PKN1–3 requires phosphorylation of the activation loop by PDK1 (phosphoinositide-dependent kinase 1) as well as turn motif phosphorylation for full catalytic activity. Similar to other AGC kinases, PKN1–3 are structurally conserved in the activation loop, as well as C-terminal segments termed the hydrophobic and turn motif. The PKN enzymes are ~100 kDa proteins and are closely related to the novel isoforms of the protein kinase C enzyme superfamily. The PKN (protein kinase N) family, also known as the PRKs (protein kinase C-related kinases), belongs to the AGC family of kinases and consists of three members: PKN1/PRK1, PKN2/PRK2 and PKN3. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent ( K i<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in k cat for PKN1 and PKN2, and a decrease in peptide K M for PKN3. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization.
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