Phage-peptide display identifies the Interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1

Burch, L. R., Scott, M. , Pohler, E., Meek, D. and Hupp, T. (2004) Phage-peptide display identifies the Interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1. Journal of Molecular Biology, 337(1), pp. 115-128. (doi:10.1016/j.jmb.2003.10.081) (PMID:15001356)

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Abstract

Phage-peptide display is a versatile tool for identifying novel protein–protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21WAF1. ZIP kinase phosphorylates p21WAF1 at Thr145 and alanine-substituted mutations in the p21WAF1 phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21WAF1 at Thr145 in vivo and can increase the half-life of p21WAF1, while the half-life of p21WAF1[T145A] is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21WAF1, and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Scott, Dr Mary
Authors: Burch, L. R., Scott, M., Pohler, E., Meek, D., and Hupp, T.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Journal of Molecular Biology
Publisher:Elsevier
ISSN:0022-2836
ISSN (Online):1089-8638
Published Online:21 February 2004

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