Abstract
To study the biological role of p73 alpha, a member of the p53 tumor suppressor family, we performed a yeast two-hybrid screen of a human cDNA library. Using a p73 alpha fragment consisting of amino acids 49-636 as bait, we found that p73 alpha is functionally associated with the human homologue of mouse and hamster homeodomain-interacting protein kinase 2 (HIPK2). The hamster homologue, also known as haHIPK2 or PKM, was used for further characterization of interactions between HIPK2 and members of the p53 protein family. Systematic yeast two-hybrid assays indicated a physical interaction between the oligomerization domains of p73 alpha and p53 (amino acid regions 345-380 and 319-360, respectively) and amino acid region 812-907 of haHIPK2. This region of haHIPK2 includes a PEST sequence, an Ubc9-binding domain, and a partial speckle retention sequence and is identical to amino acid residues 846-941 of human HIPK2 (hHIPK2). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro and immunoprecipitation assays in vivo. HIPK2 colocalized with p73 and p53 in nuclear bodies, as shown by confocal microscopy. Overexpression of HIPK2 stabilized the p53 protein and greatly increased the p73- and p53-induced transcriptional repression of multidrug-resistant and collagenase promoters in Saos2 cells but had little effect on the p73- or p53-mediated transcriptional activation of synthetic p53-responsive and p21WAF1 promoters. Stable expression of HIPK2 in U2OS cells enhanced the cisplatin response of sub-G(1) and G(2)/M populations, and it also increased the apoptotic response to cisplatin and adriamycin as demonstrated by fluorescence-activated cell sorter and 4',6-diamidino-2-phenylindole-staining analyses. HIPK2 potentiated the inhibition of colony formation by p73 and p53. These results suggest that physical interactions between HIPK2 and members of the p53 family may determine the roles of these proteins in cell cycle regulation and apoptosis.
Highlights
The tumor suppressor protein p53 is one of the most important regulators of cellular growth functions, such as cell cycle arrest, DNA repair, and apoptosis, and is mutated in about 50% of all human tumors [1]
Using a p73␣ fragment consisting of amino acids 49 – 636 as bait, we found that p73␣ is functionally associated with the human homologue of mouse and hamster homeodomaininteracting protein kinase 2 (HIPK2)
The insert sequences from a total of 42 unique library plasmids were used in a GenBank search, and five of these clones were found to be human homologues of mouse HIPK2 [46] and haHIPK2 [49]
Summary
The tumor suppressor protein p53 is one of the most important regulators of cellular growth functions, such as cell cycle arrest, DNA repair, and apoptosis, and is mutated in about 50% of all human tumors [1]. Like p53, both p73 and p63 can form homo-oligomers, bind to canonical p53 DNA binding sites, modulate transcription of p53-responsive genes, and suppress growth or induce apoptosis when overexpressed in certain human tumors (34 –37). Their polypeptide sequences appear to contain the three principal domains of p53: (a) an N-terminal transcriptional activation domain (AD), (b) a sequence-specific DNA-binding domain (DBD), and (c) an OD that mediates tetramerization. We report our investigation into the molecular bases for the differences in function among these three proteins of the p53 family
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