Silvia Soddu

Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis.

Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.

TP53INP1s and homeodomain-interacting protein kinase-2 (HIPK2) are partners in regulating p53 activity.

The TP53INP1 gene encodes two protein isoforms, TP53INP1α and TP53INP1β, located into the nucleus. Their synthesis is increased during cellular stress by p53-mediated activation of transcription. Overexpression of these isoforms induces apoptosis, suggesting an involvement of TP53INP1s in p53-mediated cell death. It was recently shown that p53-dependent apoptosis is promoted by homeodomain-interacting protein kinase-2 (HIPK2), which is known to bind p53 and induce its phosphorylation in promyelocytic leukemia protein nuclear bodies (PML-NBs). In this work we show that TP53INP1s localize with p53, PML-IV, and HIPK2 into the PML-NBs. In addition, we show that TP53INP1s interact physically with HIPK2 and p53. In agreement with these results we demonstrate that TP53INP1s, in association with HIPK2, regulate p53 transcriptional activity on p21, mdm2, pig3, and bax promoters. Furthermore, TP53INP1s overexpression induces G1 arrest and increases p53-mediated apoptosis. Although a TP53INP1s and HIPK2 additive effect was observed on apoptosis, G1 arrest was weaker when HIPK2 was transfected together with TP53INP1. These results indicate that TP53INP1s and HIPK2 could be partners in regulating p53 activity.

Growth and Differentiation Signals by the Insulin-like Growth Factor 1 Receptor in Hemopoietic Cells Are Mediated through Different Pathways

The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.

HIPK2: a multitalented partner for transcription factors in DNA damage response and development.

Protein phosphorylation is a widely diffuse and versatile post-translational modification that controls many cellular processes, from signal transduction to gene transcription. The homeodomain-interacting protein kinases (HIPKs) belong to a new family of serine-threonine kinases first identified as corepressors for homeodomain transcription factors. Different screenings for the identification of new partners of transcription factors have indicated that HIPK2, the best characterized member of the HIPK family, is a multitalented coregulator of an increasing number of transcription factors and cofactors. The aim of this review is to describe the different mechanisms through which HIPK2 regulates gene transcription.

From p63 to p53 across p73

In this review we will mainly focus on similarities and differences as well as relationships among p63, p73 and p53.

High-mobility group A1 inhibits p53 by cytoplasmic relocalization of its proapoptotic activator HIPK2

High-mobility group A1 (HMGA1) overexpression and gene rearrangement are frequent events in human cancer, but the molecular basis of HMGA1 oncogenic activity remains unclear. Here we describe a mechanism through which HMGA1 inhibits p53-mediated apoptosis by counteracting the p53 proapoptotic activator homeodomain-interacting protein kinase 2 (HIPK2). We found that HMGA1 overexpression promoted HIPK2 relocalization in the cytoplasm and inhibition of p53 apoptotic function, while HIPK2 overexpression reestablished HIPK2 nuclear localization and sensitivity to apoptosis. HIPK2 depletion by RNA interference suppressed the antiapoptotic effect of HMGA1, which indicates that HIPK2 is the target required for HMGA1 to repress the apoptotic activity of p53. Consistent with this process, a strong correlation among HMGA1 overexpression, HIPK2 cytoplasmic localization, and low spontaneous apoptosis index (comparable to that observed in mutant p53-carrying tumors) was observed in WT p53-expressing human breast carcinomas. Hence, cytoplasmic relocalization of HIPK2 induced by HMGA1 overexpression is a mechanism of inactivation of p53 apoptotic function that we believe to be novel.

The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

Background Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. Methodology/Principal Findings We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. Conclusions/Significance Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers.

Repression of the Antiapoptotic Molecule Galectin-3 by Homeodomain-Interacting Protein Kinase 2-Activated p53 Is Required for p53-Induced Apoptosis

ABSTRACT Galectin 3 (Gal-3), a member of the β-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3.

Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb

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.

Activation of p53 function in carcinoma cells by the alpha6beta4 integrin

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the α6β4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of α6β4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53 response gene, bax. In addition, we report that α6β4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these α6β4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and p53 activation by demonstrating that the clustering of α6β4 with a β4integrin-specific antibody promotes p53-dependent apoptosis in cells that express both α6β4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating p53. Moreover, given the ability of α6β4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949–960), these studies suggest that the ability of α6β4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.