Homeodomain-interacting Protein Kinase-2 Stabilizes p27kip1 by Its Phosphorylation at Serine 10 and Contributes to Cell Motility

Abstract

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 p27 kip1 . HIPK2 phosphorylates p27 kip1 in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27 kip1 phosphorylation and plays a crucial role in the stability of the protein. stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27 kip1 revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.Homeodomain-interacting protein kinases (HIPKs) 3 belong to a well conserved family of serine/threonine kinases first identified as transcriptional corepressors for homeodomain factors (1) and shown to form complexes with Groucho and histone deacetylases (2). HIPK2, the best characterized member of the HIPK family, is involved in several aspects of cell and tissue biology, including cell proliferation, apoptosis, response to DNA damage or hypoxia, differentiation, and development (for reviews, see Refs. 3 and 4). In response to DNA damage, HIPK2 was shown to promote apoptosis by its kinase activity through p53-dependent and -independent mechanisms. For example, by phosphorylating the proapoptotic oncosuppressor p53 at serine 46, HIPK2 shifts the p53 affinity from cell cycle arrestrelated promoters to apoptosis-related promoters (5-8). On the other hand, by phosphorylating the antiapoptotic corepressor C-terminal binding protein (CtBP) at serine 422, HIPK2 targets CtBP for proteasomal degradation and induces apoptosis even in p53-null cells (9). These events contribute to the cell response to UV, ionizing irradiation and several anticancer drugs and are impaired in HIPK2-depleted cells (10, 11), suggesting a role for HIPK2 as a tumor suppressor. Indeed, there are examples of HIPK2 inactivation in human cancers such as HIPK2 mutations in cases of acute myeloid leukemia (12) or HIPK2 cytoplasmic mislocalization in breast carcinomas and in leukemogenesis (13,14). In the case of breast cancer, HIPK2 mislocalization was significantly associated with overexpression of the oncogenic factor HMGA1, tumor resistance to spontaneous apoptosis (13), overexpression of the ␣6␤4 integrin, and increased anchorage-independent growth and invasion (15). In agreement with these findings, Hipk2ϩ/Ϫ and Hipk2Ϫ/Ϫ mice were shown to be more susceptible to chemical-induced carcinogenesis than Hipk2ϩ/ϩ mice (16). Furthermore, a recent report has shown that HIPK2 amplification in pilocytic astrocytomas and its overexpression in a glioma cell line resulted in an increased proliferation rate (17). These latest data might be compatible with previous observations made in non-transformed cells. Indeed, (i) embryo fibroblasts from Hipk2Ϫ/Ϫ mice have a reduced proliferation rate and altered levels of the cell cycle regulators Cyclin D a...

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1and Cyclin D1 and induces cell cycle exit and neuronal differentiation

“…Overexpression of DYRK1A increased Ser10 phosphorylation similar to HIPK2, which is known to phosphorylate p27 Kip1 at Ser10. 14 Inhibition of endogenous DYRK1A with a harmine-related DYRK1A inhibitor (AnnH31, unpublished results) reduced basal Ser10 phosphorylation compared with the untreated control ( Fig. 8C ).…”

“…In order to assess whether DYRK1A contributes to p27 Kip1 Ser10 phosphorylation in differentiating brain neurons, we cultured E12 embryos for 6 h in the presence of EGCG, harmine, and leucettine L41, 3 different and chemically unrelated DYRK1A inhibitors. 47 This time period is substantially shorter than the duration of a cell cycle, which at this stage has been estimated to last for 20–24 h. 4 , 48 Harmine, EGCG, and leucettine L41 have been profiled against large panels of protein kinases 47 and do not inhibit the other kinases that are known to phosphorylate p27 Kip1 at Ser10 (KIS, AKT, CDK5, HIPK2) 11 - 14 , 49 except for DYRK1B, which is a regulator of p27 Kip1 in cancer cells. 15 , 50 All 3 DYRK1A inhibitors substantially decreased pSer10 p27 Kip1 immunostaining in the mouse telencephalon ( Fig.…”

Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK)

“…Therefore, we performed in vitro kinase assays to assess the Ser/Thr kinase activities of the Tyr→Phe mutants towards exogenous substrates. First, we used recombinant p27 Kip1 as a substrate, because this CDK inhibitor had been shown to be a substrate of HIPK2 [ 40 ]. These assays showed that all HIPKs are capable of phosphorylating Ser10 in p27 Kip1 in vitro (Figure 4 A).…”

Emerging role of DYRK family protein kinases as regulators of protein stability in cell cycle control

“…46 Recently, HIPK2 has also been shown to phosphorylate Ser10 and stabilize p27 in asynchronously growing cell lines. 47 Further work will be necessary to uncover the contribution of the individual kinases in different cell types and different phases of the cell cycle.…”