Research
Receptor Tyrosine Kinases Dimerization
Receptor tyrosine kinases undergo ligand dependent dimerization, which activates their intrinsic protein tyrosine kinase (PTK) domains. We have determined the crystal structure of Stem cell factor (SCF) and fibroblast growth factor (FGF), two ligands of receptor tyrosine kinases. The crystal structures of FGF in complex with the extracellular ligand-binding domain of FGF-receptor (FGFR) and with a heparin sulfate oligosaccharide were also determined. The structure of the ternary FGF/heparin/FGFR complex provides a molecular view of how FGF acts in concert with heparin to induce the dimerization and activation of FGF-receptors. We have also determined the crystal structure of the catalytic PTK domain of FGFR in complex with an ATP analogue or in complex with specific PTK inhibitors of FGFR activity and function.Development of Specific Inhibitor for PTKs
These structures enabled the development of new specific inhibitor for PTKs that are currently being tested in clinical trials. Receptor tyrosine kinases undergo ligand-dependent dimerization, which activates their intrinsic protein tyrosine kinase activity resulting in autophosphorylation and subsequent interaction and recruitment of multiple cellular target proteins. The phospho rylated tyrosine residues together with their immediate flanking sequences function as binding sites for signaling molecules containing src homology 2 (SH2) domains. Many signaling proteins carry SH2 domains plus one or more small protein modules such as SH3, PH, PTB, WW or FYVE domains. These protein modules function as mediator of protein-protein or protein-lipid interactions that are critical for signal transmission.
Recruitment of Signaling Proteins
In addition to direct recruitment by RTKs, many signaling proteins are recruited by an alternative mechanism involving a family of membrane linked docking proteins such as FRS-2alpha, and ß, IRS-1 and 2, and Gab-1 and 2, among many others. Recruitment of signaling proteins by RTKs or by docking proteins leads to activation of m ultiple signaling pathways resulting in stimulation of a variety of cellular responses. The small adapter protein Grb2, for example, is bound through its SH3 domains to short, proline-rich sequences in the carboxy terminal tail of the guanine nucleotide-r eleasing factor Sos. Interaction between Grb2 and Sos with tyrosine phosphorylated RTKs or docking proteins results in translocation of Sos to the plasma membrane allowing the exchange of GDP for GTP on Ras. The activated GTP-bound form of Ras initiates t he activation of a kinase cascade composed of Raf, MAPKK, and MAPK leading to phosphorylation of prooncogene Jun on serine and threonine residues to induce transcriptional activation. These and other signaling pathways that are activated by RTKs regulate multiple cellular processes and the pleiotropic response of growth functions on multiple tissues and orgens.Schlessinger Lab Research
The extracellular ligand binding domains of receptor tyrosine kinases contain a variety of structural motifs. The cytoplasmic domain contains the protein tyrosine kinase core and regulatory regions which are subject to autophosphorylation or to phosphorylation by heterologous kinases.
In addition to the catalytic protein tyrosine kinase (PTK) core non-receptor protein tyrosine kinases contain a variety of independent domains which play a role in the regulation of PTK activity and in the control of the sub-cellular localization of these enzymes.
Both growth factor receptors and cytokine receptors that utilize cytoplasmic protein tyrosine kinases for signal transmission are activated by ligand induced receptor dimerization.
Inactive receptor monomers (green) are in equilibrium with inactive (green) or active (blue) receptor dimers. The active receptor dimers exist in a conformation compatible with trans-autophosphorylation and stimulation of the intrinsic activity of the protein tyrosine kinase domain. Ligand binding stabilizes the formation of active receptor dimers resulting in enhancement of protein tyrosine kinase activatity.
Inactive disulfide bridged insulin-receptor dimers (green) are in equilibrium with active insulin-receptor dimers (blue). Binding of insulin to the extracellular ligand-binding domain stabilizes the active dimeric state leading to activation of the protein tyrosine kinase domain of the insulin-receptor.
Ribbon representation of the structure of stem cell factor (SCF) dimer. The helices are green ribbons, the Beta-strands are orange arrows and the loop regions are grey tubes.
