Hepatoma Up-regulated Protein Research Articles

The involvement of nuclear factor-κappaB in the nuclear targeting and cyclin E1 upregulating activities of hepatoma upregulated protein

Hepatoma upregulated protein (HURP) is originally isolated during the search for the genes associated with hepatoma. HURP is upregulated in many human cancers. Culture cells exhibit transformed and invasive phenotype when ectopic HURP is introduced, revealing HURP as an oncogene candidate. Our previous studies demonstrated that Aurora-A regulated the cell transforming activities of HURP by phosphorylating HURP at four serines. To unravel how the Aurora-A/HURP cascade contributes to cell transformation, we firstly noticed that HURP shuttled between cytoplasm and nucleus. The nuclear localization activity of HURP was promoted or abolished by overexpression or knockdown of Aurora-A. Similarly, the HURP phosphorylation mimicking mutant 4E had higher nuclear targeting activity than the phosphorylation deficient mutant 4A. The HURP 4E accelerated G1 progression and upregulated cyclin E1, and the cyclin E1 upregulating and cell transforming activities of HURP were diminished when the nuclear localization signal (NLS) was removed from HURP. Furthermore, HURP employed p38/nuclear factor-κB (NF-κB) cascade to stimulate cell growth. Interestingly, NF-κB trapped HURP in nucleus by interacting with HURP 4E. At last, the HURP/NF-κB complex activated the cyclin E1 promoter. Collectively, Aurora-A/HURP relays cell transforming signal to NF-κB, and the HURP/NF-κB complex is engaged in the regulation of cyclin E1 expression.

Structure of the FP domain of Fbxo7 reveals a novel mode of protein–protein interaction

The FP (Fbxo7/PI31) domains found in the F-box protein Fbxo7 and the proteasome inhibitor PI31 mediate the homodimerization and heterodimerization of Fbxo7 and PI31. Fbxo7 is the substrate-recognition subunit of the SCF(Fbxo7) (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also interacts with proteins that are not substrates of the ubiquitin proteasome system, such as Cdk6 and PI31. Here, the crystal structure of the Fbxo7 FP domain is reported at 2.0 Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain. However, an α-helix and three β-strands in the Fbxo7 FP domain are longer than their counterparts in the PI31 FP domain. The differences in these secondary-structural elements are spatially clustered to define a more structured and extended C-terminal end of the Fbxo7 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein-protein interaction. The inter-domain interface of the Fbxo7 FP domain is defined by the α-helical surface in one protomer and the β-sheet surface in the other protomer, whereas for the PI31 domain it is defined by either the α-helical surfaces or the β-sheet surfaces in both protomers. The inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The Fbxo7 FP domain also has the potential to bind two protein partners simultaneously using the α-helical and β-sheet surfaces. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with other regulatory proteins via the FP domain.

Evaluation of urinary HURP mRNA as a marker for detection of bladder cancer: relation to bilharziasis

This study was carried out to assess the efficacy of urinary hepatoma up-regulated protein (HURP) RNA in bladder cancer diagnosis and its relation to bilharziasis. Voided urine samples and blood were collected from 344 consecutive participants: 211 patients diagnosed with bladder cancer, 71 patients with benign urological disorders and 62 healthy volunteers. Serologic assessment of schistosomiasis antibody in sera, urine cytology and estimation of HURP RNA by reverse transcription polymerase chain reaction in urothelial cells was carried out in all samples. HURP RNA expression showed a significant difference among the three investigated groups. The best cutoff point for HURP RNA was determined as 0.0132 at 78.67% sensitivity and 94% specificity. The sensitivity of urine cytology was improved when combined with HURP RNA in detection of early stage (77.3%), low grade (85.3%) and bilharzial bladder cancer (78.1%). Detection of urinary HURP RNA is a useful non-invasive test for early detection of bladder cancer and bilharzial bladder cancer and it improves sensitivity of urine cytology up to 91%.

