Stevan R Hubbard, Ph.D.

Cytokine signaling exposed
Hubbard, Stevan R
2011 Jan 12;19(1):1-2, Structure
In this issue, Lupardus et al. (2011) provide images, obtained by electron microscopy, of a complete cytokine signaling complex, offering clues into the mechanism by which binding of cytokines to their cognate receptors triggers trans-phosphorylation and activation of Janus kinases
— id: 119236, year: 2011, vol: 19, page: 1, stat: Journal Article,

Structure-guided identification of a laminin binding site on the laminin receptor precursor
Jamieson, Kelly V; Hubbard, Stevan R; Meruelo, Daniel
2011 Jan 7;405(1):24-32, Journal of molecular biology
The 37/ 67-kDa human laminin receptor (LamR) is a cell surface receptor for laminin, prion protein, and a variety of viruses. Because of its wide range of ligands, LamR plays a role in numerous pathologies. LamR overexpression correlates with a highly invasive cell phenotype and increased metastatic ability, mediated by interactions between LamR and laminin. In addition, the specific targeting of LamR with small interfering RNAs, blocking antibodies, and Sindbis viral vectors confers anti-tumor effects. We adopted a structure-based approach to map a laminin binding site on human LamR by comparing the sequences and crystal structures of LamR and Archaeoglobus fulgidus S2p, a non-laminin-binding ortholog. Here, we identify a laminin binding site on LamR, comprising residues Phe32, Glu35, and Arg155, which are conserved among mammalian species. Mutation of these residues results in a significant loss of laminin binding. Further, recombinant wild-type LamR is able to act as a soluble decoy to inhibit cellular migration towards laminin. Mutation of this laminin binding site results in loss of migration inhibition, which demonstrates the physiological role of Phe32, Glu35, and Arg155 for laminin binding activity. Mapping of the LamR binding site should contribute to the development of therapeutics that inhibit LamR interactions with laminin and may aid in the prevention of tumor growth and metastasis
— id: 116207, year: 2011, vol: 405, page: 24, stat: Journal Article,

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
2011 ;18(9):971-976, Nature structural & molecular biology
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
— id: 137017, year: 2011, vol: 18, page: 971, stat: Journal Article,

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
2011 Nov 25;286(47):40624-40630, Journal of biological chemistry
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
— id: 150244, year: 2011, vol: 286, page: 40624, stat: Journal Article,

The cytoplasmic adaptor protein Dok7 activates the receptor tyrosine kinase MuSK via dimerization
Bergamin, Elisa; Hallock, Peter T; Burden, Steven J; Hubbard, Stevan R
2010 Jul 9;39(1):100-109, Molecular cell
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
— id: 110694, year: 2010, vol: 39, page: 100, stat: Journal Article,

Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis
Cerda-Maira, Francisca A; Pearce, Michael J; Fuortes, Michele; Bishai, William R; Hubbard, Stevan R; Darwin, K Heran
2010 Sep;77(5):1123-1135, Molecular microbiology
Proteins targeted for degradation by the Mycobacterium proteasome are post-translationally tagged with prokaryotic ubiquitin-like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed 'pupylation', Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild-type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site-directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins
— id: 114585, year: 2010, vol: 77, page: 1123, stat: Journal Article,

Structural and functional studies of the Ras-associating and pleckstrin-homology domains of Grb10 and Grb14
Depetris, Rafael S; Wu, Jinhua; Hubbard, Stevan R
2009 Aug;16(8):833-839, Nature structural & molecular biology
Growth factor receptor-binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing 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 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-A resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit
— id: 101332, year: 2009, vol: 16, page: 833, stat: Journal Article,

The juxtamembrane region of EGFR takes center stage
Hubbard, Stevan R
2009 Jun 26;137(7):1181-1183, Cell
The activation process for the epidermal growth factor receptor (EGFR) involves formation of an asymmetric dimer of the tyrosine kinase domains. Jura et al. (2009) in this issue and Brewer et al. (2009) in Molecular Cell now demonstrate that the juxtamembrane region of EGFR plays a crucial role in stabilizing this dimer
— id: 100621, year: 2009, vol: 137, page: 1181, stat: Journal Article,

