Journal of Molecular Biology
CommunicationDid Protein Kinase Regulatory Mechanisms Evolve Through Elaboration of a Simple Structural Component?
Section snippets
Protein kinase structural features and functional constraints
Eukaryotic protein kinases (EPKs) phosphorylate protein substrates in important signaling pathways and thus are regulated tightly to avoid physiological catastrophes. They are influenced readily by upstream regulators due, in large part, to their conformational flexibility, which is associated with movement of the C-helix, of the activation loop and of the N-lobe relative to the C-lobe.1, 2, 3, 4, 5, 6 Hydrogen exchange studies likewise reveal conformational flexibility within the
An ancient core component of ELKs and EPKs
Two observations can be made regarding the categories of functional constraints shown in Figure 1. First, the constraints shared by ELKs, EPKs, and APKs (Figure 1(a)) center on a handful of widely dispersed residue positions, most of which are involved in catalysis. Second, relatively few residue positions are conserved only in EPKs and ELKs (Figure 1(b)), but nearly all of these interact structurally in a coherent manner either with one another or with residues also conserved in APKs. We term
A hypothetical mechanism involving the HxD-histidine and the DFG-motif
The HxD-histidine (H125Cdk2)(=Y164PKA) appears to play a central role within the ELK/EPK core component and thus within EPKs as a whole. Conserved hydrogen bonds (Figure 3(a)–(c)) link its main-chain both to the C-terminal end of the DFG-motif (at the start of the activation loop) and to the conserved aspartate in the F-helix (which is associated with substrate-binding regions). Furthermore, its side-chain hydrogen bonds both to the main-chain oxygen atom (A144Cdk2)(=T183PKA) directly preceding
The β5-glutamate, the αC-β4 region and nucleotide binding
In EPKs, DFG conformational changes may be associated with both nucleotide binding and movement of the C-helix (via the DFG-phenylalanine). The C-helix movement is itself coupled to nucleotide binding, inasmuch as upon activation the conserved glutamate in the C-helix interacts with the conserved β3-lysine that binds to and positions ATP or ADP.1 Moreover, hydrogen-exchange studies on PKA in the presence or in the absence of ADP reveal that nucleotide binding has distal structural effects;42 in
EPK constraints associated with the αC-β4-loop and the β5 and β8 strands
Within the EPKs strong selective constraints (Figure 1(c)) are imposed on residues interconnecting the αC-β4 loop, the β5 strand, the E-helix and the β8 strand (Figure 3, Figure 4). These interactions may stabilize and modulate conformational changes in the activation loop, the C-helix, and the nucleotide-binding pocket. The β8 strand may play a critical role in this regard, inasmuch as: (i) it is near the center of these interactions; (ii) it precedes the DFG-motif, and thus the activation
The F-helix aspartate links substrate-binding regions to the core component
The F-helix aspartate (D185Cdk2 in Figure 3(a))(=D220PKA) positions the main-chain both of the HxD-histidine residue (H125cdk2)(=Y164PKA), whose likely role was discussed above, and (in most EPKs) of the HRD-arginine (R126cdk2)(=R165PKA), which coordinates with a phosphorylated serine or threonine within the activation loop.2 Mutation of the F-helix aspartate to alanine in cAMP protein kinase failed to abolish catalytic activity,51 suggesting that it fails to play an important catalytic role
Conclusion
EPKs and ELKs share an ancient core component, the most distinctive features of which are the HxD-histidine, the F-helix aspartate residue, the F-helix itself, and the DFG-motif. The F-helix aspartate provides a critical link between the F-helix and the HxD-histidine. The HxD-histidine may be a focal point for signal integration, inasmuch as it both associates with key catalytic elements and influences the DFG region, which can undergo dramatic conformational changes. These, along with
Acknowledgements
This work was supported by NIH (NLM) grant LM06747 to A.F.N. and by a Cold Spring Harbor Association postdoctoral fellowship to N.K. We thank Susan Taylor for helpful discussions.
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Present address: N. Kannan, Department of Chemistry and Biochemistry, The University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0654, USA.