Nieuwe inzichten in de farmacologische inhibitie en isovormspecifieke regulatie van Proteine Kinase D2

Protein kinases are essential to life. They catalyze the phosphorylation of their substrates, which in turn alters their function. Expectedly, kinase dysregulation has been associated with multiple disorders, including cancer, and they are often targets for small-molecule inhibition.In this manuscript we studied the Protein Kinase D (PKD) family. This family belongs to the Calmodulin kinase (CAMK) group and consists of three isoenzymes in humans. While the isoenzymes are structurally highly similar, they are not always functionally redundant, and sometimes the isoenzymes even have opposing roles. The molecular basis for these isoenzyme-specific functions is not well understood. PKDs are classically activated via signaling pathways that generate diacylglycerol downstream of G-protein coupled receptors or receptor tyrosine kinases, but they are also activated in oxidative stress conditions. Remarkably, we uncovered an isoenzyme-specific regulation of PKD2 in oxidative stress through the Abl-mediated phosphorylation of a highly conserved Tyr residue in the P+1 loop (i.e. the substrate binding loop) of the activation segment. Phosphorylation specifically occurs in PKD2 due to an Abl recognition motif that is divergent between PKD1/2/3. Phosphorylation of this Tyr residue results in higher substrate turnover by PKD2. We also observed isoform-specific signaling to NF-κB in oxidative stress conditions, but this was independent of P+1 loop Tyr phosphorylation.Interestingly, while P+1 loop Tyr phosphorylation of PKD2 is dependent of upstream kinases in cells, PKDs (all isoforms) can autophosphorylate this residue in vitro. This activity is only seen in in vitro assays. This is due to the fact that PKDs associate with an autophosphorylation-inhibiting factor in cells which prohibits Tyr autophosphorylation. Interestingly, we show that the molecular determinants for Tyr autophosphorylation are different from those for Ser-autophosphorylation and trans-phosphorylation of peptide. Additionally, we show that Tyr autophosphorylation activity is dependent on the presence of an unusual Cys residue in the HxD motif of the catalytic loop. This residue is an Arg in most kinases, but is often substituted in dual-specificity kinases.Because of the role of PKD isoforms in cancer, we were also interested in the development of PKD inhibitors. In this regard, bulky PP1 analogs (e.g. 1-NM-PP1), which normally do not bind WT kinases due to clashes with the ‘gatekeeper’ amino-acid were an interesting starting point for the rational design of a potent PKD inhibitor, since they unexpectedly inhibit WT PKDs. Our structure-activity relationships (SAR) studies of 1-NM-PP-1 derivates uncovered an unusual binding mode of these type of inhibitors to PKD and resulted in the development of the most active compound of this class as of yet. These data thus provide new insights for the potential development of future PKD-specific compounds.

Jaar van publicatie:2017

Toegankelijkheid:Closed