Research

Structural Studies of Protein Tyrosine Kinases

Many important cellular signaling cascades are initiated at the cell surface by the binding of a polypeptide ligand to a transmembrane receptor possessing intrinsic tyrosine kinase activity in its cytoplasmic domain. The receptor tyrosine kinase (RTK) family includes, among others, the insulin receptor, insulin-like growth factor-1 (IGF1) receptor, epidermal growth factor receptor, fibroblast growth factor receptor and MuSK, the receptor for agrin. Ligand binding induces receptor oligomerization (growth factor receptors) or a conformational change within the receptor (insulin/IGF1 receptor), leading to autophosphorylation of specific tyrosine residues in the cytoplasmic domains of the receptors. Tyrosine autophosphorylation stimulates receptor catalytic activity and generates recruitment sites for downstream signaling proteins. RTKs are critical components in signal transduction pathways that mediate cell proliferation, differentiation, migration and metabolism, and are active during organismal development and adult homeostasis. RTKs also play primary roles in the onset or progression of pathological conditions such as diabetic retinopathy, atherosclerosis and cancer.

The Janus kinase (JAK) family comprises four non-receptor tyrosine kinases: JAK1-3 and Tyk2. These proteins signal through the JAK-STAT (signal transducer and activator of transcription) pathway following cytokine stimulation, and are important for myeloid cell development, proliferation and survival and innate and adaptive immune responses. Mutations in JAKs, particularly in JAK2, which result in constitutive activation of JAKs, are causative for myeloproliferative neoplasms (MPNs).

JAK2

Jak2 associates with the cytoplasmic regions of numerous cytokine receptors including those for growth hormone, erythropoietin, leptin, interferon gamma, interleukin-3 and interleukin-5. JAKs contain tandem protein kinase domains: a pseudokinase domain (JH2) and a tyrosine kinase domain (JH1). In collaboration with Olli Silvennoinen's lab in Finland, we recently showed that the pseuodokinase domain of JAK2 is actually an active protein kinase, despite substitution of several residues that are conserved in canonical protein kinases. A crystal structure of JAK2 JH2 revealed that this domain adopts the eukaryotic protein kinase fold, but binds Mg-ATP more tightly than a canonical protein kinase. The most prevalent MPN mutation is V617F in JAK2. A comparison of the crystal structures of wild-type JH2 and V617F indicates that the mutation stabilizes a helix in JH2 (alphaC), which facilitates trans-phosphorylation and activation of the tyrosine kinase domains (JH1).

Superposition of wild-type Jak2 JH2 and V617F. In V617F (pink), a pi-stacking interaction is observed between Phe617, Phe595 and Phe594, which stabilizes alphaC. [Bandaranayake et al., Nat. Struct. Mol. Biol. 19, 754-759 (2012)]

 

 

 

 

 

 

 

 

 

Insulin Receptor

Using x-ray crystallography as our primary experimental technique, we are attempting to understand the molecular basis for insulin receptor activation and for recruitment of downstream signaling proteins to the activated (phosphorylated) insulin receptor. Several cytoplasmic adapter proteins bind to the activated insulin receptor, including insulin receptor substrate (IRS) proteins and APS, which are positive factors in insulin signaling pathways culminating in glucose uptake. The insulin receptor is downregulated by the adapter proteins Grb14 and Grb10 as well as the tyrosine phosphatase PTP1B. We are determining crystal structures of complexes between these proteins and the insulin receptor kinase domain to elucidate the modes of interaction and the determinants of specificity. 

Model of the interaction between Grb14 and the activated insulin receptor. [Depetris et al., Nat. Struct. Mol. Biol. 16, 833-839 (2009)]

 

 

 

 

 

 

 

 

 

 

 

The KRLB region of IRS2 bound to tris-phosphorylated IRK. The N-terminal kinase lobe is colored dark gray, the C-terminal lobe is colored light gray, and the KRLB region (residues 620-634) is shown in stick representation. Atoms of the activation loop and catalytic loop of IRK are colored green and orange, respectively. [Wu et al., Nat. Struct. Mol. Biol. 15, 251-258 (2008)]

IGF1 Receptor

The IGF1 receptor is highly related in sequence and structure to the insulin receptor, but has distinct biological functions, one of which is cell survival. Therefore, this RTK is a potential target for inhibition in tumor cells. In collaboration with Dr. Todd Miller at SUNY-Stony Brook, we have determined the three-dimensional structure of the IGF1 receptor kinase domain using x-ray crystallography. Several amino acid differences between the IGF1 receptor and the insulin receptor near the ATP binding cleft might be exploited by small-molecule inhibitors to gain selectivity for the IGF1 receptor over the insulin receptor. To this end, structural studies of inhibitors bound to the IGF1 receptor kinase are being pursued.

 

Structure of the kinase domain (light and dark gray) of the IGF1 receptor in complex with a small-molecule inhibitor. The ATP-competitive inhibitor is shown in (semi-transparent) sphere representation. The kinases formed a crystallographic dimer in which a tyrosine in the activation loop (green) is bound in the active site (orange) of the other kinase domain (and vice versa), providing a view of trans-autophosphorylation in the act. [Wu et al., EMBO J. 27, 1985-1994 (2008)]

 

 

MuSK

Another RTK of interest is MuSK, or muscle-specific kinase, which is expressed exclusively in muscle cells and plays an essential role in the formation of neuromuscular synapses by promoting clustering of acetylcholine receptors. Activation of MuSK by agrin results in autophosphorylation of several tyrosines in the cytoplasmic domain of MuSK. In a collaboration with Dr. Steven Burden at the Skirball Institute (NYU School of Medicine), we have determined the crystal structure of the cytoplasmic (tyrosine kinase-containing) domain of MuSK to understand how kinase activity is regulated in this receptor. The structure reveals that MuSK is strongly autoinhibited by the kinase activation loop.

In addition to determining the crytal structure of the tyrosine kinase domain of MuSK, we have determined crystal structures of the first two immunoglobulin-like domains (Ig1-2) and the Frizzled-like cysteine-rich domain (Fz-CRD) of the MuSK extracellular region. Ig1-2 crystallized as a dimer, mediated by Ig1, and the residues in the dimer interface are critical for agrin-induced phosphorylation of the receptor. Whether these residues are important for receptor dimerization or for a heterologous interaction (e.g., with the agrin receptor, LRP4) is still under investigation.

Molecular surface representation of dimeric Ig1-2 of the MuSK ectodomain. Ig1 is colored light green/purple, and Ig2 is colored dark green/purple. The dimer interface is mediated solely by residues in Ig1. [Stiegler et al., J. Mol. Biol. 364, 424-433 (2006)]

 

 

 

 

 

 

Ribbon diagram of the Fz-CRD of MuSK. The disulfide bridges are shown in ball-and-stick representation with sulfur atoms colored yellow and carbon atoms colored gray. [Stiegler et al., J. Mol. Biol. 393, 1-9 (2009)]

Dok7 is a cytoplasmic adaptor protein that contains tandem PH and PTB domains in the N-terminal portion of the molecule. In muscle, Dok7 is required for MuSK activation, and the structure of the PH and PTB domains revealed that these domains form a dimer that binds to the juxtamembrane phosphotyrosine (pY553) in MuSK, facilitating phosphorylation of the kinase activation loop.

 

 

Model for the activation of MuSK by Dok7. The protomers of the Dok7 PH-PTB dimer are colored orange and yellow, and the two tyrosine kinase domains of the MuSK dimer are colored light and dark green. Phosphorylated Y553 is shown in sphere representation and colored red. [Bergamin et al., Mol. Cell 38, 100-109 (2010)]