Summary Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity, but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation loop phosphorylation. Since N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.

Biochemical and structural analysis of two features of kinase structure, the “R-spine” and “Shell,” afford a detailed insight into the regulation of eukaryotic protein kinases.

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 epigenome and three-dimensional (3D) –genomic architecture are emerging as key factors in the dynamic regulation of different transcriptional programs required for neuronal functions. Here we utilize an activity-dependent tagging system in mice to determine the epigenetic state, 3D-genome architecture, and transcriptional landscape of engram cells over the lifespan of memory formation and recall. Our findings reveal that memory encoding leads to an epigenetic priming event, marked by increased accessibility of enhancers without corresponding transcriptional changes. Memory consolidation subsequently results in spatial reorganization of large chromatin segments and promoter-enhancer interactions. Finally, with reactivation, engram neurons utilize a subset of de novo long-range interactions, where primed enhancers were brought in contact with their respective promoters to up-regulate genes involved in local protein translation in synaptic compartments. Collectively, our work elucidates the comprehensive transcriptional and epigenomic landscape across the lifespan of memory formation and recall in the hippocampal engram ensemble. The formation and preservation of long-term memories depends on coordinated gene expression and synthesis of synaptic proteins 1 . These molecular processes act within a specific population of neurons, referred to as engram cells 2 – 4 . Recent approaches using activity-dependent expression of reporters, provided a framework for exploring the engram ensemble 5 – 8 , but the molecular mechanisms that govern memory storage and retrieval remain poorly understood. Specifically, epigenetic modifications and 3D -genomic architecture are emerging as a key factors in dynamic regulation of gene expression 9 – 17 , and there is an increasing appreciation of their importance in neuronal function, development and disease 14 , 16 , 18 Here, we utilized the Targeted Recombination in Active Populations (TRAP) mouse model 5 , 6 , in which activated neurons expressing the Activity Regulated Cytoskeleton Associated Protein, ( Arc ) gene, are permanently tagged in an inducible manner. Activated neurons during memory encoding, consolidation and recall were sorted and subjected to nuclear RNA sequencing (nRNA-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and chromosome conformation capture (Hi-C). Our data demonstrates that memory encoding leads to a genome-wide increase in chromatin accessibility, without expected changes in gene expression. Furthermore, we demonstrate that late phase of memory consolidation was associated with re-localization of large chromatin segments (sub-compartments) from...