co-chaperone Aha1 is the activator of the Hsp90 ATPase activity (Panaretou et al., 1998Panaretou B. Prodromou C. Roe S.M. O’Brien R. Ladbury J.E. Piper P.W. Pearl L.H. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (625) Google Scholar). It is also the most common co-chaperone whose interaction is affected by phosphorylation, acetylation, and SUMOylation of Hsp90 (Mollapour et al., 2011Mollapour M. Tsutsumi S. Truman A.W. Xu W. Vaughan C.K. Beebe K. Konstantinova A. Vourganti S. Panaretou B. Piper P.W. et al.Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity.Mol. Cell. 2011; 41: 672-681Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, Mollapour et al., 2014Mollapour M. Bourboulia D. Beebe K. Woodford M.R. Polier S. Hoang A. Chelluri R. Li Y. Guo A. Lee M.J. et al.Asymmetric Hsp90 N domain SUMOylation recruits Aha1 and ATP-competitive inhibitors.Mol. Cell. 2014; 53: 317-329Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, Xu et al., 2012Xu W. Mollapour M. Prodromou C. Wang S. Scroggins B.T. Palchick Z. Beebe K. Siderius M. Lee M.J. Couvillon A. et al.Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.Mol. Cell. 2012; 47: 434-443Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). A major gap in our knowledge is how intracellular signals to the co-chaperone Aha1 dictate its interaction with Hsp90. Our study demonstrates that c-Abl tyrosine kinase phosphorylates a single tyrosine residue, Y223, in human Aha1 (hAha1). This, in turn, appears to promote its association with human Hsp90α (hHsp90α) and modify chaperoning of kinase clients, heat shock factor, glucocorticoid receptor (GR), and CFTR. Tyrosine phosphorylation of hAha1 is also a pre-requisite for its ubiquitination and degradation in the proteasome. Hsp90 chaperone function can be inhibited by small molecules that bind to the N-domain ATP-binding pocket, precluding ATP binding and hydrolysis. There are 16 different Hsp90 inhibitors that are currently undergoing clinical evaluation in cancer patients (Neckers and Workman, 2012Neckers L. Workman P. Hsp90 molecular chaperone inhibitors: are we there yet?.Clin. Cancer Res. 2012; 18: 64-76Crossref PubMed Scopus (747) Google Scholar). Co-chaperones and PTMs can affect the efficacy of Hsp90 inhibitors (Walton-Diaz et al., 2013Walton-Diaz A. Khan S. Bourboulia D. Trepel J.B. Neckers L. Mollapour M. Contributions of co-chaperones and post-translational modifications towards Hsp90 drug sensitivity.Future Med. Chem. 2013; 5: 1059-1071Crossref PubMed Scopus (49) Google Scholar). Here, we report that the pharmacologic inhibition of c-Abl prevents hAha1 interaction with hHsp90α and hypersensitizes renal cell carcinoma (RCC) to Hsp90 inhibitors in vitro and ex vivo. Hsp90 and the majority of its co-chaperones are phospho-proteins (Walton-Diaz et al., 2013Walton-Diaz A. Khan S. Bourboulia D. Trepel J.B. Neckers L. Mollapour M. Contributions of co-chaperones and post-translational modifications towards Hsp90 drug sensitivity.Future Med. Chem. 2013; 5: 1059-1071Crossref PubMed Scopus (49) Google Scholar). To determine whether Aha1 is subject to tyrosine phosphorylation, hAha1-FLAG was transiently expressed in HEK293 cells and by using a pan-anti-phospho-tyrosine antibody (4G10), we readily detected the tyrosine phosphorylation of hAha1 (Figure 1A). hAha1 has seven tyrosine residues (Figure 1B), which were individually mutated to non-phosphorylatable phenylalanine and transiently expressed in HEK293 cells. Individual mutation of Y81, Y99, Y223, and Y333 to phenylalanine significantly reduced the tyrosine phosphorylation of hAha1 (Figure S1A). We identified Y223 within the c-Abl recognition motif I/V/L-YXXP/F (Ubersax and Ferrell, 2007Ubersax J.A. Ferrell Jr., J.E. Mechanisms of specificity in protein phosphorylation.Nat. Rev. Mol. Cell Biol. 2007; 8: 530-541Crossref PubMed Scopus (1000) Google Scholar) (Figure 1B). Therefore, we