Homing in: Mechanisms of Substrate Targeting by Protein Kinases

Abstract

Protein phosphorylation is the most common reversible post-translational modification in eukaryotes. Humans have over 500 protein kinases, of which more than a dozen are established targets for anticancer drugs. All kinases share a structurally similar catalytic domain, yet each one is uniquely positioned within signaling networks controlling essentially all aspects of cell behavior. Kinases are distinguished from one another based on their modes of regulation and their substrate repertoires. Coupling specific inputs to the proper signaling outputs requires that kinases phosphorylate a limited number of sites to the exclusion of hundreds of thousands of off-target phosphorylation sites. Here, we review recent progress in understanding mechanisms of kinase substrate specificity and how they function to shape cellular signaling networks.

Keywords: enzyme specificity; linear sequence motif; protein interactions; protein kinase; protein phosphorylation.

Figure 1

Overview of the major types of kinase-substrate interactions. Kinases target their substrates through a combination of catalytic domain interactions both proximal and distal to the active site, interactions of short linear sequence motifs with protein interaction modules, and indirect interactions mediated by adaptor or scaffold proteins.

Figure 2

Canonical and non-canonical kinase-substrate interactions. (A) Canonical peptide binding mode (PDB:1O6K). Substrate peptide binds in an extended conformation, with amino acid side-chains accessing distinct pockets. Kinase residues that determine phosphoacceptor residue specificity are shown in space fill, including the phosphorylatable residue in the Gly loop [8] (green) and the DFG+1 residue [7] in the activation loop (purple). (B) Atypical interactions between the kinase catalytic cleft and peptide substrates. The Haspin-histone H3 peptide complex, in which the peptide backbone makes a 180° turn near the phosphoacceptor, is shown at left (PDB: 4OUC). In the PKCι-Par3 peptide complex shown at right (PDB: 4DC2) the peptide backbone makes two β-turns N-terminal to the phosphoacceptor residue. (C) A model for PKCβ bound to tubulin, in which basic residues in the substrate distal in primary sequence are close to the phosphoacceptor residue in the 3D structure. (D) LIMK interacts with cofilin through a hydrophobic interaction centered on the α_G helix of the kinase, in which placement of the phosphoacceptor requires a structured substrate (PDB: 5HVK). The Met115 residue on cofilin is critical for LIMK recognition.

Figure 3

MAPK docking interactions. Substrate D-site motifs (left) bind to a groove located on the opposite face of the kinase from the catalytic cleft. D-sites contain a cluster of basic residues upstream of two or three hydrophobic residues, the spacing of which can select for distinct MAPKs. ERK and p38 MAPKs can also bind DEF-sites (right) through a pocket adjacent to the active site, with different isozymes targeting distinct motifs. Logos for D-sites and DEF-sites were generated from published data [20, 25], using EnoLogos. ERK2 structures (top) were based on the co-crystal structure with the HePTP D-site peptide (PDB: 2FYS) and an HDX-MS model of a bound DEF-site peptide [21].

Figure 4

Substrate quality in biological context. (A) Higher quality substrates (i.e. those that are more efficiently phosphorylated by a kinase) are phosphorylated rapidly and to higher stoichiometry even at lower kinase activity. (B) Varying quality of mTOR substrates rationalizes their differential sensitivity to the mTORC1 inhibitor rapamycin. (C) Increasing CDK activity during the cell cycle contributes to the timing of substrate phosphorylation, with high quality substrates phosphorylated early (in G1/S phase) and lower quality substrates phosphorylated late (in G2/M phase).