RESUMEN
Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 "known" substrates and two independent and larger oriented peptide array libraries (OPALs) of â¼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Humanos , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Fosforilación , Fosfoserina/metabolismo , Proteínas Proto-Oncogénicas c-akt/química , Curva ROC , Especificidad por SustratoRESUMEN
The discovery and characterization of molecular interactions is crucial towards a better understanding of complex biological processes. Particularly protein-protein interactions (i.e., PPIs), which are responsible for a variety of cellular functions from intracellular signaling to enzyme-substrate specificity, have been studied broadly over the past decades. Position-specific scoring matrices (PSSM) in particular are used extensively to help determine interaction specificity or candidate interaction motifs at the residue level. However, not all studies successfully report their results as a candidate interaction motif. In many cases, this may be due to a lack of suitable tools for simple analysis and motif generation. Peptide Specificity Analyst (PeSA) was developed with the goal of filling this information gap and providing an easy to use software to aid peptide array analysis and motif generation. PeSA utilizes two models of motif creation: (1) frequency-based using a user-defined peptide list, and (2) weight-based using experimental binding results. The ability to produce motifs effortlessly will make studying, interpreting and disseminating peptide specificity results in an effortless and straightforward process.
Asunto(s)
Péptidos/análisis , Péptidos/química , Programas Informáticos , Histona Metiltransferasas/análisis , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina , Humanos , Péptidos/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Especificidad por SustratoRESUMEN
Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands. Here, in this chapter, I describe the broad use of SPOT peptide arrays for examining SH2 domain specificity. An orientated peptide array library (OPAL) approach can uncover both favorable and non-favorable residues, thus providing an in-depth analysis to SH2 specificity. Moreover, I discuss the application of SPOT arrays for paneling SH2 ligand binding with physiological peptides.