Faculty Bibliography

When insulin-like growth factor-1 (IGF1) binds to its receptor, a physical constraint is released that allows the two transmembrane helices to come together to facilitate activation of the receptor.

Structural and biochemical studies by Ferrao et al. (2014) in this issue demonstrate that dimerization of the kinase domain of IRAK4 is crucial for its activation, but with conditions: after-not before-receptor recruitment and before-not after-autophosphorylation.

Janus kinase-2 (JAK2) mediates signaling by various cytokines, including erythropoietin and growth hormone. JAK2 possesses tandem pseudokinase and tyrosine-kinase domains. Mutations in the pseudokinase domain are causally linked to myeloproliferative neoplasms (MPNs) in humans. The structure of the JAK2 tandem kinase domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. Using molecular dynamics simulations of protein-protein docking, we produced a structural model for the autoinhibitory interaction between the JAK2 pseudokinase and kinase domains. A striking feature of our model, which is supported by mutagenesis experiments, is that nearly all of the disease mutations map to the domain interface. The simulations indicate that the kinase domain is stabilized in an inactive state by the pseudokinase domain, and they offer a molecular rationale for the hyperactivity of V617F, the predominant JAK2 MPN mutation.

APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.

MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in the formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.

JAK (Janus kinase) 2 plays a critical role in signal transduction through several cytokine receptors. JAKs contain a typical tyrosine kinase domain preceded by a pseudokinase [JH2 (JAK homology 2)] domain which has been considered to be catalytically inactive. Identification of activating mutations in the JH2 domain of JAK2 as the major cause for polycythaemia vera and other MPNs (myeloproliferative neoplasms) demonstrate the critical regulatory function for this domain, but the underlying mechanisms have remained elusive. We have performed biochemical and functional analysis on the JH2 domain of JAK2. The results indicate that JH2 functions as an active protein kinase and phosphorylates two residues in JAK2 (Ser523 and Tyr570) that have been shown previously to be negative regulatory sites for JAK2 activity. The crystal structure of the JAK2 JH2 domain provides an explanation for the functional findings and shows that JH2 adopts a prototypical kinase fold, but binds MgATP through a non-canonical mode. The structure of the most prevalent pathogenic JH2 mutation V617F shows a high level of similarity to wild-type JH2. The most notable structural deviation is observed in the N-lobe alphaC-helix. The structural and biochemical data together with MD (molecular dynamics) simulations show that the V617F mutation rigidifies the alphaC-helix, which results in hyperactivation of the JH1 domain through an as yet unidentified mechanism. These results provide structural and functional insights into the normal and pathogenic function of the JH2 domain of JAK2.

Unlike prototypical receptor tyrosine kinases (RTKs), which are single-chain polypeptides, the insulin receptor (InsR) is a preformed, covalently linked tetramer with two extracellular alpha subunits and two membrane-spanning, tyrosine kinase-containing beta subunits. A single molecule of insulin binds asymmetrically to the ectodomain, triggering a conformational change that is transmitted to the cytoplasmic kinase domains, which facilitates their trans-phosphorylation. As in prototypical RTKs, tyrosine phosphorylation in the juxtamembrane region of InsR creates recruitment sites for downstream signaling proteins (IRS [InsR substrate] proteins, Shc) containing a phosphotyrosine-binding (PTB) domain, and tyrosine phosphorylation in the kinase activation loop stimulates InsR's catalytic activity. For InsR, phosphorylation of the activation loop, which contains three tyrosine residues, also creates docking sites for adaptor proteins (Grb10/14, SH2B2) that possess specialized Src homology-2 (SH2) domains, which are dimeric and engage two phosphotyrosines in the activation loop.