A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory ␣C-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the ␣C-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation. P rotein kinases, whose genes represent one of the largest gene families (1), have evolved to be dynamic molecular switches that regulate most biological processes (2). Typically in a basal inactive state, they are dynamically assembled into an active conformation by a complex set of regulatory events that can include recruitment to membranes, dimerization, phosphorylation, and/or translocation to the nucleus. Because protein kinases are associated with so many diseases, especially cancers, we have a large collection of structures, which allows us to explore their dynamic properties at the molecular level.We previously identified two highly conserved structural entities known as the hydrophobic spines that are common to all kinases (2, 3). The first spine to be identified is referred to as the regulatory spine, or R-spine, and the assembled R-spine is a hallmark signature of every active kinase (3). The R-spine consists of four residues, RS1 to RS4, two from the C-lobe and two from the N-lobe. Each R-spine residue comes from a critical part of the kinase. RS1 is the histidine residue from the HRD motif in the catalytic loop. RS2 is the phenylalanine from the DFG motif in the activation segment. RS3 is a conserved aliphatic residue from the ␣C-helix, and RS4 is an aliphatic residue from the 4-strand. Assembly of the R-spine, which is typically mediated by a highly regulated set of events, results in the formation of the active conformation of the kinase.The second spine, known as the catalytic spine, or C-spine, consists of a series of hydrophobic residues and is completed after ATP binds (2). Two of the conserved residues in the C-spine are from the N-lobe, and six are from the C-lobe. The binding of the a...
The synchronized motions of inner core residues allosterically modulate the activity of the protein kinase A catalytic subunit.
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.
The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of “communities” composed of congruently dynamic residues that make up the protein kinase core. Girvan–Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures.
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