bacterially expressed and purified N-terminally His6-tagged hAha1, as well as the seven individual non-phosphorylatable hAha1 mutants. These purified proteins were bound to Ni-NTA agarose and used as substrates in an in vitro kinase assay, which included pure and active c-Abl-glutatione S-transferase (GST). Under these conditions, c-Abl-GST efficiently phosphorylated hAha1 and individual non-phosphorylatable tyrosine mutants except for the Y223F (Figure 1C). These results provide strong evidence that c-Abl directly phosphorylates Y223-hAha1, and this is the only tyrosine residue in hAha1 that is targeted by c-Abl (Figure 1C). Further evidence for this process, in vivo, was obtained by transiently co-transfecting HEK293 cells with c-Abl plasmid and either with FLAG-tagged hAha1 or non-phosphorylatable Y223F mutant. Overexpression of c-Abl increased the tyrosine phosphorylation of hAha1. However, this increase in tyrosine phosphorylation is abolished with hAha1-Y223F mutant (Figure 1D). We also performed similar experiments with 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin, 5-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,4-imidazolidinedione (DPH), which binds to the myristoyl binding site of c-Abl and leads to activation of its kinase activity (Yang et al., 2011Yang J. Campobasso N. Biju M.P. Fisher K. Pan X.Q. Cottom J. Galbraith S. Ho T. Zhang H. Hong X. et al.Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site.Chem. Biol. 2011; 18: 177-186Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). HEK293 cells transiently expressing wild-type hAha1-FLAG or Y223F mutant were treated with 20 μM DPH showed an increase in tyrosine phosphorylation of hAha1, but not of the Y223F mutant (Figure 1E). We further confirmed this data by transiently expressing FLAG-hAha1 and Y223F mutant in a c-Abl deficient (c-Abl−/−) murine embryo fibroblasts (MEF) cell line and the wild-type MEF cell line (c-Abl+/+) (Figure 1F). Tyrosine phosphorylation of hAha1 was significantly reduced in c-Abl deficient MEF cells (Figure 1F). This reduction was at the same level of Y223F mutant expressed either in the c-Abl+/+ or c-Abl−/− MEF cells (Figure 1F). With the exception of the Wee1 tyrosine kinase, the yeast Saccharomyces cerevisiae does not have a bona fide tyrosine kinase offering a null background for the expression of mammalian tyrosine kinase c-Abl. We expressed FLAG-tagged hAha1 and the Y223F mutant in wild-type yeast strain W303; expression of these alleles was under the control of the native promoter of yeast AHA1 (yAHA1). We also co-expressed full-length c-Abl, controlled by the galactose inducible promoter of GAL1. Tyrosine phosphorylation of hAha1 was not observed in wild-type yeast (Figure 1G). However, after inducing c-ABL1 expression by growing the cells on galactose media, we detected tyrosine phosphorylation of wild-type hAha1. The non-phosphorylatable Y223F mutant remained unmodified (Figure 1G). Taken together, our data provide strong evidence that c-Abl targets and phosphorylates hAha1-Y223. Subsequently, we investigated whether phosphorylation of the hAha1-Y223 occurred before or after binding to hHsp90. The presence of endogenous hHsp90 and lack of efficient knockdown of hHsp90 precluded addressing this issue in mammalian cell lines. Therefore, we utilized our in vitro kinase assay to explore the dynamics of hAha1-Y223 phosphorylation. Bacterially expressed and purified FLAG-hAha1 was immobilized on anti-FLAG M2 affinity gel. As shown earlier, (Figure 1C), purified and active c-Abl-GST phosphorylates hAha1 (Figure 1H). This was detected using a pan-anti-phosphotyrosine antibody. Immunoprecipitation of FLAG-hAha1 did not co-immunoprecipitate c-Abl-GST, suggesting that the interaction between hAha1 and c-Abl is transient (Figure 1H). We carried out a similar in vitro kinase assay, modified with the addition of hHsp90α, initially, followed