Site recognition and substrate screens for PKN family proteins

International audienceThe protein kinase C related kinases (PRKs also referred to as PKNs) are a kinase family important in diverse functions including migration and cytokinesis. Here, we have re-evaluated and compared specificity for PKN1 and PKN3, and assessed predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and 3 phosphorylate serine residues in sequence contexts that have an arginine at position -3. In contrast, PKN1 and 3 do not tolerate Arg in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP170; a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR, was identified. This identified threonine 654 in EGFR as the PKN1 phosphorylation site and this retains an arginine at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr-654 is shown to be modulated by PKN in vivo