Review
Feature Review
Protein kinases: evolution of dynamic regulatory proteins

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Eukayotic protein kinases evolved as a family of highly dynamic molecules with strictly organized internal architecture. A single hydrophobic F-helix serves as a central scaffold for assembly of the entire molecule. Two non-consecutive hydrophobic structures termed “spines” anchor all the elements important for catalysis to the F-helix. They make firm, but flexible, connections within the molecule, providing a high level of internal dynamics of the protein kinase. During the course of evolution, protein kinases developed a universal regulatory mechanism associated with a large activation segment that can be dynamically folded and unfolded in the course of cell functioning. Protein kinases thus represent a unique, highly dynamic, and precisely regulated set of switches that control most biological events in eukaryotic cells.

Section snippets

Protein kinases: major signal transmitters in eukaryotic cells

Protein phosphorylation was discovered as a regulatory mechanism by Krebs and Fischer in the late 1950s through their classic studies of glycogen phosphorylase and their subsequent discovery of phosphorylase kinase [1]. The second protein kinase to be discovered was cAMP-dependent protein kinase (PKA) [2]. The tremendous diversity of the protein kinase family was later revealed when Src, the transforming protein from Rous sarcoma virus, was discovered to be a kinase that phosphorylates tyrosine

Internal architecture of protein kinases

Because the kinases are so important, not only for biology, but also for disease phenotypes, many kinase structures have now been solved (Box 1). What can we learn from this structural kinome that we could not learn from in depth analysis of a single kinase? By comparing many protein kinase structures, and searching, in particular, for spatially conserved residues, the internal architecture that allows for the assembly of an active protein kinase was revealed. It is an architecture that enables

Activation/inactivation of a protein kinase

One of the most important features that distinguish protein kinases from ELKs, and from many other more classical metabolic enzymes, is that they are highly regulated in a manner that typically involves a dynamic reorganization of the molecule 22, 27. The complicated machinery that is added to the simpler ELK scaffold to achieve these two functions is exceptional [23], not only for its complexity, but also for its dynamic properties. The function of EPKs is not simply to efficiently turn over

Gatekeepers, spines and kinase inhibitors

An important role in protein kinase activation can be played by a so-called “gatekeeper” residue [36] positioned between the C- and the R-spine (Figure 7a). A survey of the human kinases reveals that 77% of all human kinases have relatively large (Leu, Met, Phe) residues in this position, whereas 21% of them, mostly tyrosine kinases, have smaller gatekeeper residues (Thr, Val) [37]. Mutagenesis of large gatekeepers to a smaller side chain allowed the engineering of mutant kinases that could

Completing the protein kinase core

As we discussed earlier, the F-helix and two hydrophobic spines serve as a scaffold for the assembly of the active protein kinase core. Is this sufficient for optimal catalysis? In some cases, it might be, but in most cases organizing the activation segment is an essential priming step, but is not sufficient for creating the most efficient catalyst. Often, there is also a requirement for optimal orientation of the N-lobe. Each kinase will achieve this in a different way, but there are two

Dynamic integration of the kinase for catalysis

Once the kinase is activated by assembly of the R-spine and the N-lobe is correctly positioned, the enzyme is poised for catalysis. Identification of the C-spine also provides a new framework for considering how the catalytic machinery is coordinated. Unlike many other enzymes such as the proteases where there is a catalytic triad that has evolved in both convergent and divergent ways that allow for the same positioning of the three catalytic residues, protein kinases that phosphorylate Ser,

Evolution of the eukaryotic protein kinases

If we return now to the ELKs, and compare them structurally and functionally to the EPKs, we can better appreciate what is so unique about the EPKs, and how they have evolved to be such dynamic molecular switches. As a result of evolution, two additional segments have been added to the conserved core of the EPKs to achieve fast and efficient regulation (Figure 8). The first is the insertion of the activation segment between β9 and the F-helix. The second EPK-specific segment is the

Pseudokinases

About 10% of the human kinome contains most of the major sequence motifs that define the eukaryotic protein kinase structure, specifically the activation loop and the helical GHI subdomain, as discussed above, but harbors mutations in positions that were thought to be crucially important for catalysis [53]. In the beginning, these were collectively referred to as “pseudokinases” and were assumed to be inactive. As was demonstrated recently, however, pseudokinases come in many different flavors.

Concluding remarks and future perspectives

Recognizing the protein kinase internal architecture and the importance of a hydrophobic scaffold creates a new framework for analysis of the function and regulation of this biologically important enzyme family. The unique feature of these enzymes is that they are highly regulated, and their activation creates a conserved conformation that integrates the hydrophobic scaffold. The scaffold can be broken in a variety of ways, and the activation, typically achieved by phosphorylation of the

Acknowledgments

Research is supported by the National Institutes of Health, grant GM19301 for S.S.T.

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