Abstract
SummaryEukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the “DFG+1” residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.
Highlights
Proper signal transmission by protein kinases requires that they phosphorylate specific substrates at defined sites
Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue
We show that a residue located in the kinase activation segment, which we term the ‘‘downstream of a conserved Asp-Phe-Gly (DFG)+1’’ residue, acts as a major determinant for serine-threonine phosphorylation site specificity
Summary
Proper signal transmission by protein kinases requires that they phosphorylate specific substrates at defined sites. Protein kinases tend to phosphorylate substrates in the context of consensus sequence motifs that have complementarity to the kinase active site (Pinna and Ruzzene, 1996). Such kinase phosphorylation site motifs play an important role in targeting kinases to specific substrates within the cell, as well as directing kinases to phosphorylate specific sites on their substrates. Ser-Thr kinases possess conserved signature residues within the catalytic domain important for accommodating a small, aliphatic phosphoacceptor residue at the active site (Taylor et al, 1995). Substrate specificity studies have predominantly been conducted in vitro, evidence is emerging indicating that
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