Site recognition and substrate screens for PKN family proteins

Alejandra Collazos, Nicholas Michael, Richard D H Whelan, Gavin Kelly, Harry Mellor, Leon C H Pang, Nick Totty, Peter J Parker

Research output: Contribution to journalArticle (Academic Journal)

16 Citations (Scopus)

Abstract

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); 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 (epidermal growth factor receptor), was identified. This identified Thr⁶⁵⁴ in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr⁶⁵⁴ is shown to be modulated by PKN in vivo.
Original languageEnglish
Pages (from-to)535-43
Number of pages9
JournalInternational Journal of Biochemistry and Cell Biology
Volume438
Issue number3
DOIs
Publication statusPublished - 2011

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    Collazos, A., Michael, N., Whelan, R. D. H., Kelly, G., Mellor, H., Pang, L. C. H., Totty, N., & Parker, P. J. (2011). Site recognition and substrate screens for PKN family proteins. International Journal of Biochemistry and Cell Biology, 438(3), 535-43. https://doi.org/10.1042/BJ20110521