Oncogenic Mutations in B-Raf Some Losses Yield Gains

AbstractA study by Wan et al. in this issue of Cell demonstrates that the majority of oncogenic mutations in the B-Raf protein kinase result in increased catalytic activity, through disruption of the autoinhibited state of the kinase domain. Surprisingly, several mutations lead to impaired B-Raf kinase activity, yet these mutants are nevertheless capable of stimulating downstream signaling through transactivation of C-Raf

Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations

Background: Janus kinases (JAKs; JAK1 to JAK3 and tyrosine kinase 2) mediate cytokine signals in the regulation of hematopoiesis and immunity. JAK2 clinical mutations cause myeloproliferative neoplasms and leukemia, and the mutations strongly concentrate in the regulatory pseudokinase domain Janus kinase homology (JH) 2. Current clinical JAK inhibitors target the tyrosine kinase domain and lack mutation and pathway selectivity. Objective: We sought to characterize mechanisms and differences for pathogenic and cytokine-induced JAK2 activation to enable design of novel selective JAK inhibitors. Methods: We performed a systematic analysis of JAK2 activation requirements using structure-guided mutagenesis, cell-signaling assays, microscopy, and biochemical analysis. Results: Distinct structural requirements were identified for activation of different pathogenic mutations. Specifically, the predominant JAK2 mutation, V617F, is the most sensitive to structural perturbations in multiple JH2 elements (C helix [aC], Src homology 2-JH2 linker, and ATP binding site). In contrast, activation of K539L is resistant to most perturbations. Normal cytokine signaling shows distinct differences in activation requirements: JH2 ATP binding site mutations have only a minor effect on signaling, whereasJH2aCmutations reduce homomeric (JAK2-JAK2) erythropoietin signaling and almost completely abrogate heteromeric (JAK2-JAK1) IFN-gamma signaling, potentially by disrupting a dimerization interface on JH2. Conclusions: These results suggest that therapeutic approaches targeting the JH2 ATP binding site and aC could be effective in inhibiting most pathogenic mutations. JH2 ATP site targeting has the potential for reduced side effects by retaining erythropoietin and IFN-gamma functions. Simultaneously, however, we identified the JH2 aC interface as a potential target for pathway-selective JAK inhibitors in patients with diseases with unmutated JAK2, thus providing new insights into the development of novel pharmacologic interventions.Peer reviewe

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P2) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P2

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.</p

Mechanistic Insights into Regulation of JAK2 Tyrosine Kinase

JAK2 is a member of the Janus kinase (JAKs) family of non-receptor protein tyrosine kinases, which includes JAK1–3 and TYK2. JAKs serve as the cytoplasmic signaling components of cytokine receptors and are activated through cytokine-mediated trans-phosphorylation, which leads to receptor phosphorylation and recruitment and phosphorylation of signal transducer and activator of transcription (STAT) proteins. JAKs are unique among tyrosine kinases in that they possess a pseudokinase domain, which is just upstream of the C-terminal tyrosine kinase domain. A wealth of biochemical and clinical data have established that the pseudokinase domain of JAKs is crucial for maintaining a low basal (absence of cytokine) level of tyrosine kinase activity. In particular, gain-of-function mutations in the JAK genes, most frequently, V617F in the pseudokinase domain of JAK2, have been mapped in patients with blood disorders, including myeloproliferative neoplasms and leukemias. Recent structural and biochemical studies have begun to decipher the molecular mechanisms that maintain the basal, low-activity state of JAKs and that, via mutation, lead to constitutive activity and disease. This review will examine these mechanisms and describe how this knowledge could potentially inform drug development efforts aimed at obtaining a mutant (V617F)-selective inhibitor of JAK2

Structural basis for the interaction of the adaptor protein grb14 with activated ras.

Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V). The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity

Deer Island Treatment Plant Performance (2007-09)

KCa3.1 (also known as SK4 or IK1) is a mammalian intermediate-conductance potassium channel that plays a critical role in the activation of T cells, B cells, and mast cells, effluxing potassium ions to maintain a negative membrane potential for influxing calcium ions. KCa3.1 shares primary sequence similarity with three other (low-conductance) potassium channels: KCa2.1, KCa2.2, and KCa2.3 (also known as SK1-3). These four homotetrameric channels bind calmodulin (CaM) in the cytoplasmic region, and calcium binding to CaM triggers channel activation. Unique to KCa3.1, activation also requires phosphorylation of a single histidine residue, His358, in the cytoplasmic region, which relieves copper-mediated inhibition of the channel. Near the cytoplasmic C-terminus of KCa3.1 (and KCa2.1-2.3), secondary-structure analysis predicts the presence of a coiled-coil/heptad repeat. Here, we report the crystal structure of the C-terminal coiled-coil region of KCa3.1, which forms a parallel four-helix bundle, consistent with the tetrameric nature of the channel. Interestingly, the four copies of a histidine residue, His389, in an 'a' position within the heptad repeat, are observed to bind a copper ion along the four-fold axis of the bundle. These results suggest that His358, the inhibitory histidine in KCa3.1, might coordinate a copper ion through a similar binding mode

Comparison of Grb14^RA-PH-H-Ras structure with other RA domain-GTPase structures.

<p>(A) Stereo diagram of a superposition (aligned on Ras) of the structures (Cα traces) of Grb14^RA-PH-H-Ras and RalGDS^RA-H-Ras (PDB code 1LFD) [18]. Grb14^RA is colored green, RalGDS^RA is colored dark green, H-Ras is colored cyan (for Grb14^RA) or dark blue (for RalGDS^RA). Mg-GTP is shown bound to H-Ras in the Grb14^RA-PH structure. Select secondary-structure elements in the RA domains are labeled. (B) Stereo diagram of a superposition (aligned on Ras/Rap1) of the structures of Grb14^RA-PH-H-Ras and Raf1^RA-Rap1 (PDB code 1GUA) [26]. Raf1^RA is colored dark green and Rap1 is colored dark blue. Val88 of Raf1^RA is shown in stick and surface representation.</p

Crystal structure of Grb14^RA-PH-H-Ras and comparison to other RA-PH structures.

<p>(A) Ribbon diagram and molecular surface representation of the Grb14^RA-PH-H-Ras complex. The Grb14 RA domain is colored green, with β2 colored dark green, the PH domain is colored orange, with the phosphoinositide-binding β1-β2 loop colored magenta, and the RA-PH linker is colored gray. H-Ras is colored cyan, with switch 1 (residues 32-38) colored purple and switch 2 (residues 59-67) colored magenta. GTP is shown in stick representation with carbon atoms colored yellow, oxygen atoms red, nitrogen atoms blue, and phosphorus atoms black. The Mg²⁺ ion is colored brown. Select secondary-structure elements are labeled, along with the N- and C-termini. The view on the right is rotated by 90° as indicated. (B) Stereo diagram of a superposition of the structures (Cα traces) of Grb14^RA-PH, Grb10^RA-PH (PDB code 3HK0) [12], and RIAM^RA-PH (PDB code 3TCA) [17], in the same orientation as in the left panel of (A). Grb14^RA-PH is colored by domain as in (A), Grb10^RA-PH is colored blue (all) and RIAM^RA-PH is colored magenta (all). The N- and C-termini are labeled.</p