pocket of the kinase, undergo a thermodynamic advantage, hindering their release into the surrounding aqueous medium. We also considered the possibility that intracellular TBID could irreversibly inactivate HIPK-2 by preventing the phosphorylation of its up-regulatory tyrosine, an event occurring only during translation. In our cell model, however, we couldn��t RN-1734 detect any phospho-Tyr signal in HIPK-2 immunoprecipitated from either untreated or treated cells. To reinforce the view that endogenous HIPK-2 is inhibited upon cell treatment with TBID, advantage has been also taken of p53 Ser46, a known target of the kinase. As shown in Figure 6B, TBID treatment markedly reduces the phosphorylation level of this residue, without affecting the amount of p53, under conditions devoid of cell toxicity. To note that, although p53 Ser46 is not targeted exclusively by HIPK2, other putative phosphorylating agents of this residue, notably DYRK2 and PKC, are nearly unaffected by the inhibitor under conditions where HIPK2 is.70% inhibited.This observation, in conjunction with the similar dose dependency of HIPK2 activity inhibition and decrease of p53 Ser46 phosphorylation, support the view that the reduction of p53 Ser46 phosphorylation is mainly due to HIPK2 inhibition. It should be noted in this connection that the concentration required for half maximal inhibition is two orders of magnitude higher in cells than it is in vitro. This is not unusual among MEDChem Express ONO-4059 (hydrochloride) protein kinase inhibitors as exemplified elsewhere and may be accounted for by massive sequestration of lipophilic compounds to cellular structures and to the fact that ATP competitive inhibitors have to cope with a very high ATP concentration within the cell. Collectively taken, the data presented fill a gap in the field of signal transduction mediated by protein phosphorylation by making available for the first time a specific and cell permeable inhibitor for HIPK2, a protein kinase whose emerging role as regulator of cell growth and apoptosis in various tissues and whose implication in the mode of action of chemotherapeutic agents is rising remarkable interest. The only compound used so far as an HIPK2 inhibitor in fact was developed to inhibit different classes of protein kinases and its efficacy to inhibit HIPK2 activity is questionable, as clearly shown here and
