Determining protein kinase substrate specificity by parallel solution-phase assay of large numbers of peptide substrates

PROTOCOL

Determining protein kinase substrate specificity by parallel solution-phase assay of large numbers of peptide substrates

Benjamin E. Turk1, Jessica E. Hutti2, and Lewis C. Cantley2

2006 Nature Publishing Group http://www.nature.com/natureprotocols

1Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. 2Department of Systems Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA. Correspondence should be addressed to B.E.T. ([email protected]). Phone: [phone]. Fax: [phone].

Published online 27 June 2006; doi:10.1038/nprot.2006.57

We describe here a protocol for determining the activity of protein kinases on a large set of peptide substrates. Biotin-tagged peptides are arrayed in multiwell plates and incubated in solution with the kinase of interest and radiolabeled ATP. Reactions are then spotted simultaneously onto a streptavidin membrane, which is washed, dried, and analyzed by autoradiography or phosphor imaging. Differences in the extent of radiolabel incorporation into the various peptide substrates provide a measure of the sequence specificity of the kinase. This approach is a faster, more sensitive, and more generally applicable method for determining kinase phosphorylation motifs than older peptide library screening approaches based on Edman sequencing. The procedure is readily adaptable to other applications that require parallel processing of many kinase reactions, such as screening for small molecule inhibitors. In the format described here, preparation of stock plates prior to running the reactions will require about 4 days. Afterwards, the protocol takes approximately 6 hours to perform.

INTRODUCTION

Protein kinases generally display a high degree of sequence specificity at sites of phosphorylation. Studies of a protein kinase are greatly facilitated by knowing its consensus phosphorylation motif. For example, one can use this information to design peptide substrates useful for studying regulation of the kinase or in screening for inhibitors. In addition, inspecting for sequences that match the consensus can identify specific phosphorylation sites in protein substrates. In the protocol described here, the substrate specificity of a protein serine-threonine kinase is determined by simultaneously measuring its ability to phosphorylate each member of a library of about 200 peptide substrates1 (Table 1). Each peptide contains a central fixed phosphorylation site (an equi-molar mixture of serine and threonine) flanked on either side by degenerate (mixture)...