Using High-Throughput Microarrays To Profile the Specificities and Identify Novel Substrates of Protein Phosphatases

The reversible phosphorylation and dephosphorylation of proteins catalyzed by protein kinases (PK) and protein phosphatases (PP), respectively, is well recognized as a predominant means of intracellular signal transduction. Protein phosphorylation plays a critical role in the regulation of core cellular processes, and defects in phosphorylation-mediated signaling underlie more than 400 diseases including cancer, diabetes, and autoimmunity. To clearly define the biological roles of PK’s and PP’s in healthy and diseased cellular states, we need to identify and characterize their physiological substrates. In contrast to PK’s, the large majority of PP’s are poorly characterized and their physiological substrates and, consequently, their biological roles remain ill-defined. Therefore, our goal is to elucidate the substrate specificities of phosphatases and to identify their physiological substrates. Here, we present the application of a high-throughput peptide microarray to profile the substrate specificities of recombinant, purified human protein phosphatases and to identify their potential in vivo substrates. Most catalytic enzymes, including kinases and phosphatases, display intrinsic selectivity for specific substrate sequence features, which can be mimicked by peptides. Therefore, by assaying large assortments of peptides that exhibit a high degree of sequence variation, the substrate specificities of enzymes can be worked out through analysis of their ‘substrate fingerprints’. To study the substrate specificities of protein phosphatases, we chemically synthesized and printed onto glass slides a library of 500 phosphorylated peptides, which represent the sequences of the most highly conserved physiological phosphorylation-sites (P-sites) in humans, and also exhibit high sequence variation. These phosphopeptide microarrays were screened against a selection of purified recombinant phosphatases in high-throughput dephosphorylation assays. Analysis of dephosphorylation fingerprints of individual phosphatases allows for the identification of peptide sequences that serve as good substrates. Next, through the alignment of optimal substrate sequences, we can determine consensus sequence motifs that feature in favorable substrates. Finally, to identify potential in vivo substrates of the phosphatases, protein and P-site databases are searched for proteins containing the specificity determining consensus sequence motifs. Promising substrates are then investigated in vivo through co-immunoprecipitation of phosphatases and putative substrate. Using our phosphopeptide microarray, we have been able to confirm previously reported substrate consensus sequence motifs for well-studied phosphatases, determine the sequence specificities of other protein phosphatases, as well as identify potential physiological substrates that may be confirmed through future in vivo studies.