Kinase Regulation by Hydrophobic Spine Assembly in Cancer

Abstract

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...

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

“…The assembled regulatory "R spine" is the hallmark signature of an active kinase, and the disassembled-assembled R-spine switch constitutes the dynamic link between kinase inactivation-activation (5). The catalytic "C spine" is completed by binding of the ATP adenine ring that primes the kinase for catalysis (5,6). Phosphorylation of an activation loop in the large lobe forms an almost universal mechanism for EPK regulation (7,8).…”

The Ys and wherefores of protein kinase autoinhibition

“…A systematic evaluation of the biochemical properties of the R--spine residues and number of surrounding, "shell" hydrophobic residues confirmed that the hydrophobic nature of their side chains is essential for catalytic activity and provided other insights of value for understanding kinase regulation and of interest in the development of rational approaches to kinase drug discovery [23]. Interestingly, while the bottom two R--spine residues usually have aromatic side chains, the αC--helix R--spine residue side chain is usually Leu and mutation to bulkier Ile, Met or Phe is sufficient to drive kinase activity [24]. Displacement of the DFG motif, αC--helix and R--spine into positions incompatible with catalysis is part of the regulatory mechanism of many kinases and can also be induced by small molecule inhibitors of kinases.…”

Integration of signaling in the kinome: Architecture and regulation of the αC Helix

“…Once again it becomes increasingly clear that the protein kinases, like the GTPases, have evolved to be highly dynamic and regulated switches. In some cases the kinase can contribute to downstream signaling without actually transferring the phosphate while in other cases transfer of the phosphate is essential [12-15]. In either case, however, these conformational switches are highly regulated.…”

“…Instead we must consider dynamics as an integral intermediate step that links structure and function. There are two levels of dynamics: one relates to activation and the assembly of the active kinase [12] while the other is associated with the dynamics of the catalytic cycle[16]. To understand these dynamic processes one needs to look more deeply at the conserved motifs that were initially defined.…”