Abstract
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. 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. 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. 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. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcat for PKN1 and PKN2, and a decrease in peptide KM for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.
Highlights
The protein kinase N (PKN) family, known as the PRKs, belongs to the AGC family of kinases and consists of three members: PKN1/PRK1, PKN2/PRK2 and PKN3
PKN activation loop and turn motif phosphorylation analysis Full-length, FLAG-tagged human PKN1, PKN2 and PKN3 were expressed in mammalian HEK-293 cells
The tandem MS (MS/MS) data derived from the resulting fragmentation peptides for PKN1, PKN2 and PKN3 (Figure 1C) were evaluated in Agilent Spectrum Mill using a variety of criteria (Figure 1D)
Summary
The PKN (protein kinase N) family, 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 work addressed a hypothesis that interaction of lipids with the PKNs may free the protein from a compact, inhibited state, leading to enzymatic activation and downstream signalling, similar to the PKC family of kinases. To that extent, using recombinant full-length human enzymes and a synthetic peptide substrate, we determined the kinetic mechanism of PKN isoforms. PKN kinase assay The activity of all PKN proteins was monitored through the use of a highly sensitive off-chip mobility shift assay utilizing a LabChip EZ Reader II instrument (Caliper Life Sciences/PerkinElmer); this assay format has been described elsewhere [31]. Kinase activity was monitored through the phosphorylation of a 5-FAMlabelled PKN substrate peptide found through Pfizer in-house optimization efforts and derived from the amino acid sequence of GSK3α Where v is measured velocity, vo and vi are the corresponding plateaus at low and high lipid concentrations, respectively; EC50 is the concentration at which enzyme activity is stimulated by 50 %, and X is lipid concentration in log units
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