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Protein Kinase Structure and Function
Summary: Susan Taylor and her colleagues are studying the structure, dynamics, and localization of cAMP-dependent protein kinase, a prototype for the protein kinase superfamily. Understanding the dynamic behavior of these proteins, how they behave individually, and how their structure and dynamics are altered as part of a large molecular assembly are fundamental questions for signal transduction today.
Protein phosphorylation is the most important mechanism for regulation in the eukaryotic cell. It is predicted that the human genome encodes more than 600 protein kinases. Although very diverse in how they receive and transmit signals, all protein kinases share a conserved catalytic core. While it is essential to understand how these enzymes function as catalysts, it is equally important to understand how they are regulated. Subcellular localization is also critical; the enzyme must be in the correct part of the cell at the correct time to mediate its proper biological response. Catalysis, regulation, and localization are all mediated protein:protein interactions.
Studying the structure and function of cAMP-dependent protein kinase (PKA), one of the simplest members of the protein kinase family, has been our primary focus. Our initial chemical studies led eventually to our solving the crystal structure of PKA in 1991. This first protein kinase structure continues to serve as a template for the entire family of serine-, threonine-, and tyrosine-specific protein kinases. An underlying theme of our laboratory is the use of biophysical, biochemical, and biological approaches to probe PKA structure and function at both the atomic and cellular levels.
Catalytic Subunit
By solving nearly a dozen structures of the catalytic (C) subunit complexed with various substrates and inhibitors, and as an apoenzyme, we have not only contributed to understanding how the enzyme recognizes substrates but also defined the conformational flexibility of the enzyme. The structure of the C subunit bound to analogs of balanol, a natural product inhibitor of the AGC family of protein kinases and a competitive inhibitor of ATP, shows exquisite complementarity to the active site and provides a foundation for rational design of novel inhibitors for other protein kinases.
Through the various crystal structures, we have defined "open" and "closed" conformations, as well as intermediate states. We recently solved the structure of an ADP:AlF3 complex with a substrate peptide. This is an excellent model for the transition state. Eleven nearly invariant residues cluster around the active-site cleft, mostly interacting with the phosphates of ATP or facilitating phosphoryl transfer. The fully closed conformation, with the tip of the glycine-rich loop hydrogen-bonding to the δ-phosphate of ATP, most likely mimics the transition-state intermediate and is necessary for optimal catalysis; however, the cleft must open to release the nucleotide. Opening the active-site cleft, not phosphoryl transfer, is the rate-limiting step of catalysis. Tethering it in any way will render the enzyme inactive.
The crystal structures provide a static view of various conformational states, but understanding how the domains move in solution and how those motions correlate with functional steps of catalysis requires solution methods. We engineered a series of mutant proteins in which cysteines are selectively introduced and then labeled with a fluorescent probe. Time-resolved fluorescence anisotropy provides a tool for measuring local motion. This approach has revealed novel isoform-induced changes in the mobility of the amino-terminal helix and its accessibility to membranes. These mutant enzymes not only allow us to map different surfaces but also help us to understand the intricate network of interacting residues that link the active site to other regions of the protein. David Johnson (University of California, Riverside) collaborates in these fluorescence studies.
We recently showed that PKA, like AKT, can be phosphorylated on its activation loop Thr by the phospholipid-dependent protein kinase, PDK1. The structural changes that occur as a consequence of this phosphorylation are being explored, as is the specificity of PDK1. Close to this Thr is a reactive cys that can be glutathionylated under stress conditions.
Regulatory Subunits
Solving the structure of a truncated regulatory subunit, RIα, provided a first glimpse of the tandem cAMP-binding domains. The subsequent solution of an RIIβ structure, coupled with site-specific and deletion mutagenesis, is allowing us to dissect the subdomains of the cAMP-binding motif. This is an ancient signaling motif conserved from bacteria to humans. It is also an ancient allosteric motif in which cAMP binding is coupled to a DNA-binding motif (CAP), to kinase regulation (PKA and PKG), to ion channels, and to a guanine nucleotide exchange factor (EPAC).
The amino terminus is multifunctional. It is responsible for maintaining the R subunit as a stable dimer and also serves as a docking site that allows the R subunit and holoenzymes to bind to anchoring proteins that localize the enzyme to specific sites in the cell. We have just solved by nuclear magnetic resonance (NMR) spectroscopy the structure of the dimerization/docking domain (D/D) of RIα, which reveals a novel helix bundle. The D/D domain does not interact directly with the C subunit, but dimerization does enhance the cooperativity of cAMP-mediated activation. The dynamic features of the RIα and RIIβ subunits have been mapped by hydrogen/deuterium (H/D) exchange coupled with mass spectrometry. Docking interfaces are predicted, and we are analyzing structures of R:C complexes. In collaboration with Jill Trewhella (Los Alamos National Laboratory), we have used small-angle x-ray scattering to characterize the free R subunits and the holoenzymes.
A-Kinase–Anchoring Proteins
Although PKA was one of the first protein kinases to be discovered, the importance of its subcellular localization has only recently been recognized. Using a yeast two-hybrid screen, we identified two novel dual-specific A-kinase–anchoring proteins (D-AKAPs) that bind both RI and RII. D-AKAP1 targets PKA to either mitochondria or endoplasmic reticulum via amino-terminal motifs; D-AKAP2 has two putative RGS domains and is linked via its carboxyl-terminal PDZ motif to cotransporters. The biochemical and physiological roles of these AKAPs are being explored at several levels, including nuclear magnetic resonance (NMR), x-ray crystallography, and H/D exchange. Subcellular localization is being characterized using electron microscopy and by expressing proteins that are fused to green fluorescent proteins.
Trafficking Signals and Fluorescence Strategies
In collaboration with Roger Tsien's laboratory (HHMI, University of California, San Diego), we discovered a nuclear export signal (NES) in the heat-stable PKA-specific inhibitor (PKI). Active nuclear export, mediated by an NES, is now recognized to be widespread. With the Tsien laboratory, we have developed new fluorescent probes for measuring kinase activity and localization. A major goal is to understand the role of targeting in PKA signaling.
Last updated: August 28, 2007
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