Overexpression Of E3 Ubiquitin Ligase CHIP Induces Mitotic Catastrophe In K562 Cells

Background:CHIP(carboxyl terminus of Hsc70/Hsp70-interacting protein) was identified during screening a library with a cDNA fragment coding tetratricopeptide repeat (TPR) motifs in 1999. CHIP is highly conserved across species. Subsequent studies revealed that CHIP possesses three TPR domains at its amino terminus, which have been found to interact with Hsp70 and Hsp90. CHIP contains U-box domain at the carboxyl-terminal region. U-box resembles that of the RING-finger domain, which is responsible for E3 activities of many ubiquitin ligases. Separating the TPR and U-box domains in CHIP is a central domain rich in charged residues and also containing 2 possible nuclear localizing signals. The function of CHIP's charged domain is not yet known. The special characteristics of CHIP make it become the bridge of molecular chaperone system and ubiquitin-proteasome pathway. CHIP remodels of Hsp90 chaperone complexes and mediates some clients of Hsp90 degradation through ubiquitin-proteasome pathway, such as ErbB2/neu, AR, ER, GR and mutant p53. Nor are they limited to Hsp90 clients; the cystic fibrosis transmembrane conductance receptor, an Hsp70 client, undergoes CHIP-dependent degradation that is Hsp70 dependent, and luciferase undergoes CHIP-dependent ubiquitylation in vitro when it is misfolded and bound by Hsp70. CHIP involves in degradation of accumulation of misfolded protein aggregates that are the signature of several degenerative diseases, such as tau andα-synuclein.Heat shock proteins (Hsps) are kinds of important molecular chaperone of the cells, expect for involving the modulation of protein conformation, stability and kinases activities, these proteins could offer protection when cells are in various stress condition. Among these Hsps, Hsp90 is a chaperone protein with special client proteins which include oncogenic proteins (mutant p53, Bcr-Abl); kinase proteins (v-Src,ErbB2,Raf-1,Akt,Cdk4,Cdk6,et al); cell membrane growth factor receptors (EGFR,PDGF,IGFR, et al) and nucleus receptors (androgen receptor, estrogen receptor). Hsp90's association is required for the stability and function of client proteins. When the function of Hsp90 is inhibited, the client proteins separate from Hsp90 and degradate through ubiquitin-proteasome pathway.Mitosis is a highly intricate and tightly controlled progression; many proteins are involved in cell cycle events including centrosome cycle, spindle checkpoint, microtubule-kinetochore attachment, spindle assembly and chromosome condensation. The regulation of M-phase progression relies predominantly on two post-translational mechanisms: protein phosphorylation and proteolysis. These are intimately intertwined in that the proteolytic machinery is controlled by phosphorylation, whereas several mitotic kinases are downregulated by degradation. The ubiquitin-proteasome pathway is involved in the regulation of mitotic progression.Since Bcr-Abl kinase which is the client of Hsp90 is the ideal target protein of chronic myelogenous leukemia (CML) treatment, whether the cochaperone/E3 ubiquitin ligase CHIP mediates the degradation of Bcr-Abl throuth ubiquitin-proteasome pathway is unknown yet. In our study, stable overexpression models in CML K562 cells were established via Lifofectamine-mediated wild type CHIP and its TPR or U-box deletion mutants gene transfection, and the function of CHIP in Bcr-Abl degradation was investigated. In addition, our preliminary data also showed that overexpression wild type CHIP induced mitotic abnormity. It is rational to assume that CHIP may exert its effect on inducing mitotic abnormity via affecting the localization and expression of mitotic related proteins. To confirm our hypothesis and elucidate the possible mechanisms, the localization and expression of mitotic related proteins was detected; proteomics analysis was used to indentify the possible substrates of CHIP.Experiment design and results:1. Establishment of K562-CHIP stable transfectant and its biological characteristicsStable overexpression models in CML K562 cells were established via Lifofectamine-mediated wild type CHIP and its TPR or U-box deletion mutants gene transfection. Followed G418 pressure selection, stable transfected cell clones were obtained by limited dilution. Exogenous overexpression of CHIP was identified by Western blotting and Immunofluorescence staining. The results showed that overexpression wild type CHIP did not inhibit cell proliferation, but slightly increased cell ratio of G2/M phase. The results of Wright-Giemsa staining demonstrated that the giant cells and abnormal mitotic cells were remarkably increased in K562-CHIP cells, and the cell ratio of M-phase increased. There was no markedly difference in anticancer drugs FK228 or Taxol induced cytotoxicity between K562-CHIP and K562 cells. CHIP had no effect on the stability of Bcr-Abl kinase protein. HDAC inhibitor FK228-induced Bcr-Abl degradation did not enhanced by CHIP.2. Overexpression of E3 ubiquitin ligase CHIP induces mitotic catastrophe in K562 cellsThe results of Hoechest 33258 stainning demonstrated that overexpression of CHIP induced mitotic abnormity in K562 cells, including aberrant nuclear morphology, lagging chromosome and multipolar spindle, et al. Overexpression of CHIP distrupted the centromere localization of chromosomal passenger protein AuroraB and Survivin in aberrant nuclear cells. CHIP inhibited the kinetochore proteins CENP-E and BubR1 located in outer plate of the centromere, while the localization of Plk1 kinase and kinetochore protein Bub1 did not influented by CHIP. In addition, the expression of AuroraA kinase increased in K562-CHIP cells. Multipolar, monopolar and asymmetrical spindles were observed in K562-CHIP cells. Abnormal mitotic morphology cannot be detected in K562- U-box cells. We first demonstrated that CHIP was present in centrosome in K562 and MCF-7 cells, it also can move to spindle and midbody during mitotic progression.3. Proteomic assay to identify the differentially expressed proteins induced by wild type CHIPThe protein profile of K562 and K562-CHIP cells was determined by 2-DE and MALDI-TOF-MS. In total, 39 proteins with significant alteration between K562 and K562-CHIP cells were identified, which included 20 down-regulated protein spots and 19 up-regulated spots. The results were searched in SwissProt and NCBInr database, and 20 different proteins were identified. According to the biological function, the identified proteins were classified into six clusters, including metabolism, cytoskeleton, transcription, protein folding, protein degradation and immunity regulation.Conclusions:1. Overexpression wild type CHIP did not inhibit cell proliferation and the sensitivity to FK228 or Taxol induced cytotoxicity, but slightly increased cell ratio of G2/M phase. Notably the giant cells and abnormal mitotic cells were remarkably increased in K562-CHIP cells.2. CHIP had no effect on the stability of Bcr-Abl kinase protein. HDAC inhibitor FK228-induced Bcr-Abl degradation did not enhanced by CHIP, indicating that CHIP selectively mediates the degradation of Hsp90 client proteins.3. Overexpression of CHIP induced mitotic abnormity in K562 cells, including aberrant nuclear morphology, lagging chromosome and multipolar spindle. CHIP distrupted the centromere localization of chromosomal passenger protein AuroraB and Survivin in aberrant nuclear cells. CHIP inhibited the kinetochore proteins CENP-E and BubR1 located in outer plate of the centromere, while the localization of Plk1 kinase and kinetochore protein Bub1 did not influented by CHIP. In addition, the expression of AuroraA kinase increased in K562-CHIP cells. Multipolar, monopolar and asymmetrical spindles were observed in K562-CHIP cells. Abnomal mitotic morphology cannot be detected in K562- U-box cells.4. We first demonstrated that CHIP was present in centrosome in K562 and MCF-7 cells by Immunofluorescence staining, it also can move to spindle and midbody during mitotic progression.