Prognostic Value of Discs Large Homolog 7 Transcript Levels in Prostate Cancer

Hypoxia has been associated with malignant progression, metastasis and resistance to therapy. Hence, we studied expression of hypoxia–regulated genes in 100 prostate cancer (CaP) bulk tissues and 71 adjacent benign tissues. We found 24 transcripts significantly overexpressed (p≤0.02). Importantly, higher transcript levels of disc large (drosophila) homolog-associated protein 5 (DLGAP5)/discs large homolog 7 (DLG7)/hepatoma up-regulated protein (HURP), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with higher Gleason score and more advanced systemic progression. Since the products of HMMR and CCNB1 have been identified recently as molecular markers of CaP progression, we postulated that DLG7 has prognostic value too. To test this hypothesis, we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within six years of surgery) and controls (no progression within eight years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CI, 0.68 to 0.8). Overall, our data indicate that expression of DLG7, a hypoxia-controlled gene, holds prognostic potential in high-risk CaP; this also demonstrates that variation of oxygen tension may constitute a tool for identification of novel biomarkers for CaP.

Abstract B39: Role of hepatoma upregulated Protein (HURP) in prostate cancer tumorigenesis.

Abstract Hepatoma up-regulated protein (HURP) is a cell cycle-regulated and microtubule-associated protein. It functions as a Ran GTPase effector and is involved in the stabilization of the mitotic kinetochore fibers. The expression of this protein is elevated in different tumor types and associated with tumor progression and aggressiveness. Also, a significant elevation of HURP expression was reported in normal stem cells including haematopoietic stem cells, mesenchymal stem cells, and mouse embryonic stem cells but not in differentiated cells, an evidence for HURP as stemness maintenance factor. The aim of this study is to analyze the oncogenic potential of HURP in prostate cancer (PCa). We analyzed the expression level of HURP in PCa patients and found the transcripts of HURP were overexpressed in PCa. The elevated expression of HURP in PCa was found to be associated with higher Gleason score, systemic progression and poor prognosis. Furthermore, using a case-control cohort study we demonstrated the overall prognostic ability of HURP mRNA in advanced PCa, as tested in receiver operating characteristic analysis [AUC 0.74 (95% CL, 0.68 to 0.8)]. In addition, we analyzed the expression level of HURP protein in tumor tissue and in prostate benign and cancer cell lines; we found that the level of HURP protein was almost 5-fold higher (p<0.005) relative to control (cystoprostatectomy) tissue and in cancer cell lines compared to the benign cells. Overall, these data suggest a strong relationship between the elevated expression of HURP and the development and progression of PCa. Next, we set out a tetracyclin-regulated transient expression system model to study the effects of HURP overexpression in LNCaP cells. Data obtained from western blot revealed that HURP overexpression was associated with the phosphorylation of C-Jun-N-Terminal kinase (JNK) and the induction of both Fra1 and c-JUN proteins as well as with the suppression of pro-apoptotic protein Bcl-Xs, and p53 without influencing the negative regulator MDM2. Also, the overexpression of HURP in CaP cell lines was found to enhance the expression of the anti-apoptotic protein Bcl-2 without influencing the anti-apoptotic protein. Conversely, viral titer of shHURP was found to inhibit the basal phosphorylation of JNK, to suppress the basal level of cyclin A, to increase the degradation of the inhibitor of NF-κB, to decrease the expression of PKC. Furthermore, down regulation of HURP was found to enhance the expression of p53 and to abolish its negative regulator MDM2, suggesting a central role for p53 in the modulation of HURP-induced cell proliferation and tumor progression. Also, the overexpression of HURP promoted the expression of the stemness markers Oct3/4 and Nanog, an evidence for the involvement of HURP in the maintenance of stemness properties in PCa cells. This idea is supported by the reported role of Nanog and Oct3/4 in promoting PCa stem cell characteristics as well as prostate resistance to androgen deprivation, and by our results showing decreased p53, with demonstrated roles in reprogramming, dedifferentiation, self-renewal, and pluripotency. Overall, our data suggest a potential role of HURP in PCa tumorigenesis. In addition, these findings suggest that HURP's stemness properties can contribute to its role in the etiology of PCa. Support: DOD PC094680 (CRG), PCF Creativity Award (CRG). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B39. Citation Format: Abdelouahid El-Khattouti, Tangeng Ma, James R. Kent, Ingrid Espinoza, Christian R. Gomez. Role of hepatoma upregulated Protein (HURP) in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B39.