Crystal structure of the frizzled-like cysteine-rich domain of the receptor tyrosine kinase MuSK
Stiegler, Amy L; Burden, Steven J; Hubbard, Stevan R
2009 Oct 16;393(1):1-9, Journal of molecular biology
Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 A resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ
— id: 102933, year: 2009, vol: 393, page: 1, stat: Journal Article,

Crystal structure of the human laminin receptor precursor
Jamieson, Kelly V; Wu, Jinhua; Hubbard, Stevan R; Meruelo, Daniel
2008 Feb 8;283(6):3002-3005, Journal of biological chemistry
The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR
— id: 76763, year: 2008, vol: 283, page: 3002, stat: Journal Article,

Lrp4 is a receptor for Agrin and forms a complex with MuSK
Kim, Natalie; Stiegler, Amy L; Cameron, Thomas O; Hallock, Peter T; Gomez, Andrea M; Huang, Julie H; Hubbard, Stevan R; Dustin, Michael L; Burden, Steven J
2008 Oct 17;135(2):334-342, Cell
Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin
— id: 93378, year: 2008, vol: 135, page: 334, stat: Journal Article,

How IRE1 reacts to ER stress
Ron, David; Hubbard, Stevan R
2008 Jan 11;132(1):24-26, Cell
The long-awaited structure of the effector portion of IRE1, the endoplasmic reticulum stress transducer, is published in this issue of Cell (Lee et al., 2008). This structure provides new insight into the mysterious coupling of kinase and endoribonuclease activities in the oldest, most-conserved branch of the unfolded protein response in eukaryotes
— id: 76457, year: 2008, vol: 132, page: 24, stat: Journal Article,

Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor
Wu, Jinhua; Li, Wanqing; Craddock, Barbara P; Foreman, Kenneth W; Mulvihill, Mark J; Ji, Qun-sheng; Miller, W Todd; Hubbard, Stevan R
2008 Jul 23;27(14):1985-1994, EMBO journal
The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)- imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus
— id: 80313, year: 2008, vol: 27, page: 1985, stat: Journal Article,

Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2
Wu, Jinhua; Tseng, Yolanda D; Xu, Chong-Feng; Neubert, Thomas A; White, Morris F; Hubbard, Stevan R
2008 Mar;15(3):251-258, Nature structural & molecular biology
Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast two-hybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2
— id: 76468, year: 2008, vol: 15, page: 251, stat: Journal Article,

Receptor tyrosine kinases: mechanisms of activation and signaling
Hubbard, Stevan R; Miller, W Todd
2007 Apr;19(2):117-123, Current opinion in cell biology
Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands - mainly growth factors - play key roles in processes such as cellular growth, differentiation, metabolism and motility. Recent progress has been achieved towards an understanding of the precise (and varied) mechanisms by which RTKs are activated by ligand binding and by which signals are propagated from the activated receptors to downstream targets in the cell.
— id: 72808, year: 2007, vol: 19, page: 117, stat: Journal Article,

Structural basis for phosphotyrosine recognition by suppressor of cytokine signaling-3
Bergamin, Elisa; Wu, Jinhua; Hubbard, Stevan R
2006 Aug;14(8):1285-1292, Structure
Suppressor of cytokine signaling (SOCS) proteins are indispensable negative regulators of cytokine-stimulated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. SOCS proteins (SOCS1-7 and CIS) consist of a variable N-terminal region, a central Src homology-2 (SH2) domain, and a C-terminal SOCS box. The N-terminal region in SOCS1 and SOCS3 includes the so-called kinase inhibitory region that has been shown to inhibit the catalytic activity of JAK2. Here, we present a crystal structure at 2.0 A resolution of the N-terminally extended SH2 domain of SOCS3 in complex with its phosphopeptide target on the cytokine receptor gp130. The structure reveals that major insertions in the EF and BG loops of the SOCS3 SH2 domain are responsible for binding to gp130 with high affinity and specificity. In addition, the structure provides insights into the possible mechanisms by which SOCS3 and SOCS1 inhibit JAK2 kinase activity
— id: 68986, year: 2006, vol: 14, page: 1285, stat: Journal Article,

