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Eukaryotic 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.

Original publication

DOI

10.1016/j.molcel.2013.11.013

Type

Journal article

Journal

Mol Cell

Publication Date

09/01/2014

Volume

53

Pages

140 - 147

Keywords

Binding Sites, Crystallography, X-Ray, HEK293 Cells, Humans, Kinetics, Peptides, Phosphorylation, Protein-Serine-Threonine Kinases, Substrate Specificity, p21-Activated Kinases