by c-Abl. This promoted a hAha1-c-Abl-hHsp90α complex formation, but did not increase the tyrosine phosphorylation of hAha1 (Figure 1H). Addition of calf-intestinal alkaline phosphatase (CIAP) caused dephosphorylation of hAha1 and de-stabilized the hAha1-c-Abl-Hsp90α complex (Figure 1H, last lane). Also, c-Abl does not phosphorylate hHsp90α (Figure S1B). Taken together, our data suggest that c-Abl has the ability to phosphorylate hAha1-Y223 even in the absence of hHsp90α. This fosters formation of a hAha1-c-Abl-hHsp90α complex. Our data suggest a model where tyrosine phosphorylation of hAha1 promotes interaction, while a possible dephosphorylation disrupts interaction with hHsp90. However, we sought to examine whether phosphorylation of hAha1 serves any additional functions. First, we characterized the intracellular distribution of hAha1 as the result of phosphorylation of Y223. HEK293 cells were transiently co-transfected with hHsp90α-HA, wild-type hAha1-FLAG, and non-phosphorylatable Y223F or phosphomimetic Y223E mutants. We performed immunofluorescence microscopy, staining the cells with anti-FLAG antibody and co-staining with anti-HA antibody and DAPI. There were 82% of cells expressing the hAha1-Y223F mutant that showed nuclear-cytoplasmic localization and 18% with cytoplasmic localization, whereas 100% of cells expressing the Y223E mutant displayed cytoplasmic localization (Figures 2A, 2B, and S2; Table S1). Protein phosphorylation often serves as a signal prompting ubiquitination and subsequent proteasome-mediated degradation of the substrate. Supporting this possibility, HEK293 cells transiently expressing the wild-type hAha1-FLAG or non-phosphorylatable hAha1-Y223F-FLAG mutant were treated with 5 μM proteasome inhibitor, MG132, for 6 hr (Figure 2C). hAha1-FLAG proteins were immunoprecipitated with anti-FLAG M2 affinity gel and then salt-stripped with 0.5 M NaCl prior to analysis by immunoblotting (Figure 2C). Ubiquitination of hAha1 was significantly decreased in hAha1-Y223F samples treated with MG132 (Figure 2C). This finding suggests that phosphorylation of Y223 is a prerequisite for ubiquitination and degradation of hAha1 in the proteasome. It is noteworthy that salt-stripping of our samples prevented the interaction of proteins with hAha1, and the efficacy of the salt-stripping procedure was confirmed by checking for the co-precipitation of hHsp90 (data not shown). Therefore, only ubiquitinated hAha1 is visualized in Figure 2B, as opposed to ubiquitinated proteins in complex with hAha1. Finally, the rate of hAha1 protein degradation was determined by cycloheximide (CHX) chase analysis. The non-phosphorylatable hAha1-Y223F mutant was markedly more stable compared to the wild-type hAha1 (Figure 2D). Conversely, the phosphomimetic Y223E mutant was highly unstable supporting a model where c-Abl mediated phosphorylation of Y223 leads to hAha1 ubiquitination and degradation in the proteasome. Aha1 interacts with a diverse range of proteins (Sun et al., 2015Sun L. Hartson S.D. Matts R.L. Identification of proteins associated with Aha1 in HeLa cells by quantitative proteomics.Biochim. Biophys. Acta. 2015; 1854: 365-380Crossref PubMed Scopus (10) Google Scholar). To better understand the effect of phosphorylation on the global interactions of hAha1, we applied a quantitative liquid chromatography-tandem mass spectrometry (LC–MS/MS) proteomics approach, comparing the interactomes of FLAG-hAha1-Y223E to FLAG-hAha1-Y223F (Figure S3A; Table S2). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001737. We identified 99 candidate partners of hAha1, 84% (83/99) of which demonstrated preferential binding to the phosphomimetic isoform of hAha1 (Figures S3B and S3C; Table S2). Significant enrichment of several Gene Ontology (GO) terms was observed, including metabolism, ribosomal components, and transcription/translation (Figure S3B). Interestingly, binding to major chaperones, including human Hsp70 (hHsp70) and hHsp90 was significantly increased upon hAha1 phosphorylation. hAha1 is known to interact and activate hHsp90 ATPase activity (Panaretou et al., 2002Panaretou B. Siligardi G. Meyer P. Maloney A. Sullivan J.K. Singh S. Millson S.H. Clarke P.A. Naaby-Hansen S. Stein R. et al.Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1.Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Phosphorylation of hHsp90 can affect its binding to hAha1 (Mollapour et al., 2011Mollapour M. Tsutsumi S. Truman A.W. Xu W. Vaughan C.K. Beebe K. Konstantinova A. Vourganti S. Panaretou B. Piper P.W. et al.Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity.Mol. Cell. 2011; 41: 672-681Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, Soroka et al., 2012Soroka J. Wandinger S.K. Mäusbacher N. Schreiber T. Richter K. Daub H. Buchner J. Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.Mol. Cell. 2012; 45: 517-528Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Xu et al., 2012Xu W. Mollapour M. Prodromou C. Wang S. Scroggins B.T. Palchick Z. Beebe K. Siderius M. Lee M.J. Couvillon A. et al.Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.Mol. Cell. 2012; 47: 434-443Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), therefore, we queried whether phosphorylation of hAha1 impacts binding to hHsp90. FLAG-tagged wild-type hAha1, non-phosphorylatable Y223F, and phosphomimetic Y223E mutants were transiently expressed in HEK293 cells. hAha1 proteins were isolated with anti-FLAG M2 affinity gel and interaction with hHsp90 and co-chaperones was examined by western blot analysis. In agreement with our proteomic study, the non-phosphorylatable hAha1-Y223F association with Hsp90 was completely abrogated (Figure 3A). Conversely, the interaction of hAha1-Y223E mutant with hHsp90α was significantly stronger than the wild-type hAha1 (Figure 3A). In order to directly demonstrate that the phosphorylation of hAha1 promotes its interaction with hHsp90, we treated HEK293 cells transiently expressing wild-type hAha1-FLAG or Y223F mutant with 20 μM DPH and then isolated these proteins by anti-FLAG M2 affinity gel. The hHsp90 was co-immunoprecipitated with hAha1-FLAG, and this interaction was enhanced in DPH treated cells (Figure 3B). However, we did observe hAha1-Y223F association with hHsp90 even in DPH treated cells (Figure 3B). Similar results were also obtained from c-Abl−/− MEF cells (Figure 3C), therefore providing convincing evidence that c-Abl mediated phosphorylation of hAha1 promotes its interaction with hHsp90. We further validated our proteomic data by observing hHsp70 interaction with the phosphomimetic hAha1 mutant Y223E, presumably mediated by hHsp90 (Figure 3D). We have previously shown that the PP5 co-chaperone is also found in complexes containing hAha1 and Hsp90 (Xu et al., 2012Xu W. Mollapour M. Prodromou C. Wang S. Scroggins B.T. Palchick Z. Beebe K. Siderius M. Lee M.J. Couvillon A. et al.Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.Mol. Cell. 2012; 47: 434-443Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The phosphomimetic hAha1-Y223E mutant moderately enhanced the amount of PP5 found in these complexes, in comparison to the amount of PP5 that was co-immunprecipitated with the wild-type hAha1 (Figure 3D). In contrast, PP5 is absent in complexes that contain the hAha1-Y223F mutant (Figure 3D). It is noteworthy that neither the wild-type hAha1 nor the phospho mutants were able to form a complex with the other co-chaperones (HOP, p23, CHIP, and Cdc37), (Figure S3C). ATPase activity of hHsp90 is essential for its chaperone function and hAha1 is a potent stimulator of this activity (Panaretou et al., 2002Panaretou B. Siligardi G. Meyer P. Maloney A. Sullivan J.K. Singh S. Millson S.H. Clarke P.A. Naaby-Hansen S. Stein R. et al.Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1.Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). We transiently expressed hHsp90α-HA in the prostate cancer PC3 cell line, wild-type hAha1-FLAG, and the Y223 phospho mutants in HEK293 cells. Proteins were immunoprecipitated, salt stripped, and competed off the HA or FLAG affinity beads with the relevant peptides (Figure S4A). The quality of the isolated proteins was examined by Coomassie staining of the SDS-PAGE (Figure S4B). These purified proteins were quantified and used at a ratio of 1:2, hHsp90α:hAha1 in the PiPer Phosphate Assay Kit (Thermo Fisher Scientific), (Experimental Procedures) in the presence of ATP as substrate as previously described (Kamal et al., 2003Kamal A. Thao L. Sensintaffar J. Zhang L. Boehm M.F. Fritz L.C. Burrows F.J. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors.Nature. 2003; 425: 407-410Crossref PubMed Scopus (1205) Google Scholar). We measured the ATPase activity of isolated hHsp90α in vitro as previously described (Kamal et al., 2003Kamal A. Thao L. Sensintaffar J. Zhang L. Boehm M.F. Fritz L.C. Burrows F.J. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors.Nature. 2003; 425: 407-410Crossref PubMed Scopus (1205) Google Scholar) (Figures 3E, S4C, and S4D). Addition of 10 μM ganetespib inhibited ATPase activity (Figures 3E and S4D). Addition of wild-type hAha1, comprised of phospho- and non-phosphorylated Y223, stimulated ATPase activity of the hHsp90α 5-fold higher than that of hHsp90α, alone, while hAha1-Y223F caused minimal increase in ATPase activity (1.5-fold increase) (Figures 3F and S4E). In contrast, the phosphomimetic hAha1-Y223E mutant stimulated hHsp90α ATPase by 8.5-fold (Figures 3F and S4E). ATP turnover was expressed as mmol Pi per mol per minute for hHsp90α alone and in the presence of hAha1 (wild-type and Y223 mutants), (0.84 = hHsp90α), (4.22 = hHsp90α + wild-type hAha1), (1.26 = hHsp90α + hAha1-Y223F), and (7.15 = hHsp90α and hAha1-Y223E), (Figure S4E). These data are comparable to previously reported ATPase turnover rates of hHsp90α (Kamal et al., 2003Kamal A. Thao L. Sensintaffar J. Zhang L. Boehm M.F. Fritz L.C. Burrows F.J. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors.Nature. 2003; 425: 407-410Crossref PubMed Scopus (1205) Google Scholar). Also, overexpression of wild-type hAha1 or the phospho mutants did not affect the Hsp90 binding to ATP-agarose (Figure S4F). It is noteworthy that isolated hAha1 and the mutant proteins had no contaminating ATPase activity (Figure S4D). We previously demonstrated that hAha1 co-exists in an hHsp90-kinase client complex (Xu et al., 2012Xu W. Mollapour M. Prodromou C. Wang S. Scroggins B.T. Palchick Z. Beebe K. Siderius M. Lee M.J. Couvillon A. et al.Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.Mol. Cell. 2012; 47: 434-443Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Therefore, we assessed the impact of Y223F and Y223E mutations on hAha1 interaction with kinase clients. We transiently expressed FLAG-tagged wild-type hAha1 and the Y223 phospho mutants in HEK293 cells. Following immunoprecipitation with anti-FLAG M2 affinity gel, we observed hAha1 in complex with the endogenous active (phospho-S89) c-Abl, Raf-1, c-Src, Wee1, and Ulk1 kinases, but not Cdk4 (Figure 3G). These kinases formed a stronger interaction with the phosphomimetic Y223E mutant than did the wild-type hAha1 (Figure 3G). Conversely, association of the non-phosphorylatable Y223F mutant with c-Abl, Raf-1, c-Src, Wee1, and Ulk1 was completely abolished (Figure 3G). Overexpression of hAha1 compromises the folding of CFTR (Koulov et al., 2010Koulov A.V. LaPointe P. Lu B. Razvi A. Coppinger J. Dong M.Q. Matteson J. Laister R. Arrowsmith C. Yates 3rd, J.R. Balch W.E. Biological and structural basis for Aha1 regulation of Hsp90 ATPase activity in maintaining proteostasis in the human disease cystic fibrosis.Mol. Biol. Cell. 2010; 21: 871-884Crossref PubMed Scopus (129) Google Scholar, Wang et al., 2006Wang X. Venable J. LaPointe P. Hutt D.M. Koulov A.V. Coppinger J. Gurkan C. Kellner W. Matteson J. Plutner H. et al.Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis.Cell. 2006; 127: 803-815Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). To examine the effects of Y223 phospho mutants on Hsp90 chaperone function, we assessed their impact on steady-state expression of CFTR protein in mammalian cells. HEK293 cells were transiently co-transfected with CFTR and FLAG-hAha1, or Y223F or Y223E mutants. Empty plasmid pcDNA3 (C) was used as a negative control. Western blot analysis of these samples using anti-CFTR antibody detected a doublet (Figure 3H), with the upper band representing the mature Golgi-processed glycoform of CFTR found at the cell surface and the lower band an immature core-glycosylated protein (Figure 3H). Overexpression of the non-phosphorylatable Y223F mutant did not reduce the stability of CFTR (Figure 3H), confirming its inability to bind and activate hHsp90. In contrast, overexpression of the phosphomimetic hAha1-Y223E mutant displayed a significant reduction of CFTR protein (Figure 3H). The enhanced interaction of Y223E-hAha1 mutant with hHsp90 increases chaperone activity and, therefore, reduces the expression of CFTR. Finally, we transiently expressed hAha1-FLAG and Y223 phosphomutants in HEK293 cells and then stimulated the cells with 10 μM dexamethasone for 1 hr, representing time 0. Cells were then washed and incubated in dexamethasone free media. GR activity was monitored by western blot analysis using anti-phospho-Ser211-GR antibody. Overexpression of the non-phospho-hAha1-Y223F mutant maintained the GR activity even 16 hr post dexamethasone treatment (Figure 3I). However, overexpression of the phosphomimetic hAha1-Y223E caused a rapid reduction in GR activity (Figure 3I). These data confirm the hypothesis that phosphorylation of hAha1-Y223 increases binding to Hsp90 and compromises the chaperoning and the activity of the clients that are “difficult” to fold. The budding yeast S. cerevisiae was used to further investigate the impact of hAha1 Y223 phosphorylation on Hsp90 chaperone function. Aha1 is an evolutionarily conserved co-chaperone, and it has been previously shown that deletion of yAHA1 renders cells temperature-sensitive on glucose (YPED) and respiratory growth media (YPEG) (Panaretou et al., 1998Panaretou B. Prodromou C. Roe S.M. O’Brien R. Ladbury J.E. Piper P.W. Pearl L.H. ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (625) Google Scholar). Since we did not observe tyrosine phosphorylation of hAha1 in yeast (data not shown), we hypothesized that yAha1 acts through a different mechanism to initiate its interaction with yHsp90. Therefore, the same effect should be displayed when either hAha1 or the non-phosphorylatable hAha1-Y223F mutant is expressed in yeast. This turned out to be the case, as overexpression of hAHA1 and the Y223F mutant, under control of the yAHA1 native promoter on a centromeric (single copy) plasmid, reverted the temperature sensitivity phenotype of the yaha1 knockout cells on both YPED and YPEG media (Figure 4A). Furthermore, both forms of hAha1 bound to yHsp90 similarly to wild-type yAha1 (Figure 4B). Expression of the phosphomimetic Y223E mutant did not revert the temperature-sensitivity of yaha1Δ cells on YPEG (Figure 4A) and, compared to the wild-type yAha1, its association to yHsp90 was enhanced (Figure 4B). These data indicate that expression of Y223E in yeast has a dominant-negative effect on initiating its bind" @default.
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• W1708752746 date "2015-08-01" @default.
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• W1708752746 title "c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells" @default.
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