Structural Basis for Phosphotyrosine Recognition by the Src Homology-2 Domains of the Adapter Proteins SH2-B and APS
Hu, Junjie; Hubbard, Stevan R
2006 Aug 4;361(1):69-79, Journal of molecular biology
SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS
— id: 65226, year: 2006, vol: 361, page: 69, stat: Journal Article,

EGF receptor activation: push comes to shove
Hubbard, Stevan R
2006 Jun 16;125(6):1029-1031, Cell
A study by Zhang et al. (2006) in this issue of Cell provides compelling evidence that the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) is activated by the formation of an asymmetric dimer, with one kinase domain in the EGF-mediated dimer activating the other through an allosteric mechanism
— id: 65227, year: 2006, vol: 125, page: 1029, stat: Journal Article,

Crystal structure of the agrin-responsive immunoglobulin-like domains 1 and 2 of the receptor tyrosine kinase MuSK
Stiegler, Amy L; Burden, Steven J; Hubbard, Stevan R
2006 Dec 1;364(3):424-433, Journal of molecular biology
Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed exclusively in skeletal muscle, where it is required for formation of the neuromuscular junction. MuSK is activated by agrin, a neuron-derived heparan sulfate proteoglycan. Here, we report the crystal structure of the agrin-responsive first and second immunoglobulin-like domains (Ig1 and Ig2) of the MuSK ectodomain at 2.2 A resolution. The structure reveals that MuSK Ig1 and Ig2 are Ig-like domains of the I-set subfamily, which are configured in a linear, semi-rigid arrangement. In addition to the canonical internal disulfide bridge, Ig1 contains a second, solvent-exposed disulfide bridge, which our biochemical data indicate is critical for proper folding of Ig1 and processing of MuSK. Two Ig1-2 molecules form a non-crystallographic dimer that is mediated by a unique hydrophobic patch on the surface of Ig1. Biochemical analyses of MuSK mutants introduced into MuSK(-/-) myotubes demonstrate that residues in this hydrophobic patch are critical for agrin-induced MuSK activation
— id: 70021, year: 2006, vol: 364, page: 424, stat: Journal Article,

Structural basis for inhibition of the insulin receptor by the adaptor protein grb14
Depetris, Rafael S; Hu, Junjie; Gimpelevich, Ilana; Holt, Lowenna J; Daly, Roger J; Hubbard, Stevan R
2005 Oct 28;20(2):325-333, Molecular cell
Grb14, a member of the Grb7 adaptor protein family, possesses a pleckstrin homology (PH) domain, a C-terminal Src homology-2 (SH2) domain, and an intervening stretch of approximately 45 residues known as the BPS region, which is unique to this adaptor family. Previous studies have demonstrated that Grb14 is a tissue-specific negative regulator of insulin receptor signaling and that inhibition is mediated by the BPS region. We have determined the crystal structure of the Grb14 BPS region in complex with the tyrosine kinase domain of the insulin receptor. The structure reveals that the N-terminal portion of the BPS region binds as a pseudosubstrate inhibitor in the substrate peptide binding groove of the kinase. Together with the crystal structure of the SH2 domain, we present a model for the interaction of Grb14 with the insulin receptor, which indicates how Grb14 functions as a selective protein inhibitor of insulin signaling
— id: 58752, year: 2005, vol: 20, page: 325, stat: Journal Article,

Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: molecular yardsticks for analyzing catalytic mechanism and inhibitor design
Hines, Aliya C; Parang, Keykavous; Kohanski, Ronald A; Hubbard, Stevan R; Cole, Philip A
2005 Aug;33(4):285-297, Bioorganic chemistry
Bisubstrate analogs have the potential to provide enhanced specificity for protein kinase inhibition and tools to understand catalytic mechanism. Previous efforts led to the design of a peptide-ATP conjugate bisubstrate analog utilizing aminophenylalanine in place of tyrosine and a thioacetyl linker to the gamma-phosphate of ATP which was a potent inhibitor of the insulin receptor kinase (IRK). In this study, we have examined the contributions of various electrostatic and structural elements in the bisubstrate analog to IRK binding affinity. Three types of changes (seven specific analogs in all) were introduced: a Tyr isostere of the previous aminophenylalanine moiety, modifications of the spacer between the adenine and the peptide, and deletions and substitutions within the peptide moiety. These studies allowed a direct evaluation of the hydrogen bond strength between the anilino nitrogen of the bisubstrate analog and the enzyme catalytic base Asp and showed that it contributes 2.5 kcal/mol of binding energy, in good agreement with previous predictions. Modifications of the linker length resulted in weakened inhibitory affinity, consistent with the geometric requirements of an enzyme-catalyzed dissociative transition state. Alterations in the peptide motif generally led to diminished inhibitory potency, and only some of these effects could be rationalized based on prior kinetic and structural studies. Taken together, these results suggest that a combination of mechanism-based design and empirical synthetic manipulation will be necessary in producing optimized protein kinase bisubstrate analog inhibitors
— id: 65228, year: 2005, vol: 33, page: 285, stat: Journal Article,

Structural Characterization of a Novel Cbl Phosphotyrosine Recognition Motif in the APS Family of Adapter Proteins
Hu, Junjie; Hubbard, Stevan R
2005 May 13;280(19):18943-18949, Journal of biological chemistry
The Cbl adapter proteins typically function to down-regulate activated protein tyrosine kinases and other signaling proteins by coupling them to the ubiquitination machinery for degradation by the proteasome. Cbl proteins bind to specific tyrosine-phosphorylated sequences in target proteins via the tyrosine kinase-binding (TKB) domain, which comprises a four-helix bundle, an EF-hand calcium-binding domain, and a non-conventional Src homology-2 domain. The previously derived consensus sequence for phosphotyrosine recognition by the Cbl TKB domain is NXpY(S/T)XXP (X denotes lesser residue preference), wherein specificity is conferred primarily by residues C-terminal to the phosphotyrosine. Cbl is recruited to and phosphorylated by the insulin receptor in adipose cells through the adapter protein APS. APS is phosphorylated by the insulin receptor on a C-terminal tyrosine residue, which then serves as a binding site for the Cbl TKB domain. Using x-ray crystallography, site-directed mutagenesis, and calorimetric studies, we have characterized the interaction between the Cbl TKB domain and the Cbl recruitment site in APS, which contains a sequence motif, RA(V/I)XNQpY(S/T), that is conserved in the related adapter proteins SH2-B and Lnk. These studies reveal a novel mode of phosphopeptide interaction with the Cbl TKB domain, in which N-terminal residues distal to the phosphotyrosine directly contact residues of the four-helix bundle of the TKB domain
— id: 52545, year: 2005, vol: 280, page: 18943, stat: Journal Article,

EGF receptor inhibition: attacks on multiple fronts
Hubbard, Stevan R
2005 Apr;7(4):287-288, Cancer cell
The epidermal growth factor receptor (EGFR) drives tumor growth in a subset of human epithelial carcinomas. A crystallographic study by Li et al. in this issue of Cancer Cell provides the molecular basis for inhibition of EGFR by cetuximab (Erbitux), a monoclonal antibody that has been approved by the Food and Drug Administration as a therapeutic for advanced-stage colorectal cancers. Cetuximab targets one of the ligand binding domains of EGFR, thus preventing ligand activation of the receptor
— id: 52544, year: 2005, vol: 7, page: 287, stat: Journal Article,