Aurora kinase inhibitors reveal mechanisms of HURP in nucleation of centrosomal and kinetochore microtubules

The overexpression of Aurora kinases in multiple tumors makes these kinases appealing targets for the development of anticancer therapies. This study identified two small molecules with a furanopyrimidine core, IBPR001 and IBPR002, that target Aurora kinases and induce a DFG conformation change at the ATP site of Aurora A. Our results demonstrate the high potency of the IBPR compounds in reducing tumorigenesis in a colorectal cancer xenograft model in athymic nude mice. Human hepatoma up-regulated protein (HURP) is a substrate of Aurora kinase A, which plays a crucial role in the stabilization of kinetochore fibers. This study used the IBPR compounds as well as MLN8237, a proven Aurora A inhibitor, as chemical probes to investigate the molecular role of HURP in mitotic spindle formation. These compounds effectively eliminated HURP phosphorylation, thereby revealing the coexistence and continuous cycling of HURP between unphosphorylated and phosphorylated forms that are associated, respectively, with microtubules emanating from centrosomes and kinetochores. Furthermore, these compounds demonstrate a spatial hierarchical preference for HURP in the attachment of microtubules extending from the mother to the daughter centrosome. The finding of inequality in the centrosomal microtubules revealed by these small molecules provides a versatile tool for the discovery of new cell-division molecules for the development of antitumor drugs.

Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

Mitotic regulators exhibiting gain of function in tumor cells are considered useful cancer therapeutic targets for the development of small-molecule inhibitors. The human Aurora kinases are a family of such targets. In this study, from a panel of 105 potential small-molecule inhibitors, two compounds Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in vitro, in vivo single cell and in silico studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development.

Knockdown of HURP inhibits the proliferation of hepacellular carcinoma cells via downregulation of gankyrin and accumulation of p53

We determined earlier that the hepatoma upregulated protein (HURP) is overexpressed in hepatocellular carcinoma (HCC), but the role of this protein during cancer development and progression remains unknown. Here, we observed that the overexpression of HURP in HEK293 cells promoted the ubiquitination of p53 and its degradation by the proteasome. In contrast, HURP knockdown using short-hairpin RNA reversed these effects. Knockdown of HURP promoted the accumulation of p53 in SK-Hep-1 cells (p53+/−), and these cells showed reduced proliferation, while the p53-mutant Mahlavu cells were not affected. HURP knockdown did not affect the proliferation of H1299 lung carcinoma cells and Hep3B HCC cells which lack p53. Knockdown of HURP also sensitized SK-Hep-1 cells to cisplatin. On the other hand, the expression of exogenous p53 in H1299 and Hep3B cells was decreased following overexpression of HURP, and these cells showed decreased sensitivity to cisplatin-induced apoptosis. Importantly, overexpression of HURP promoted the proliferation of HEK293 cells in an anchorage-independent manner, and inoculation of SK-Hep-1 cancer cells that expressed short-hairpin RNA to knockdown HURP resulted in smaller tumors in nude mice. Gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, was found to be upregulated following HURP expression, and gankyrin knockdown decreased the HURP-mediated downregulation of p53. Notably, we detected a positive correlation between elevated HURP and gankyrin protein levels in HCC patients (r2=0.778; N=9). Taken together, these results indicate that HURP represents an oncogene that may play a role in HCC progression and chemoresistance.

