Protein folding creates structure-based, noncontiguous consensus phosphorylation motifs recognized by kinases.

Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C βI (PKCβI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCβI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.