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The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells. [Display omitted] [ABSTRACT FROM AUTHOR]

The faithful inheritance of chromosomes is essential for the propagation of organisms. In eukaryotes, central to this process is the mitotic spindle. Recently, we have identified TRIM8 as a gene aberrantly expressed in gliomas whose expression reduces the clonogenic potential in the patients' glioma cells. TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. To gain insights into the TRIM8 functions, we characterized the TRIM8 interactome in primary mouse embryonic neural stem cells using proteomics. We found that TRIM8 interacts with KIFC1, and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. By exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability. [ABSTRACT FROM AUTHOR]

Abstract: HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354. [Copyright &y& Elsevier]

HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27kip1. HIPK2 phosphorylates p27kip1 in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27kip1 phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27kip1 phosphorylation at serine 10 and of p27kip1 stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27kip1 revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53. [ABSTRACT FROM AUTHOR]

Abstract: Over the past decade several investigators have reported on the physical interaction of serine/threonine kinases of the homeodomain interacting-protein family (HIPKs) with increasing number of nuclear factors and on their localization in different nuclear sub-compartments. Although we are still far from a global understanding of the molecular consequences of HIPK subnuclear compartmentalization, the spatial description of particular interactions and posttranslational modifications promoted by these kinases on key cellular regulators might provide relevant insights. Here we will discuss the possible implications of the HIPK subnuclear localization in the regulation of gene transcription and in the cell response to stress. [Copyright &y& Elsevier]

Summary: Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage. [Copyright &y& Elsevier]

Abstract: Tp53 is frequently mutated or inactivated in glioblastomas. Due to the impairment of p53 activity, glioblastomas show a high degree of radioresistance. In an attempt to convert the radioresistant phenotype to a more radiosensitive one, we evaluated the efficacy of the combination of Adp53 gene transfer and X-ray irradiation. The combination of Adp53, at low multiplicity in order to mimic the low in vivo efficiency of virus-mediated gene delivery, with X-ray irradiation resulted in a marked decrease of glioblastoms cell survival. Interestingly, Adp53 was able to induce low dose (<2Gy) hypersensitivity. The data suggest the possibility for the development of new therapeutic strategies. [Copyright &y& Elsevier]

Presents a study showing that the TP53INP1 gene localize with p53, PML-IV and HIPK2 into the PML-NB. Physical interaction of the gene with HIPK2 and p53; Role of TP53INP1, in association with HIPK2, regulate p53 transcriptional activity on p21, mdm2, pig3 and bax promoters; Impact of TP53INP1 overexpression on p53-mediated apoptosis.

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins. [Copyright &y& Elsevier]