Crystal structure of a complex between protein tyrosine phosphatase 1B and the insulin receptor tyrosine kinase
Li, Shiqing; Depetris, Rafael S; Barford, David; Chernoff, Jonathan; Hubbard, Stevan R
2005 Nov;13(11):1643-1651, Structure
Protein tyrosine phosphatase 1B (PTP1B) is a highly specific negative regulator of insulin receptor signaling in vivo. The determinants of PTP1B specificity for the insulin receptor versus other receptor tyrosine kinases are largely unknown. Here, we report a crystal structure at 2.3 A resolution of the catalytic domain of PTP1B (trapping mutant) in complex with the phosphorylated tyrosine kinase domain of the insulin receptor (IRK). The crystallographic asymmetric unit contains two PTP1B-IRK complexes that interact through an IRK dimer interface. Rather than binding to a phosphotyrosine in the IRK activation loop, PTP1B binds instead to the opposite side of the kinase domain, with the phosphorylated activation loops sequestered within the IRK dimer. The crystal structure provides evidence for a noncatalytic mode of interaction between PTP1B and IRK, which could be important for the selective recruitment of PTP1B to the insulin receptor
— id: 62350, year: 2005, vol: 13, page: 1643, stat: Journal Article,

Juxtamembrane autoinhibition in receptor tyrosine kinases
Hubbard, Stevan R
2004 Jun;5(6):464-471, Nature reviews. Molecular cell biology
— id: 43031, year: 2004, vol: 5, page: 464, stat: Journal Article,

Oncogenic mutations in B-Raf: some losses yield gains
Hubbard, Stevan R
2004 Mar 19;116(6):764-766, Cell
A 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
— id: 43032, year: 2004, vol: 116, page: 764, stat: Journal Article,

Agrin/MuSK signaling: willing and Abl
Burden, Steven J; Fuhrer, Christian; Hubbard, Stevan R
2003 Jul;6(7):653-654, Nature neuroscience
— id: 65229, year: 2003, vol: 6, page: 653, stat: Journal Article,

Structural Basis for Recruitment of the Adaptor Protein APS to the Activated Insulin Receptor
Hu, Junjie; Liu, Jun; Ghirlando, Rodolfo; Saltiel, Alan R; Hubbard, Stevan R
2003 Dec;12(6):1379-1389, Molecular cell
The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology 2 (SH2) domain of APS. Here, we present the crystal structure of the APS SH2 domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel dimeric configuration of the APS SH2 domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH2 domains. The APS SH2 dimer engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH2 domain and a second phosphotyrosine, pTyr-1162, coordinated by two lysine residues in beta strand D. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its dimeric receptor
— id: 40090, year: 2003, vol: 12, page: 1379, stat: Journal Article,

Structural and biochemical evidence for an autoinhibitory role for tyrosine 984 in the juxtamembrane region of the insulin receptor
Li, Shiqing; Covino, Nicole D; Stein, Evan G; Till, Jeffrey H; Hubbard, Stevan R
2003 Jul 11;278(28):26007-26014, Journal of biological chemistry
Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of alpha-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in kcat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus
— id: 39243, year: 2003, vol: 278, page: 26007, stat: Journal Article,

Structural Basis for Dimerization of the Grb10 Src Homology 2 Domain. IMPLICATIONS FOR LIGAND SPECIFICITY
Stein, Evan G; Ghirlando, Rodolfo; Hubbard, Stevan R
2003 Apr 11;278(15):13257-13264, Journal of biological chemistry
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
— id: 34133, year: 2003, vol: 278, page: 13257, stat: Journal Article,

Protein tyrosine kinases: autoregulation and small-molecule inhibition
Hubbard, Stevan R
2002 Dec;12(6):735-741, Current opinion in structural biology
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
— id: 39343, year: 2002, vol: 12, page: 735, stat: Journal Article,

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
2002 Sep;10(9):1187-1187, Structure
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
— id: 32906, year: 2002, vol: 10, page: 1187, stat: Journal Article,

Structure and autoregulation of the insulin-like growth factor 1 receptor kinase
Favelyukis S; Till JH; Hubbard SR; Miller WT
2001 Dec;8(12):1058-1063, Nature structural biology
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
— id: 24974, year: 2001, vol: 8, page: 1058, stat: Journal Article,

Theme and variations: juxtamembrane regulation of receptor protein kinases
Hubbard SR
2001 Sep;8(3):481-482, Molecular cell
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
— id: 24975, year: 2001, vol: 8, page: 481, stat: Journal Article,

