Abstract:Recent studies of proteins containing kinase-like domains that lack catalytic residue(s) classically required for phosphotransfer, termed pseudokinases, have uncovered important roles in cell signalling across the kingdoms of life. Additionally, mutations within pseudokinase domains are known to underlie human diseases, suggesting that these proteins may represent new and unexplored therapeutic targets. To date, few pseudokinases have been studied in intricate detail, but as described in the present article an… Show more
“…Concomitantly, many members of the protein kinase superfamily have become important therapeutic targets, in part due to their inherent druggability [9], but also because they play rate-limiting roles in many human diseases, including inflammation, heart disease and cancer [10]. Protein kinases are ubiquitous and evolutionary related, possessing canonical amino acid motifs that are positioned at the catalytic interface, trapping divalent metal cofactors and positioning protein substrate directly adjacent to the ATP phosphoryl donor, which is thought to bind prior to substrate in the catalytic cycle [11,12]. This conserved mode of ligand binding implies that the development of specific probe compounds to interrogate protein kinase catalytic function is a challenge, and much remains to be learnt about the chemical and protein ligand sensitivity of ‘open’ and ‘closed’ (lying between ‘inactive’ and ‘active’) conformational states that exist within dynamic populations of kinase signaling complexes [13].…”
cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R2C2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.
“…Concomitantly, many members of the protein kinase superfamily have become important therapeutic targets, in part due to their inherent druggability [9], but also because they play rate-limiting roles in many human diseases, including inflammation, heart disease and cancer [10]. Protein kinases are ubiquitous and evolutionary related, possessing canonical amino acid motifs that are positioned at the catalytic interface, trapping divalent metal cofactors and positioning protein substrate directly adjacent to the ATP phosphoryl donor, which is thought to bind prior to substrate in the catalytic cycle [11,12]. This conserved mode of ligand binding implies that the development of specific probe compounds to interrogate protein kinase catalytic function is a challenge, and much remains to be learnt about the chemical and protein ligand sensitivity of ‘open’ and ‘closed’ (lying between ‘inactive’ and ‘active’) conformational states that exist within dynamic populations of kinase signaling complexes [13].…”
cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R2C2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.
“…A classification scheme based on differences in nucleotide binding has recently been proposed (8). Since excellent reviews on pseudokinases are available that focus on their noncatalytic roles in signaling pathways (9)(10)(11)(12), we will focus here on a new way of thinking about kinase and pseudokinase regulation as driven by hydrophobic spine assembly and the potential noncatalytic functions for bona fide kinases. Specifically, we wish to extend ideas about pseudokinases by asking (i) do all kinases, in fact, have bifunctional properties with both catalytic and noncatalytic functions, (ii) can we uncouple and independently explore these two functions, and (iii) are both functions essential for signaling or in some cases is the noncatalytic function sufficient?…”
Protein kinases are thought to mediate their biological effects through their catalytic activity. The large number of pseudokinases in the kinome and an increasing appreciation that they have critical roles in signaling pathways, however, suggest that catalyzing protein phosphorylation may not be the only function of protein kinases. Using the principle of hydrophobic spine assembly, we interpret how kinases are capable of performing a dual function in signaling. Its first role is that of a signaling enzyme (classical kinases; canonical), while its second role is that of an allosteric activator of other kinases or as a scaffold protein for signaling in a manner that is independent of phosphoryl transfer (classical pseudokinases; noncanonical). As the hydrophobic spines are a conserved feature of the kinase domain itself, all kinases carry an inherent potential to play both roles in signaling. This review focuses on the recent lessons from the RAF kinases that effectively toggle between these roles and can be "frozen" by introducing mutations at their hydrophobic spines.
“…1. Direct modulation of this sort appears to be particularly important for pseudokinases [7], which are amongst the best-studied pseudoenzymes and the subject of research in the authors’ laboratories.Fig. 1.Pseudoenzymes can allosterically regulate a partner protein’s catalytic activity.…”
Section: How Do Catalytically Inert Enzymes Impact On Signalling Netwmentioning
confidence: 99%
“…The deviations in ‘pseudoactive’ site geometry raise the attractive idea that pseudoenzymes might have evolved distinct features that will enable their specific targeting by small molecules in preference to their catalytically active cousins. Of particular interest in the field of signalling is that of the >50 protein pseudokinases found in vertebrates [7], half have already been implicated in one disease or another [6]. This furnishes enough potential drug candidates to keep pharma busy for years, and we predict that the pseudophosphatase, pseudoprotease and pseudo-deubiquitinase fields are also likely to rise to pharmaceutical prominence over the next decade.…”
Section: Can Pseudoenzymes Be Therapeutically Targeted?mentioning
Pseudoenzymes are catalytically deficient variants of enzymes that are represented in all major enzyme families. Their regulatory functions in signalling pathways are shedding new light on the non-catalytic functions of active enzymes, and are suggesting new ways to target cellular signalling mechanisms with drugs.
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