Determination of Peptide Substrate Motifs for Protein Kinases Using a “One-Bead One-Compound” Combinatorial Library Approach

Abstract

Protein phosphorylation is one of the more than 100 known posttranslational modifications of proteins (1–4). There are more than 240 protein phosphorylation sites reported (5), and many protein kinases have been cloned and expressed. The sites of phosphorylation are usually serine, threonine, tyrosine, or occastonally histrdine. The two major classes of protein kinases are serme-threonine protein kmase and protein tyrosine kinase. Substrate motifs for several serine-threonine kinases are known (5), and they are often confined to a linear motif. In contrast, little is known about the substrate speclficity of protein tyrosine kinases (PTKs) and peptide substrates based on the autophosphorylation stie of these enzymes are often very inefficient, with a K _m in the high micromolar to millimolar range. It was not until recently when combinatorial peptide library approaches were used that some novel and relatively more efficient peptide substrates for some PTKs were discovered (6–8). Songyang et al. (6) synthesized a biased random peptide library with the following general structure: MXXXXYXXXXAKKK, where X = all 15 amino acids except Tyr, Trp, Cys, Ser, and Thr. The peptide mixtures were phosphorylated in vitro with a specific PTK and unlabeled ATP. The phospho-peptides were then isolated by a ferric chelation column. The eluted peptides were then sequenced concurrently (6; see also Chapter 11). Therefore, the resulting motif is a summation of the sequences of all the peptides recovered. We (7,8), on the other hand, used the “one-bead one-compound” library method by incubating a totally random peptide-bead library (e.g., XXXXXXX, where X = all 19 amino acids except Cys) with [γ-³²P]ATP and a specific protein kmase. The [³²P]-labeled peptide-beads were then localized by autoradrography, isolated, and microsequenced individually. Peptide substrates with multiple motifs can be identified using this method. Although we limit the following discussion on methodologies for determining phosphorylation motifs, the “one-bead one-compound” library approach, in principle, can also be applied to the determination of substrate motifs for other post-translational modifications, such as glycosylation, ribosylation, and methylation.