Mechanism-based design of a protein kinase inhibitor
Parang K; Till JH; Ablooglu AJ; Kohanski RA; Hubbard SR; Cole PA
2001 Jan;8(1):37-41, Nature structural biology
Protein kinase inhibitors have applications as anticancer therapeutic agents and biological tools in cell signaling. Based on a phosphoryl transfer mechanism involving a dissociative transition state, a potent and selective bisubstrate inhibitor for the insulin receptor tyrosine kinase was synthesized by linking ATPgammaS to a peptide substrate analog via a two-carbon spacer. The compound was a high affinity competitive inhibitor against both nucleotide and peptide substrates and showed a slow off-rate. A crystal structure of this inhibitor bound to the tyrosine kinase domain of the insulin receptor confirmed the key design features inspired by a dissociative transition state, and revealed that the linker takes part in the octahedral coordination of an active site Mg2+. These studies suggest a general strategy for the development of selective protein kinase inhibitors
— id: 20816, year: 2001, vol: 8, page: 37, stat: Journal Article,

The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors
Stein EG; Gustafson TA; Hubbard SR
2001 Mar 30;493(2-3):106-111, FEBS letters
Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin-like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin- and/or IGF1-mediated signaling
— id: 20817, year: 2001, vol: 493, page: 106, stat: Journal Article,

Crystallographic and solution studies of an activation loop mutant of the insulin receptor tyrosine kinase: insights into kinase mechanism
Till JH; Ablooglu AJ; Frankel M; Bishop SM; Kohanski RA; Hubbard SR
2001 Mar 30;276(13):10049-10055, Journal of biological chemistry
The tyrosine kinase domain of the insulin receptor is subject to autoinhibition in the unphosphorylated basal state via steric interactions involving the activation loop. A mutation in the activation loop designed to relieve autoinhibition, Asp-1161 --> Ala, substantially increases the ability of the unphosphorylated kinase to bind ATP. The crystal structure of this mutant in complex with an ATP analog has been determined at 2.4-A resolution. The structure shows that the active site is unobstructed, but the end of the activation loop is disordered and therefore the binding site for peptide substrates is not fully formed. In addition, Phe-1151 of the protein kinase-conserved DFG motif, at the beginning of the activation loop, hinders closure of the catalytic cleft and proper positioning of alpha-helix C for catalysis. These results, together with viscometric kinetic measurements, suggest that peptide substrate binding induces a reconfiguration of the unphosphorylated activation loop prior to the catalytic step. The crystallographic and solution studies provide new insights into the mechanism by which the activation loop controls phosphoryl transfer as catalyzed by the insulin receptor
— id: 20815, year: 2001, vol: 276, page: 10049, stat: Journal Article,

PROTEIN TYROSINE KINASE STRUCTURE AND FUNCTION
Hubbard SR; Till JH
2000 ;69:373-398, Annual review of biochemistry
— id: 11520, year: 2000, vol: 69, page: 373, stat: Journal Article,

SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors
Laird AD; Vajkoczy P; Shawver LK; Thurnher A; Liang C; Mohammadi M; Schlessinger J; Ullrich A; Hubbard SR; Blake RA; Fong TA; Strawn LM; Sun L; Tang C; Hawtin R; Tang F; Shenoy N; Hirth KP; McMahon G; Cherrington
2000 Aug 1;60(15):4152-4160, Cancer research
Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials
— id: 20814, year: 2000, vol: 60, page: 4152, stat: Journal Article,

Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity
Plotnikov AN; Hubbard SR; Schlessinger J; Mohammadi M
2000 May 12;101(4):413-424, Cell
To elucidate the structural determinants governing specificity in fibroblast growth factor (FGF) signaling, we have determined the crystal structures of FGF1 and FGF2 complexed with the ligand binding domains (immunoglobulin-like domains 2 [D2] and 3 [D3]) of FGF receptor 1 (FGFR1) and FGFR2, respectively. Highly conserved FGF-D2 and FGF-linker (between D2-D3) interfaces define a general binding site for all FGF-FGFR complexes. Specificity is achieved through interactions between the N-terminal and central regions of FGFs and two loop regions in D3 that are subject to alternative splicing. These structures provide a molecular basis for FGF1 as a universal FGFR ligand and for modulation of FGF-FGFR specificity through primary sequence variations and alternative splicing
— id: 11676, year: 2000, vol: 101, page: 413, stat: Journal Article,

