High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth

Abstract

The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with di erent transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomaininteracting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory e ect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory e ect is independent from the interaction with the HMGI(Y) protein. Oncogene (2001) 20, 6132 ± 6141.

The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4

“…The deletion of HIPK2 in mice is pleiotropic, but supports a role of the kinase in cell survival (Isono et al, 2006;Wei et al, 2007). In addition to regulating transcription factors, HIPK2 binds, phosphorylates and regulates the activity of the chromatin protein HMGA1 (Pierantoni et al, 2001(Pierantoni et al, , 2007Zhang and Wang, 2007), and it also acts on the Polycomb group protein Pc2 . The regulation of HMGA1 and Pc2 suggests that HIPK2 may also act at the level of epigenetic control.…”

“…For immunoprecipitation of HIPK2, cellular extracts were prepared using a specific protocol that efficiently releases this protein (Pierantoni et al, 2001). Endogenous HIPK2 was detected by western blot using a rabbit polyclonal serum (Moller et al, 2003).…”

HMGA proteins promote ATM expression and enhance cancer cell resistance to genotoxic agents

“…All the other transfections were performed by using Lipofectamine 2000 (Invitrogen), as suggested by the manufacturer. The pCEFL-HA-HMGA1, pCEFL-HA-HMGA2, pCEFL-HA-HMGA1 (1-43) and pCEFL-HA-HMGA2 (1-73) vectors were previously described (Fedele et al, 2006;Pierantoni et al, 2001;Pentimalli et al, 2008). The anti-HMGA1 antisense construct was described elsewhere (Berlingieri et al, 2002).…”

“…Antibodies used were anti-FLAG M2 (Sigma, St Louis, MO, USA), anti-HA Clone 12CA5 (Roche, Branford, CT, USA), anti-P-ATM-substrate (phospho-Ser/Thr antibody), anti-CHK2pThr68 (Cell Signalling Technology, Danvers, MA, USA), anti-ATM S1981p (Rockland, Philadelphia, PA, USA), anti-p53 DO-1, anti-p-p53 (Ser15), anti-ATM and anti-vinculin (7F9) (Santa Cruz, Santa Cruz, CA, USA). Anti-HMGA1 and anti-HMGA2 polyclonal antibodies were described elsewhere (Pierantoni et al, 2001;Fedele et al, 2006).…”

Body language: the function of PML nuclear bodies in apoptosis regulation

“…In humans and mice the family comprises three Function of PML in apoptosis regulation TG Hofmann and H Will members, HIPK1-3, which were originally identified as corepressors for homeodomain transcription factors. 89 HIPKs belong to the dual-specificity tyrosine phosphorylation-regulated kinase family of protein kinases 90 and function in transcriptional regulation, 91,92 growth suppression 93 and apoptosis. 24,25 HIPK2, which mediates p53 phosphorylation and ultraviolet (UV)-induced apoptosis 24,25 (for details, see section on 'Regulation of p53 activity in PML-NBs') was found to interact with TRADD when overexpressed in 293T cells.…”