Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain.

The dual-specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis-auto-phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological-like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme).

HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation.

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.

HIPK2 controls cytokinesis and prevents tetraploidization by phosphorylating histone H2B at the midbody.

Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody.

HIPK2 regulation by MDM2 determines tumor cell response to the p53-reactivating drugs nutlin-3 and RITA.

In the past few years, much effort has been devoted to show the single-target specificity of nongenotoxic, p53 reactivating compounds. However, the divergent biological responses induced by the different compounds, even in the same tumor cells, demand additional mechanistic insights, whose knowledge may lead to improved drug design or selection of the most potent drug combinations. To address the molecular mechanism underlying induction of mitotic arrest versus clinically more desirable apoptosis, we took advantage of two MDM2 antagonists, Nutlin-3 and RITA, which respectively produce these two outcomes.

HIPKs: Jack of all trades in basic nuclear activities.

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.

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.

Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication.

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens.

A pRb-independent mechanism preserves the postmitotic state in terminally differentiated skeletal muscle cells.

In skeletal muscle differentiation, the retinoblastoma protein (pRb) is absolutely necessary to establish definitive mitotic arrest. It is widely assumed that pRb is equally essential to sustain the postmitotic state, but this contention has never been tested. Here, we show that terminal proliferation arrest is maintained in skeletal muscle cells by a pRb-independent mechanism.

HPV E7 expression in skeletal muscle cells distinguishes initiation of the postmitotic state from its maintenance.

The E7 oncogene is an essential tool used by papillomaviruses to interfere with the cell cycle and cellular differentiation. We investigated the effects of E7 expression on both cellular functions in skeletal muscle cells, a terminally differentiating system. When expressed in myoblasts, E7 impaired differentiation only partially, but allowed continuation of DNA synthesis during and after differentiation.