Molecular determinants of Hsp90 recognition of Src kinase revealed by deep mutational scanning
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE218190 | GEO | 2023/05/22
REPOSITORIES: GEO
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Project description:Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, on the kinase specific co-chaperone CDC37, and the client itself. Removal of regulatory phosphorylation from client kinases and release from the HSP90-CDC37 system depends on a Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here we present the cryoEM structure of the oncogenic client kinase BRAFV600E bound to HSP90-CDC37, and structures of complexes of PP5 with that. Together with proteomic analysis of its phosphatase activity, our results reveal how PP5 is activated by recruitment to HSP90 complexes to dephosphorylate client proteins.
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Project description:Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, on the kinase specific co-chaperone CDC37, and the client itself. Removal of regulatory phosphorylation from client kinases and release from the HSP90-CDC37 system depends on a Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here we present the cryoEM structure of the oncogenic client kinase BRAFV600E bound to HSP90-CDC37, and structures of complexes of PP5 with that. Together with proteomic analysis of its phosphatase activity, our results reveal how PP5 is activated by recruitment to HSP90 complexes to dephosphorylate client proteins.
2023-03-11 | PXD033678 | Pride
Project description:The molecular chaperone Hsp90 is involved in the stability and activity of its client proteins which largely comprise of kinases. Phosphorylation of Hsp90 by these kinase clients provides a reciprocal regulatory mechanism between these proteins, however the mechanism of regulating the cellular pathway remains elusive. Here, we show that the serine/threonine kinase Atg1(yeast)/ULK1(mammalian) phosphorylates a conserved serine in the amino-domain of Hsp90 and reduces its ATPase activity hence lowers chaperone function. Broadly this modification negatively impacts the chaperoning of the kinase clients including Atg1/ULK1, yet enhances the activity of the non-kinase clients such as the heat shock factor and the steroid hormone receptors. Interestingly, ATG1/ULK1 mediated phosphorylation of the Hsp90 is essential for initiation of autophagy since yeast expressing a non-phosphorylatable Hsp90 were unable to undergo autophagy and the phosphomimetic Hsp90 mutants were underwent autophagy even in the absence of stimulus. Our findings provide a new paradigm where kinase client mediated phosphorylation of Hsp90 not only regulates the chaperone function but it is essential to initiate and regulate the relevant signaling pathway.
2023-09-08 | PXD038924 | Pride
Project description:Cdc37 is a core cochaperone of the Hsp90 machinery. It is involved in an early stage of the chaperone cycle to activate nascent forms of protein kinases as well as to stabilize mature forms of a subset of protein kinases. Overall, the chaperone cycle is quite complex and involves several steps. Progression, through these steps is regulated by the incorporation of several other cochaperones, ATP hydrolysis and posttranslational modifications on both Cdc37 and Hsp90. We show that phosphorylation of Cdc37 at Y298 by Yes kinase results in partial unfolding of its C-terminal domain, without affecting its ability to form binary or ternary complexes with kinases and Hsp90. Unfolding, unmasks a phosphopeptide sequence that exhibits high affinity for SH2 domains of non-receptor tyrosine kinases (nRTKs), resulting in their recruitment in the chaperone complex. In turn, the high local concentration of nRTKs potentiates Hsp90 phosphorylation at Y197, which results in dissociation of this early kinase-recruitment complex
2018-01-22 | PXD008375 | Pride
Project description:Heat shock protein 90 (Hsp90) is essential for the stability and the function of many client proteins, such as ERB2, C-RAF, CDK4, HIF-1 aplha and AKT. Recent reports demonstrated that inhibition of Hsp90 modulates multiple functions required for survival of human cancer, such as myeloma (Mitsiades et al, Blood:107, 1092, 2006), The aim of this study is evaluate the effect of Hsp90 inhibition, and to identify molecular pathways responsible for anti-proliferative effect on ATL cells. For Hsp90 inhibition, Geldanamycin derivates, 17AAG (17-allylamino -17-demethoxygeldanamycin) and 17DMAG (17-(dimethylaminoethylamino) 17-demethoxygeldanamycin) were used in this study. Interleukin 2-independent ATL cell lines (MT-2 and MT-4) and an interleukin 2-dependent ATL cell line (TaY-E10) were incubated, with or without Hsp90 inhibitors.