HURP Regulates Chromosome Congression by Modulating Kinesin Kif18A Function

Chromosome biorientation and congression during mitosis require precise control of microtubule dynamics [1-4]. Thedynamics of kinetochore microtubules (K-MTs) are regulated by a variety of microtubule-associated proteins (MAPs) [4-9]. Recently, a MAP known as HURP (hepatoma upregulated protein) was identified [10-12]. During mitosis, Ran-guanosine 5'-triphosphate (RanGTP) releases HURP from the importin β inhibitory complex and allows it to localize to the kinetochore fiber (k-fiber) [12, 13]. HURP stabilizes k-fibers and promotes chromosome congression [12, 14, 15]. However, the molecular mechanism underlying the role of HURP in regulating chromosome congression remains elusive. Here, we show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP(278)) of HURP induces a series of mitotic defects that mimic the effects of Kif18A depletion. In addition, coimmunoprecipitation and bimolecular fluorescence complementation assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative results from live-cell imaging analyses illustrate that HURP regulates Kif18A localization and dynamics at the plus end of K-MTs. Lastly, misaligned chromosomes in HURP(278)-overexpressing cells can be partially rescued by the overexpression of Kif18A. Our results demonstrate in part the regulatory mechanism for Kif18A during chromosome congression and provide new insights into the mechanism of chromosome movement at the metaphase plate.

The tyrosine kinase inhibitor sorafenib sensitizes hepatocellular carcinoma cells to taxol by suppressing the HURP protein

The hepatoma upregulated protein (HURP) represents a putative oncogene that is overexpressed in many human cancers, especially hepatocellular carcinoma (HCC). HURP plays an important role during mitotic spindle formation, a process that is targeted by various anti-cancer drugs like taxol. However, the role of HURP during the establishment of taxol chemoresistance in HCC remains unclear. In this study, we observed that high HURP protein level correlates with taxol resistance in HCC cells. Following HURP knockdown, HCC cells show a more sensitive response to taxol treatment. Notably, sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, inhibits HURP expression primarily at the transcriptional level and sensitizes HCC cells to sub-lethal doses of taxol. By using real-time PCR and chromatin immunoprecipitation assays, we observed that the NF-κB family member c-Rel represents a putative transcription factor that activates HURP gene expression. In addition, the inhibitory effect of sorafenib on HURP expression was attributed to a reduced translation and nuclear translocation of c-Rel. Accordingly, downregulation of c-Rel using short-hairpin RNA was shown to reduce HURP protein level and enhance taxol-induced cell death. Taken together, our results indicate that HURP acts as a novel survival protein that protects HCC cells against taxol-induced cell death. In addition, the regulation of HURP gene expression by NF-κB signaling appears to be critical for the response of HCC cells to taxol.

Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase

The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)-dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.

Hepatitis B virus X protein prevents apoptosis of hepatocellular carcinoma cells by upregulating SATB1 and HURP expression

Protein X from hepatitis B virus (HBV) appears to play a critical role in the development of hepatocellular carcinoma (HCC). The hepatoma upregulated protein (HURP) is also upregulated in a majority of HCC cases, therefore suggesting that HURP represents an oncogene. In this study, we describe a link between the viral protein HBx, HURP, and the establishment of cisplatin chemoresistance in HCC cells. Hep3B cells which express HBx displayed increased levels of HURP mRNA and protein, and showed resistance to cisplatin-induced apoptosis. Knockdown of HURP in HBx-expressing cells reversed this effect and sensitized Hep3B cells to cisplatin. Interestingly, SATB1, a global gene regulator which is often overexpressed in malignant breast cancer, was also induced following expression of HBx. The anti-apoptotic effect of HBx was shown to require activation of the p38/MAPK pathway in Hep3B cells. In addition, the expression of survivin, an anti-apoptotic protein, was also upregulated by HBx in an HURP-dependent manner. Taken together, these results indicate that HBx activates the expression of HURP via the p38/MAPK pathway and the SATB1 protein, culminating with the accumulation of the anti-apoptotic protein survivin. Our findings illustrate the role of the viral protein HBx in preventing apoptosis during cancer progression and establishment of chemoresistance.

