The folding of both wild-type and mutant forms of the cystic-fibrosis transmembrane-conductance regulator (CFTR), a plasma-membrane chloride-ion channel, is inefficient. Most nascent CFTR is retained in the endoplasmic reticulum and degraded by the ubiquitin proteasome pathway. Aberrant folding and defective trafficking of CFTRDeltaF508 is the principal cause of cystic fibrosis, but how the endoplasmic-reticulum quality-control system targets CFTR for degradation remains unknown. CHIP is a cytosolic U-box protein that interacts with Hsc70 through a set of tetratricorepeat motifs. The U-box represents a modified form of the ring-finger motif that is found in ubiquitin ligases and that defines the E4 family of polyubiquitination factors. Here we show that CHIP functions with Hsc70 to sense the folded state of CFTR and targets aberrant forms for proteasomal degradation by promoting their ubiquitination. The U-box appeared essential for this process because overexpresion of CHIPDeltaU-box inhibited the action of endogenous CHIP and blocked CFTR ubiquitination and degradation. CHIP is a co-chaperone that converts Hsc70 from a protein-folding machine into a degradation factor that functions in endoplasmic-reticulum quality control.

The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466, 1991) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUF1. Using antisera specific for these polypeptides, we demonstrate that the 37- and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.

BACKGROUND Fusion genes are chromosomal aberrations in malignancies that can be used as prognostic markers as well as therapeutic targets. The FGFR3-TACC3 (F3-T3) was initially discovered as an oncogenic molecule in glioblastoma and bladder cancer and subsequently found in many other cancer types. Based on clinical evidence, F3-T3 was found in glioblastoma patients before and after TMZ and radiotherapy treatment, suggesting that targeting F3-T3 is a valid strategy for glioblastoma treatment. MATERIAL AND METHODS We profiled the proteins that interacted with F3-T3 fusion protein in U-251 MG cells with F3-T3 through 2-D liquid chromatography-tandem mass spectrometry. To validate the result of proteomic analysis, we performed reverse immunoprecipitation by pulling down Hsp90 or Cdc37 in U-251 MG cells stably expressing F3-T3. To inhibit the association between F3-T3 and the Hsp90-Cdc37 complex, we treated U-251 MG and LN-229 cells stably expressing F3-T3 with Hsp90 inhibitors or siRNA of Cdc37. We applied the CCK8 assay to evaluate the sensitivity of glioblastoma cells stably expressing F3-T3, wild-type FGFR3, kinase-dead F3-T3 (K508R), and empty vectors to TMZ. Immunoblot and immunofluorescence staining were used to detect DNA damage marker pH2AX. The drug combination effect index was analyzed using software CalcuSyn. U-251 MG cells stably expressing F3-T3 infected with luciferase virus were intracranially injected in nude mice. The experimental group was administered with temozolomide (5mg/kg/day) by oral gavage, Hsp90 inhibitor Onalespib (30mg/kg/day) by tail vein injection or the combination of the two for indicated days. RESULTS We identified the proteins that showed increased binding ratios to F3-T3 over full-length FGFR3, the molecular chaperone proteins encoded by the genes HSP90AB1, HSP90AA1, and CDC37 emerged as 5th, 6th, and 7th on the top ten list, showing an approximately 4-fold increase in normalized spectral counts. Using Hsp90 inhibitors or Cdc37 siRNA disrupted the formation of the F3-T3/Hsp90/Cdc37 complex. Disruption of Hsp90-Cdc37 chaperoning caused a ubiquitination-mediated degradation of the glycosylated form of F3-T3 and abrogated the maturation of nascent F3-T3, resulting in suppression of F3-T3 signaling pathways. Additionally, our results provide evidence that the F3-T3 signaling pathway confers drug resistance to TMZ induced DNA damage. However, the resistance of TMZ was disrupted in glioblastoma cells harboring kinase-dead F3-T3 (K508R). We also demonstrated Hsp90 inhibitor significantly sensitized glioblastoma cells harboring the F3-T3 fusion gene to TMZ treatment and improved survival of xenograft model bearing F3-T3 tumor in vivo. CONCLUSION F3-T3 is a strong Hsp90 client that shows strong addiction to the Hsp90-Cdc37 chaperone system. Combination therapy with Hsp90 inhibitor overcomes the TMZ resistance conferred by F3-T3.