Highlights d Stress-inducible TIMP2 is a bona fide co-chaperone of extracellular HSP90 (eHSP90) d TIMP2 regulates HSP90 chaperone function and interaction with client MMP2 d Secreted co-chaperones TIMP2 and AHA1 displace each other on the eHSP90:MMP2 complex d TIMP2-AHA1 competition impacts client MMP2 activity and matrix gelatinolysis
SummaryThe tissue inhibitor of metalloproteinases 2 (TIMP-2) is a specific endogenous inhibitor of matrix metalloproteinase 2 (MMP-2), which is a key enzyme that degrades the extracellular matrix and promotes tumor cell invasion. Although the TIMP-2:MMP-2 complex controls proteolysis, the signaling mechanism by which the two proteins associate in the extracellular space remains unidentified. Here we report that TIMP-2 is phosphorylated outside the cell by secreted c-Src tyrosine kinase. As a consequence, phosphorylation at Y90 significantly enhances TIMP-2 potency as an MMP-2 inhibitor and weakens the catalytic action of the active enzyme. TIMP-2 phosphorylation also appears to be essential for its interaction with the latent enzyme proMMP-2 in vivo. Absence of the kinase or non-phosphorylatable Y90 abolishes TIMP-2 binding to the latent enzyme, ultimately hampering proMMP-2 activation. Together, TIMP-2 phosphorylation by secreted c-Src represents a critical extracellular regulatory mechanism that controls the proteolytic function of MMP-2.
SUMMARY 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 the 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.
Summary The serine/threonine protein phosphatase-5 (PP5) regulates multiple cellular signaling networks. A number of cellular factors, including heat shock protein-90 (Hsp90) promote the activation of PP5. However, it is unclear whether post-translational modifications also influence PP5 phosphatase activity. Here, we show an “on/off switch” mechanism for PP5 regulation. The casein kinase-1 δ (CK1δ) phosphorylates T362 in the catalytic domain of PP5, which activates and enhances phosphatase activity independent of Hsp90. Overexpression of the phosphomimetic T362E-PP5 mutant hyperdephosphorylates the substrates such as the co-chaperone Cdc37 and the glucocorticoid receptor in cells. Our proteomic approach identified the tumor suppressor von Hippel-Lindau protein (VHL) to interact and ubiquitinate K185/K199-PP5 for proteasomal degradation in a hypoxia- and prolyl hydroxylation-independent manner. Finally, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines and patient tumors exhibit elevated PP5 levels. Down-regulation of PP5 causes ccRCC cells to undergo apoptosis, suggesting a prosurvival role for PP5 in kidney cancer.
The molecular chaperone Heat shock protein 90 (Hsp90) is essential for the folding, stability, and activity of several drivers of oncogenesis. Hsp90 inhibitors are currently under clinical evaluation for cancer treatment, however their efficacy is limited by lack of biomarkers to optimize patient selection. We have recently identified the tumor suppressor tuberous sclerosis complex 1 (Tsc1) as a new co-chaperone of Hsp90 that affects Hsp90 binding to its inhibitors. Highly variable mutations of TSC1 have been previously identified in bladder cancer and correlate with sensitivity to the Hsp90 inhibitors. Here we showed loss of TSC1 leads to hypoacetylation of Hsp90-K407/K419 and subsequent decreased binding to the Hsp90 inhibitor ganetespib. Pharmacologic inhibition of histone deacetylases (HDACs) restores acetylation of Hsp90 and sensitizes Tsc1-mutant bladder cancer cells to ganetespib, resulting in apoptosis. Our findings suggest that TSC1 status may predict response to Hsp90 inhibitors in patients with bladder cancer, and co-targeting HDACs can sensitize tumors with Tsc1 mutations to Hsp90 inhibitors.
Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone that comprises about 1-3% of the total cellular protein. Over the last decade, Hsp90 has been detected and studied in the extracellular space (extracellular or eHsp90) of normal and neoplastic cells. Once outside the cell, eHsp90 has been shown to interact with extracellular client proteins and promote their stabilization and function. Cell conditioned media are routinely collected to detect and quantify eHsp90, and determine its interactions with extracellular clients. Finally, targeting specifically the eHsp90 with pharmacologic inhibitors or antibodies that are unable to cross the plasma membrane has been beneficial in inhibiting tumor cell motility and invasion.
INTRODUCTION AND OBJECTIVES: Xp11.2 translocation renal cell carcinoma (Xp11.2 tRCC) is a newly defined subset of RCC which is characterized by various translocations involving chromosome Xp11.2. All the Xp11.2 translocations produce chimeric TFE3 genes, which retain the coding sequences for the basic helix-loop-helix leucine zipper structure (bHLH-Zip) through which TFE3 binds to DNA. It is suggested that the TFE3 fusion proteins encoded by the chimeric TFE3 genes work as constitutively active transcription factors resulting in Xp11.2 tRCC development. We aim to clarify the cancer-causing molecular mechanism of Xp11.2 tRCC by analyzing the transcriptional function of the chimeric TFE3 proteins.METHODS: 1) We performed comprehensive gene expression analysis by RNA sequencing (RNA-seq) to identify the gene clusters whose expressions were changed predominantly by chimeric TFE3 proteins using cell lines that expressed chimeric TFE3 proteins (PRCC-TFE3, PSF-TFE3, NONO-TFE 3) in a doxycycline-dependent manner or wild-type TFE3. 2) We performed chromatin immunoprecipitation sequencing (Chip-seq) and mapped chimeric TFE3 and wild type TFE3 binding to the genome.RESULTS: WT-TFE3 and PRCC-TFE3 mapping on the genome revealed sharp peaks in the promoter regions, while PSF-TFE3 and NONO-TFE3 showed distinctly broader peaks. RNA-seq analysis demonstrated that PSF-TFE3 and NONO-TFE3 protein induction caused distinct gene expression changes compared to wild type TFE3 induction. In addition, gene expression changes caused by PRCC-TFE3 protein induction were also different compared to wild type TFE3 protein induction, despite the fact that mapping patterns in promoter regions were similar in both cases.CONCLUSIONS: Our key new finding is that PSF-TFE3 and NONO-TFE3 fusions show clearly different binding patterns to the genome, and these chimeric TFE3 proteins cause distinct gene expression changes compared to wild-type TFE3. These findings suggest that chimeric TFE3 proteins contribute to carcinogenesis of renal cell carcinoma as constitutively active transcription factors that induce altered binding patterns on the genome, which result in gene expression profiles that are distinct from wild-type TFE3.
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