2009-05-27 | GSE12287 | GEO
Project description:Heat shock protein 90 (Hsp90) is essential for the stability and the function of many client proteins, such as ERB2, C-RAF, CDK4, HIF-1 aplha and AKT. Recent reports demonstrated that inhibition of Hsp90 modulates multiple functions required for survival of human cancer, such as myeloma (Mitsiades et al, Blood:107, 1092, 2006), The aim of this study is evaluate the effect of Hsp90 inhibition, and to identify molecular pathways responsible for anti-proliferative effect on ATL cells. For Hsp90 inhibition, Geldanamycin derivates, 17AAG (17-allylamino -17-demethoxygeldanamycin) and 17DMAG (17-(dimethylaminoethylamino) 17-demethoxygeldanamycin) were used in this study. Interleukin 2-independent ATL cell lines (MT-2 and MT-4) and an interleukin 2-dependent ATL cell line (TaY-E10) were incubated, with or without Hsp90 inhibitors. Experiment Overall Design: Three ATL cell lines(TaY-E10, MT-2, and MT-4) were cultured in the presence or absence of Hsp90 inhibitors(17-AAG and 17-DMAG).
2009-06-08 | E-GEOD-12287 | biostudies-arrayexpress
Project description:Hsp90 is a molecular chaperone involved in the regulation and maturation of kinases and transcription factors. In Caenorhabditis elegans, it contributes to the development of fertility, maintenance of muscle structure, the regulation of heat-shock response and dauer state. To understand the consequences of Hsp90-depletion, we studied Hsp90 RNAi-treated nematodes by DNA microarrays and mass spectrometry. We find that upon development of phenotypes the levels of chaperones and Hsp90 cofactors are increased, while specific proteins related to the innate immune response are depleted. In microarrays, we further find many differentially expressed genes related to gonad and larval development. These genes form an expression cluster that is regulated independently from the immune response implying separate pathways of Hsp90-involvement. Using fluorescent reporter strains for the differentially expressed immune response genes skr-5, dod-24 and clec-60 we observe that their activity in intestinal tissues is influenced by Hsp90-depletion. Instead, effects on the development are evident in both gonad arms. After Hsp90-depletion, changes can be observed in early embryos and adults containing fluorescence-tagged versions of SEPA-1, CAV-1 or PUD-1, all of which are downregulated after Hsp90-depletion. Our observations identify molecular events for Hsp90-RNAi induced phenotypes during development and immune responses, which may help to separately investigate independent Hsp90-influenced processes that are relevant during the nematode’s life and development. We used microarrays to detail the global programme of gene expression after RNAi-mediated Hsp90 knock-down.
2017-10-17 | GSE105030 | GEO
Project description: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 also increases Hsp90 ATPase activity,enhancesHsp90 interaction with kinase clients,and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a new regulatory paradigm, we 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 bothin vitro and ex vivo.
2017-03-28 | MSV000080713 | MassIVE
Project description:The molecular chaperone Hsp90 stabilizes and activates client proteins. Co-chaperones and post-translational modifications tightly regulate Hsp90 function and consequently lead to activation of clients. However, it is unclear whether this process occurs abruptly or gradually in the cellular context. We show that casein kinase-2 phosphorylation of the co-chaperone Folliculin-interacting protein-1 (FNIP1) on priming serine-938 and subsequent relay phosphorylation on serine-939, 941, 946, and 948 promotes its gradual interaction with Hsp90. This leads to incremental inhibition of Hsp90 ATPase activity and gradual activation of both kinase and non-kinase clients. We further demonstrate that serine/threonine protein phosphatase-5 (PP5) dephosphorylates FNIP1, allowing addition of O-GlcNAc (O-linked N-acetylglucosamine) to the priming serine-938. This process antagonizes phosphorylation of FNIP1, preventing its interaction with Hsp90, and consequently promotes FNIP1 lysine-1119 ubiquitination and proteasomal degradation. These findings provide a mechanism for gradual activation of the client proteins through intricate crosstalk of post-translational modifications of the co-chaperone FNIP1.
2019-01-31 | PXD012229 | Pride
Project description:SUMMARY Mps1 protein kinase is required for accurate chromosome segregation during the mitotic checkpoint. The molecular chaperone Heat Shock Protein 90 (Hsp90) has been linked to Mps1 activity, however the molecular mechanismsunderlying this regulatory process remain elusive. We report that Mps1 directly phosphorylates a conserved threonine residue (T101 in yeast Hsp90 and T115 in human Hsp90) in the N domain of Hsp90. This phosphorylation regulates chaperone function by reducingHsp90 ATPase activity andfosteringits association with kinase client proteins including Mps1. Phosphorylation of T101is also essential for the mitotic checkpoint because it confers Mps1 stability and activity.Additionally,Mps1 phosphorylation of Hsp90 sensitizes cells to Hsp90 inhibitors and elevated Mps1 levels confer tumor selectivity on Hsp90 drugs.Finally,we identified Cdc14 as the phosphatasethat dephosphorylatesT101 and disruptsthe Mps1-Hsp90 interaction. This leads to degradation of Mps1,thusproviding a mechanism for its inactivation and mitotic exit.
2016-01-27 | PXD001969 | Pride