Mars and tousled-like kinase act in parallel to ensure chromosome fidelity in Drosophila

BackgroundHigh levels of Hepatoma Up-Regulated Protein (HURP) and Tousled-Like Kinase (TLK) transcripts are found in hepatocellular carcinoma. HURP overexpression induces anchorage-independent growth of 293-T cells and enhances a rough-eye phenotype resulting from tlk overexpression in Drosophila. In addition, both HURP and Mars, a Drosophila HURP sequence homologue, promote polymerization of mitotic spindles. Thus, the genetic interaction of mars with tlk might be required for accurate chromosome segregation.MethodsTo reveal whether chromosome fidelity was decreased, the frequency of gynandromorphy, an individual with both male and female characteristics, and of non-disjunction were measured in the progeny from parents with reduced mars and/or tlk activities and analyzed by Student's t-test. To show that the genetic interaction between mars and tlk is epistatic or parallel, a cytological analysis of embryos with either reduced or increased activities of mars and/or tlk was used to reveal defects in mitotic-spindle morphology and chromosome segregation.ResultsA significant but small fraction of the progeny from parents with reduced mars activity showed gynandromorphy and non-disjunction. Results of cytological analysis revealed that the decrease in chromosome fidelity was a result of delayed polymerization of the mitotic spindle, which led to asynchronous chromosome segregation in embryos that had reduced mars activity. By removing one copy of tousled-like kinase (tlk) from flies with reduced mars activity, chromosome fidelity was further reduced. This was indicated by an increased in the non-disjunction rate and more severe asynchrony. However, the morphology of the mitotic spindles in the embryos at metaphase where both gene activities were reduced was similar to that in mars embryos. Furthermore, tlk overexpression did not affect the morphology of the mitotic spindles and the cellular localization of Mars protein.ConclusionChromosome fidelity in progeny from parents with reduced mars and/or tlk activity was impaired. The results from cytological studies revealed that mars and tlk function in parallel and that a balance between mars activity and tlk activity is required for cells to progress through mitosis correctly, thus ensuring chromosome fidelity.

Mars, aDrosophilaprotein related to vertebrate HURP, is required for the attachment of centrosomes to the mitotic spindle during syncytial nuclear divisions

The formation of the mitotic spindle is controlled by the microtubule organizing activity of the centrosomes and by the effects of chromatin-associated Ran-GTP on the activities of spindle assembly factors. In this study we show that Mars, a Drosophila protein with sequence similarity to vertebrate hepatoma upregulated protein (HURP), is required for the attachment of the centrosome to the mitotic spindle. More than 80% of embryos derived from mars mutant females do not develop properly due to severe mitotic defects during the rapid nuclear divisions in early embryogenesis. Centrosomes frequently detach from spindles and from the nuclear envelope and nucleate astral microtubules in ectopic positions. Consistent with its function in spindle organization, Mars localizes to nuclei in interphase and associates with the mitotic spindle, in particular with the spindle poles, during mitosis. We propose that Mars is an important linker between the spindle and the centrosomes that is required for proper spindle organization during the rapid mitotic cycles in early embryogenesis.

Hepatoma Up-Regulated Protein Is Required for Chromatin-induced Microtubule Assembly Independently of TPX2

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.

Hurp Deficiency in Mice Leads to Female Infertility Caused by an Implantation Defect

First revealed in cancer studies, HURP (hepatoma up-regulated protein) is a cell cycle-associated gene with elevated expression in the G(2)/M phase. Cell culture studies have revealed that HURP is an essential factor required for spindle formation and chromosome congression during mitosis. However, the function of HURP in an in vivo context has not been explored. We generated a Hurp knock-out (Hurp(-/-)) mouse to investigate the role of HURP in development under normal physiological conditions. Hurp(-/-) mice develop normally and are indistinguishable from their wild-type littermates. Interestingly, breeding experiments revealed that Hurp(-/-) females are completely infertile, whereas the males reproduce normally. Ovulation, fertilization, and pre-implantation embryo development are normal; however, the Hurp(-/-) females are unable to form implantation sites due to an inability to undergo the decidual reaction. This is caused by a defect in endometrial stromal proliferation that leads to implantation failure. Additionally, HURP expression in the uterus coincides with the implantation stage and can be induced by estrogen treatment. Our results demonstrate for the first time that HURP affects endometrial stromal proliferation during implantation but is dispensable during normal development in mice; specifically, HURP has an essential function in uterine biology.

