PKA: Lessons learned after twenty years

Highlights

• We discovered a hydrophobic spine architecture in protein kinases.

• This architecture defines highly dynamic nature of protein kinases.

• We report diverse quaternary structures for different isoforms of protein kinase A.

Abstract

The first protein kinase structure, solved in 1991, revealed the fold that is shared by all members of the eukaryotic protein kinase superfamily and showed how the conserved sequence motifs cluster mostly around the active site. This structure of the PKA catalytic (C) subunit showed also how a single phosphate integrated the entire molecule. Since then the EPKs have become a major drug target, second only to the G-protein coupled receptors. Although PKA provided a mechanistic understanding of catalysis that continues to serve as a prototype for the family, by comparing many active and inactive kinases we subsequently discovered a hydrophobic spine architecture that is a characteristic feature of all active kinases. The ways in which the regulatory spine is dynamically assembled is the defining feature of each protein kinase. Protein kinases have thus evolved to be molecular switches, like the G-proteins, and unlike metabolic enzymes which have evolved to be efficient catalysis. PKA also shows how the dynamic tails surround the core and serve as essential regulatory elements. The phosphorylation sites in PKA, introduced both co- and post-translationally, are very stable. The resulting C-subunit is then packaged as an inhibited holoenzyme with cAMP-binding regulatory (R) subunits so that PKA activity is regulated exclusively by cAMP, not by the dynamic turnover of an activation loop phosphate. We could not understand activation and inhibition without seeing structures of R:C complexes; however, to appreciate the structural uniqueness of each R₂:C₂ holoenzyme required solving structures of tetrameric holoenzymes. It is these tetrameric holoenzymes that are localized to discrete sites in the cell, typically by A Kinase Anchoring Proteins where they create discrete foci for PKA signaling. Understanding these dynamic macromolecular complexes is the challenge that we now face. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Introduction

As we look back over the past two decades since the first protein kinase structure was solved, we find that we are entering a new era in our understanding not only of cAMP-dependent Protein Kinase (PKA) but also of all protein kinases. Although our most comprehensive understanding of the protein kinases at the molecular level has come from crystal structures, we are now coming to appreciate that these are surprisingly dynamic molecules that have specifically evolved to be highly regulated molecular switches and not efficient catalysts [1]. Thus our conventional ways of looking at enzymes may have to be revisited when we think about these regulated switches that work on proteins, not peptides, and are part of dynamic and typically oscillating macromolecular complexes. Furthermore, although the eukaryotic protein kinases (EPKs) all share a conserved kinase core, they are regulated in highly dynamic ways by the tails and linker segments that flank that kinase core. These dynamic flanking regions are frequently an integral part of the active kinase; however, unlike the conserved core, they differ in each kinase. Almost every protein kinase is also regulated in some way by phosphorylation whether it is mediated by auto-phosphorylation or by a heterologous kinase. Some of these phosphorylation sites lie in the core, specifically in the activation loop, while other sites are in the tails and linkers. These phosphates can play very different roles.

So at this point in time, just over twenty years since the first protein kinase structure was solved, we find ourselves with an enormous amount of information about this enzyme family, which has also become a major drug target for the pharmaceutical and biotechnology industries. We are also now entering a new era of computation where we can carry out simulations of proteins that reach μs to ms time scales, which lie in the range where most catalytic and regulatory functions occur. This allows us for the first time to actually experimentally validate the simulations. Advances in NMR spectroscopy where we can explore the residue-specific dynamics of a protein in solution are also revealing new insights about the dynamic properties of this enzyme family [2], [3], [4]. Instead of being limited to the static structures that are defined in a crystal lattice, we can now begin to explore in a far more comprehensive way how these proteins behave in solution. Most importantly we are coming to appreciate that these molecules have evolved to do something different from metabolic enzymes that we have studied so intensely in the past. Whereas the metabolic enzymes have evolved to be efficient catalysts that turn over large amounts of small molecule substrates, the protein kinases, like the GTPases, have evolved to be molecular switches that initiate a cascade of downstream signaling events. Often they function as part of a macromolecular complex under single turnover conditions where the substrate and kinase are in a 1:1 complex. While efficient catalysis is important for a metabolic enzyme, dynamic and precise regulation is essential for a switch. Oscillating between off and on states is essential for almost every protein kinase.