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47


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

Wynne, Joseph P; Wu, Jinhua; Su, Wenjuan; Mor, Adam; Patsoukis, Nikolaos; Boussiotis, Vassiliki A; Hubbard, Stevan R; Philips, Mark R
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)P(2)) 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)P(2).
PMCID:3471229
PMID: 23045549
ISSN: 0021-9525
CID: 180090

Crystal structures of the JAK2 pseudokinase domain and the pathogenic mutant V617F

Bandaranayake, Rajintha M; Ungureanu, Daniela; Shan, Yibing; Shaw, David E; Silvennoinen, Olli; Hubbard, Stevan R
The protein tyrosine kinase JAK2 mediates signaling through numerous cytokine receptors. JAK2 possesses a pseudokinase domain (JH2) and a tyrosine kinase domain (JH1). Through unknown mechanisms, JH2 regulates the catalytic activity of JH1, and hyperactivating mutations in the JH2 region of human JAK2 cause myeloproliferative neoplasms (MPNs). We showed previously that JAK2 JH2 is, in fact, catalytically active. Here we present crystal structures of human JAK2 JH2, including both wild type and the most prevalent MPN mutant, V617F. The structures reveal that JH2 adopts the fold of a prototypical protein kinase but binds Mg-ATP noncanonically. The structural and biochemical data indicate that the V617F mutation rigidifies alpha-helix C in the N lobe of JH2, facilitating trans-phosphorylation of JH1. The crystal structures of JH2 afford new opportunities for the design of novel JAK2 therapeutics targeting MPNs.
PMCID:3414675
PMID: 22820988
ISSN: 1545-9985
CID: 174351

Agrin binds to the N-terminal region of Lrp4 protein and stimulates association between Lrp4 and the first immunoglobulin-like domain in muscle-specific kinase (MuSK)

Zhang, Wei; Coldefy, Anne-Sophie; Hubbard, Stevan R; Burden, Steven J
Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four beta-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK
PMCID:3220470
PMID: 21969364
ISSN: 1083-351x
CID: 150244

The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling

Ungureanu, Daniela; Wu, Jinhua; Pekkala, Tuija; Niranjan, Yashavanthi; Young, Clifford; Jensen, Ole N; Xu, Chong-Feng; Neubert, Thomas A; Skoda, Radek C; Hubbard, Stevan R; Silvennoinen, Olli
Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2
PMCID:4504201
PMID: 21841788
ISSN: 1545-9985
CID: 137017

The cytoplasmic adaptor protein Dok7 activates the receptor tyrosine kinase MuSK via dimerization

Bergamin, Elisa; Hallock, Peter T; Burden, Steven J; Hubbard, Stevan R
Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes
PMCID:2917201
PMID: 20603078
ISSN: 1097-4164
CID: 110694

Structural Basis for Dimerization of the Grb10 Src Homology 2 Domain. IMPLICATIONS FOR LIGAND SPECIFICITY

Stein, Evan G; Ghirlando, Rodolfo; Hubbard, Stevan R
Grb7, Grb10, and Grb14 are members of a distinct family of adapter proteins that interact with various receptor tyrosine kinases upon receptor activation. Proteins in this family contain several modular signaling domains including a pleckstrin homology (PH) domain, a BPS (between PH and SH2) domain, and a C-terminal Src homology 2 (SH2) domain. Although SH2 domains are typically monomeric, we show that the Grb10 SH2 domain and also full-length Grb10gamma are dimeric in solution under physiologic conditions. The crystal structure of the Grb10 SH2 domain at 1.65-A resolution reveals a non-covalent dimer whose interface comprises residues within and flanking the C-terminal alpha helix, which are conserved in the Grb7/Grb10/Grb14 family but not in other SH2 domains. Val-522 in the BG loop (BG3) and Asp-500 in the EF loop (EF1) are positioned to interfere with the binding of the P+3 residue of a phosphopeptide ligand. These structural features of the Grb10 SH2 domain will favor binding of dimeric, turn-containing phosphotyrosine sequences, such as the phosphorylated activation loops in the two beta subunits of the insulin and insulin-like growth factor-1 receptors. Moreover, the structure suggests the mechanism by which the Grb7 SH2 domain binds selectively to pTyr-1139 (pYVNQ) in Her2, which along with Grb7 is co-amplified in human breast cancers
PMID: 12551896
ISSN: 0021-9258
CID: 34133

Protein tyrosine kinases: autoregulation and small-molecule inhibition

Hubbard, Stevan R
Receptor and non-receptor protein tyrosine kinases (PTKs) are essential enzymes in cellular signaling processes that regulate cell growth, differentiation, migration and metabolism. The kinase activity of PTKs is tightly controlled through steric, autoregulatory mechanisms, as well as by the action of protein tyrosine phosphatases. Recent structural studies have revealed several modes of autoregulation governing the catalytic state of these enzymes. Aberrant catalytic activity of many PTKs, via mutation or overexpression, plays an important role in numerous pathological conditions, including cancer. Structural studies of the Abl tyrosine kinase domain in complex with the small-molecule inhibitor STI571 provide a molecular basis for understanding the specificity determinants of this highly successful drug used in the treatment of chronic myeloid leukemia
PMID: 12504677
ISSN: 0959-440x
CID: 39343

Crystal Structure of the MuSK Tyrosine Kinase. Insights into Receptor Autoregulation

Till, Jeffrey H; Becerra, Manuel; Watty, Anke; Lu, Yun; Ma, Yuliang; Neubert, Thomas A; Burden, Steven J; Hubbard, Stevan R
Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region
PMID: 12220490
ISSN: 0969-2126
CID: 32906

Structure and autoregulation of the insulin-like growth factor 1 receptor kinase

Favelyukis S; Till JH; Hubbard SR; Miller WT
The insulin-like growth factor 1 (IGF1) receptor is closely related to the insulin receptor. However, the unique biological functions of IGF1 receptor make it a target for therapeutic intervention in human cancer. Using its isolated tyrosine kinase domain, we show that the IGF1 receptor is regulated by intermolecular autophosphorylation at three sites within the kinase activation loop. Steady-state kinetic analyses of the isolated phosphorylated forms of the IGF1 receptor kinase (IGF1RK) reveal that each autophosphorylation event increases enzyme turnover number and decreases Km for ATP and peptide. We have determined the 2.1 A-resolution crystal structure of the tris-phosphorylated form of IGF1RK in complex with an ATP analog and a specific peptide substrate. The structure of IGF1RK reveals how the enzyme recognizes peptides containing hydrophobic residues at the P+1 and P+3 positions and how autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Although the nucleotide binding cleft is conserved between IGF1RK and the insulin receptor kinase, sequence differences in the nearby interlobe linker could potentially be exploited for anticancer drug design
PMID: 11694888
ISSN: 1072-8368
CID: 24974

Theme and variations: juxtamembrane regulation of receptor protein kinases [Comment]

Hubbard SR
Huse et al. in this issue of Molecular Cell and Wybenga-Groot et al. in the September 21, 2001 issue of Cell present biochemical and structural studies that elucidate the roles of juxtamembrane phosphorylation in a receptor serine/threonine kinase, the type I receptor for transforming growth factor beta, and in a receptor tyrosine kinase, the ephrin receptor EphB2
PMID: 11583608
ISSN: 1097-2765
CID: 24975