Structure of a Conserved Dimerization Domain within the F-box Protein Fbxo7 and the PI31 Proteasome Inhibitor

F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCF(Fbxo7) E3 ligase, and the proteasome inhibitor PI31. SCF(Fbxo7) is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7.Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel alpha/beta-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7.Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCF(Fbxo7). We present a model for FP domain-mediated dimerization of SCF(Fbxo7) and PI31.

HURP controls spindle dynamics to promote proper interkinetochore tension and efficient kinetochore capture

Through a functional genomic screen for mitotic regulators, we identified hepatoma up-regulated protein (HURP) as a protein that is required for chromosome congression and alignment. In HURP-depleted cells, the persistence of unaligned chromosomes and the reduction of tension across sister kinetochores on aligned chromosomes resulted in the activation of the spindle checkpoint. Although these defects transiently delayed mitotic progression, HeLa cells initiated anaphase without resolution of these deficiencies. This bypass of the checkpoint arrest provides a tumor-specific mechanism for chromosome missegregation and genomic instability. Mechanistically, HURP colocalized with the mitotic spindle in a concentration gradient increasing toward the chromosomes. HURP binds directly to microtubules in vitro and enhances their polymerization. In vivo, HURP stabilizes mitotic microtubules, promotes microtubule polymerization and bipolar spindle formation, and decreases the turnover rate of the mitotic spindle. Thus, HURP controls spindle stability and dynamics to achieve efficient kinetochore capture at prometaphase, timely chromosome congression to the metaphase plate, and proper interkinetochore tension for anaphase initiation.

Enhanced transformation and chemosensitivity of NIH3T3 cells transduced with hepatoma up-regulated protein

Hepatoma up-regulated protein (HURP) is a recently identified novel cell-cycle-regulated gene. The HURP gene is overexpressed in human hepatocellular carcinoma and transitional cell carcinoma. The cellular function of HURP is not fully understood. In this study, the NIH3T3 cells transduced with the exogenous HURP gene manifested the general characteristics of tumor cells, which had higher growth rate in low-serum media and advanced ability of colony formation on agarose-based plates. Transduced HURP was capable of specifically enhancing the chemosensitivity of deoxycytosine analogs, such as gemcitabine, ARA-C, and 5-AZA-CdR, but neither had an effect on the response of DNA intercalating agents, such as cisplatin, carboplatin, and doxorubicin, nor on the response of microtubule stabilizers, such as paclitaxel, docetaxel, and vinblastine. These results indicate that the HURP gene might be a potential oncogenic gene and capable of enhancing the chemosensitivity of deoxycytosine analogs in NIH3T3 cells.

Fbx7 Functions in the SCF Complex Regulating Cdk1-Cyclin B-phosphorylated Hepatoma Up-regulated Protein (HURP) Proteolysis by a Proline-rich Region

F-box proteins, components of SCF ubiquitin-ligase complexes, are believed to be responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins that have been identified to function in the SCF complexes to date mostly have substrate-binding motifs, such as WD repeats or leucine-rich repeats in their C termini. However, many F-box proteins lack recognizable substrate-binding modules; whether they can function in the SCF complexes remains unclear. We show here that Fbx7, an F-box protein without WD repeats and leucine-rich repeats, is required for the proteasome-mediated proteolysis of the hepatoma up-regulated protein (HURP). Depletion of Fbx7 by small interfering RNA leads to depression of HURP ubiquitination and accumulation of HURP abundance. In the SCF(Fbx7) complex, Fbx7 recruits HURP through its C-terminal proline-rich region in a Cdk1-cyclin B-phosphorylation dependent manner. Mutation of the multiple Cdk1-cyclin B phosphorylation sites on HURP or the proline-rich region of Fbx7 abolishes the association between Fbx7 and HURP. Thus, Fbx7 is a functional adaptor of the SCF complex with a proline-rich region as the substrate-binding module. In addition to Fbx7, data base analyses reveal two putative mammalian proline-rich region-containing F-box proteins, KIAA1783 and RIKEN cDNA 2410015K21. Taken together, these findings further expound the diverse substrate-recognition abilities of the SCF complexes.