Structural analysis of receptor tyrosine kinases
Hubbard SR
1999 ;71(3-4):343-358, Progress in biophysics & molecular biology
Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the gamma-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies
— id: 6129, year: 1999, vol: 71, page: 343, stat: Journal Article,

Crystal structure of the ARF-GAP domain and ankyrin repeats of PYK2-associated protein beta
Mandiyan V; Andreev J; Schlessinger J; Hubbard SR
1999 Dec 15;18(24):6890-6898, EMBO journal
ADP ribosylation factors (ARFs), which are members of the Ras superfamily of GTP-binding proteins, are critical components of vesicular trafficking pathways in eukaryotes. Like Ras, ARFs are active in their GTP-bound form, and their duration of activity is controlled by GTPase-activating proteins (GAPs), which assist ARFs in hydrolyzing GTP to GDP. PAPbeta, a protein that binds to and is phosphorylated by the non-receptor tyrosine kinase PYK2, contains several modular signaling domains including a pleckstrin homology domain, an SH3 domain, ankyrin repeats and an ARF-GAP domain. Sequences of ARF-GAP domains show no recognizable similarity to those of other GAPs, and contain a characteristic Cys-X(2)-Cys-X(16-17)-Cys-X(2)-Cys motif. The crystal structure of the PAPbeta ARF-GAP domain and the C-terminal ankyrin repeats has been determined at 2.1 A resolution. The ARF-GAP domain comprises a central three-stranded beta-sheet flanked by five alpha-helices, with a Zn(2+) ion coordinated by the four cysteines of the cysteine-rich motif. Four ankyrin repeats are also present, the first two of which form an extensive interface with the ARF-GAP domain. An invariant arginine and several nearby hydrophobic residues are solvent exposed and are predicted to be the site of interaction with ARFs. Site-directed mutagenesis of these residues confirms their importance in ARF-GAP activity
— id: 8587, year: 1999, vol: 18, page: 6890, stat: Journal Article,

Structural basis for FGF receptor dimerization and activation
Plotnikov AN; Schlessinger J; Hubbard SR; Mohammadi M
1999 Sep 3;98(5):641-650, Cell
The crystal structure of FGF2 bound to a naturally occurring variant of FGF receptor 1 (FGFR1) consisting of immunoglobulin-like domains 2 (D2) and 3 (D3) has been determined at 2.8 A resolution. Two FGF2:FGFR1 complexes form a 2-fold symmetric dimer. Within each complex, FGF2 interacts extensively with D2 and D3 as well as with the linker between the two domains. The dimer is stabilized by interactions between FGF2 and D2 of the adjoining complex and by a direct interaction between D2 of each receptor. A positively charged canyon formed by a cluster of exposed basic residues likely represents the heparin-binding site. A general model for FGF- and heparin-induced FGFR dimerization is inferred from the crystal structure, unifying a wealth of biochemical data
— id: 8352, year: 1999, vol: 98, page: 641, stat: Journal Article,

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain
Mohammadi M; Froum S; Hamby JM; Schroeder MC; Panek RL; Lu GH; Eliseenkova AV; Green D; Schlessinger J; Hubbard SR
1998 Oct 15;17(20):5896-5904, EMBO journal
Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation
— id: 7692, year: 1998, vol: 17, page: 5896, stat: Journal Article,

Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog
Hubbard SR
1997 Sep 15;16(18):5572-5581, EMBO journal
The crystal structure of the phosphorylated, activated form of the insulin receptor tyrosine kinase in complex with a peptide substrate and an ATP analog has been determined at 1.9 A resolution. The activation loop (A-loop) of the kinase undergoes a major conformational change upon autophosphorylation of Tyr1158, Tyr1162 and Tyr1163 within the loop, resulting in unrestricted access of ATP and protein substrates to the kinase active site. Phosphorylated Tyr1163 (pTyr1163) is the key phosphotyrosine in stabilizing the conformation of the tris-phosphorylated A-loop, whereas pTyr1158 is completely solvent-exposed, suggesting an availability for interaction with downstream signaling proteins. The YMXM-containing peptide substrate binds as a short anti-parallel beta-strand to the C-terminal end of the A-loop, with the methionine side chains occupying two hydrophobic pockets on the C-terminal lobe of the kinase. The structure thus reveals the molecular basis for insulin receptor activation via autophosphorylation, and provides insights into tyrosine kinase substrate specificity and the mechanism of phosphotransfer
— id: 8449, year: 1997, vol: 16, page: 5572, stat: Journal Article,

Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors
Mohammadi M; McMahon G; Sun L; Tang C; Hirth P; Yeh BK; Hubbard SR; Schlessinger J
1997 May 9;276(5314):955-960, Science
A new class of protein tyrosine kinase inhibitors was identified that is based on an oxindole core (indolinones). Two compounds from this class inhibited the kinase activity of fibroblast growth factor receptor 1 (FGFR1) and showed differential specificity toward other receptor tyrosine kinases. Crystal structures of the tyrosine kinase domain of FGFR1 in complex with the two compounds were determined. The oxindole occupies the site in which the adenine of adenosine triphosphate binds, whereas the moieties that extend from the oxindole contact residues in the hinge region between the two kinase lobes. The more specific inhibitor of FGFR1 induces a conformational change in the nucleotide-binding loop. This structural information will facilitate the design of new inhibitors for use in the treatment of cancer and other diseases in which cell signaling by tyrosine kinases plays a crucial role in disease pathogenesis
— id: 8448, year: 1997, vol: 276, page: 955, stat: Journal Article,

Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism
Mohammadi M; Schlessinger J; Hubbard SR
1996 Aug 23;86(4):577-587, Cell
The crystal structure of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1K) has been determined in its unliganded form to 2.0 angstroms resolution and in complex with with an ATP analog to 2.3 angstrosms A resolution. Several features distinguish the structure of FGFR1K from that of the tyrosine kinase domain of the insulin receptor. Residues in the activation loop of FGFR1K appear to interfere with substrate peptide binding but not with ATP binding, revealing a second and perhaps more general autoinhibitory mechanism for receptor tyrosine kinases. In addition, a dimeric form of FGFR1K observed in the crystal structure may provide insights into the molecular mechanisms by which FGF receptors are activated. Finally, the structure provides a basis for rationalizing the effects of kinase mutations in FGF receptors that lead to developmental disorders in nematodes and humans
— id: 7021, year: 1996, vol: 86, page: 577, stat: Journal Article,

CATALYTIC SPECIFICITY OF PROTEIN-TYROSINE KINASES IS CRITICAL FOR SELECTIVE SIGNALING
SONGYANG, Z; CARRAWAY, KL; ECK, MJ; HARRISON, SC; FELDMAN, RA; MOHAMMADI, M; SCHLESSINGER, J; HUBBARD, SR; SMITH, DP; ENG, C; LORENZO, MJ; PONDER, BAJ; MAYER, BJ; CANTLEY, LC
1995 FEB 9 ;373(6514):536-539, Nature
How do distinct protein-tyrosine kinases activate specific downstream events? Src-homology-2 (SH2) domains on tyrosine kinases or targets of tyrosine kinases recognize phosphotyrosine in a specific sequence context and thereby provide some specificity(1-3). The role of the catalytic site of tyrosine kinases in determining target specificity has not been fully investigated. Here we use a degenerate peptide library to show that each of nine tyrosine kinases investigated has a unique optimal peptide substrate. We find that the cytosolic tyrosine kinases preferentially phosphorylate peptides recognized by their own SH2 domains or closely related SH2 domains (group I; ref. 3), whereas receptor tyrosine kinases preferentially phosphorylate peptides recognized by subsets of group In SH2 domains(3). The importance of these findings for human disease is underscored by our observation that a point mutation in the RET receptor-type tyrosine kinase, which causes multiple endocrine neoplasia type 2B, results in a shift in peptide substrate specificity
— id: 87442, year: 1995, vol: 373, page: 536, stat: Journal Article,