Rapid affinity-based fingerprinting of 14-3-3 isoforms using a combinatorial peptide microarray.

The 14-3-3 proteins are a family of acidic proteins (ca. 30 kDa) expressed in all eukaryotic cells. By binding as either homoor heterodimers to a variety of phosphoserinecontaining proteins, they regulate important cellular events including cell-cycle progression, DNA damage, apoptosis, protein trafficking, signal transduction, cytoskeletal rearrangements, metabolism, and transcriptional regulation. In humans, there are seven distinct but highly homologous 14-3-3 isoforms: b, e, h, g, s, t, and z. Thus far, however, the only isoform directly linked to cancer has been 14-3-3s, which is regulated by the major tumor suppressor gene, p53. Inactivation of 14-3-3s is crucial in tumorigenesis. Consequently, there has been tremendous interest in the determination of the substrate specificity of 14-3-3–phosphopeptide binding as well as the structural basis of this interaction. The elegant work of Yaffe et al. is particularly worth noting, in which a degenerate phosphoserine-oriented peptide library was used to examine the sequence requirements for binding to 14-3-3. The results confirmed the highly conserved substrate-binding specificity amongst the 14-3-3 isoforms, thus giving rise to a consensus hexapeptide binding motif, RXXpSXP, which binds strongly to all 14-3-3 isoforms tested. In other studies, X-ray crystallography was used to provide detailed structural information of seven human 14-3-3 isoforms bound to a phosphopeptide. Interestingly, 14-3-3s, unlike the other six isoforms, was the only protein shown to preferentially form a homodimer upon substrate binding. Our ongoing interest in the use of 14-3-3s as a potential cancer target has prompted us to speculate that it might be possible to identify phosphopeptides that preferentially bind to 14-3-3s over other isoforms. Herein, we report the first peptide microarray platform made of fragmentbased, combinatorial phosphoserine/phosphothreonine-containing heptapeptide libraries (P 3P 2P 1-p(S/T)-P+1P+2P+3) for substrate-specificity determination of seven mammalian 14-3-3 proteins, and the identification of putative 14-3-3sselective motifs (Figure 1). The peptide microarray is a miniaturized screening platform that enables thousands of individually addressable peptides to be simultaneously interrogated on a glass slide. It is well-suited for high-throughput (HT) studies of protein– peptide interactions. Compared to other HT peptide-screening approaches, that is, phage display, SPOT synthesis, and combinatorial peptide libraries (positional scanning (PS), degenerate, and one-bead-one-compound (OBOC)), it offers the unique advantage that every single spot on the slide may be made addressable and quantifiable instantaneously. To obviate the current upper limit of 1000–10000 different features attainable on a microarray platform, we introduced the concept of a fragment-based combinatorial peptide microarray that enables sufficient coverage of all P 3P 2P 1p(S/T)-P+1P+2P+3 sequences with only 1000 different spotting features (500 Nand C-terminal sublibraries each; P+/ represents ten or five individual amino acids (AAs) and X+/ represents an isokinetic mixture of 14 AAs; Figure 1a). [5]