A space-filling model depicting FGF2 (orange) in complex with the extracellular ligand-binding domain of FGFR1. The D2 domain is green and the D3 domain is in cyan.
The FGF2 bound to FGFR1 shown in the Fig. 7 were pulled away from each other and rotated 90 deg. about the vertical axis. The receptor-binding molecule on FGF2 and the primary ligand-bindi ng region on FGFR1 are colored red.
Surface charge distribution of a top view of dimeric FGF2/FGFR1 complex. Blue represents positive electrostatic potential.
Molecular surface representation of a top view of a dimeric FGF2-FGFR1 complex. FGF2 is in orange and D2 of the extracellular ligand-binding domain is in green. The side chains of heparin binding reside on FGF2 and D2 are rendered in ball-and-stick. The ordered sulfate ions bound to FGF2 and D2 are also shown. (Same view as in the previous image)
The complex is viewed from the side. FGF2 is in orange, D2 is in green and D3 is in cyan. The sugar rings of the decasaccharides are in ball-and-stick.
Molecular surface representation of a ternary FGF2/ FGFR1/ Heparin complex. Only the first six sugar rings of the decasaccharides are present in ball-and-stick and the nonreducing and reducing ends are labeled.
Different signaling pathways are presented as distinct signaling cassettes (colored boxes). In several cases the signaling cassettes do not include all the known components of a given path way. Also shown, examples of stimulatory and inhibitory signals for the different pathways. For example, in addition to activation of the MAP kinase signaling cascade, Ras activates PI-3 kinase and Cdc42. Stimulation of PI-3 kinase leads to activation of PDK1 and PKB, two kinases that regulate various metabolic processes and prevent apoptotic death. In addition, PI-3 kinase activation stimulates generation of hydrogen peroxide which in turn oxidizes and blocks the action of an inhibitory protein tyrosine phosphatase (PTP). The signaling cassettes presented in the figure regulate the activity of multiple cytoplasmic targets. However, the Ras/MAP, STAT, JNK, and PI-3 kinase signaling pathways also regulate the activity of transcriptional factors by phosphorylation and by other mechanisms.
The activated PDGFR is autophosphorylated on at least eight tyrosine autophosphorylation sites. The tyrosine phosphorylation sites serve as docking sites for recruitment and activation of the Ras/MAP kinase signaling cascade (yellow), PI-3 kinase dependent signaling pathway (cyan) and PLC gamma dependent Ca+2 signal (green).
A summary of various protein modules that mediate protein-protein, protein-lipid and other interactions necessary for transmitting the signal generated at the cell membrane by activation of surface receptors. Also shown are specific ligands and binding partners of the various protein modules.
SH2 and SH3 domain containing adaptor and regulatory proteins that mediate and regulate cellular signaling pathways.
Signaling proteins containing SH2, SH3, PH or other protein modules. The protein modules play a role in the control of the intrinsic activities of host proteins, in mediating interactions with specific target molecules and in control of their subcellular localization.
A family of docking proteins linked to the cell membrane by means of myristilation anchors, transmembrane domains or by binding of PH domain to inositol phospholipids. The carboxy termini of the docking proteins contain multiple tyrosine phosphorylation sites that function as binding sites for SH2 domain containing adaptors or signaling proteins.
The two members of the FRS2 family of docking proteins FRS2 and FRS2 function as major mediators of signaling by FGF, NGF and GDNF receptors.
At least two separate molecular events are required for RTK-induced activation of signaling molecules. As many protein targets of RTKs are located at the cell membrane, translocation to th e cell membrane is essential for activation of many effector proteins. Activation of PKB (also known as Akt) by membrane translocation. PtdIns(3,4,5)P3 generated in response to growth factor stimulation serves as a binding site for the PH domains of PDK1 and PKB. Membrane translocation is accompanied by release of an autoinhibition leading to activation of PDK1 and PKB kinase activities. Full activation of PKB requires phosphorylation of PDK1 (and also by PDK2?). Activated PKB phosphorylates a variety of target proteins that prevent apoptotic death and regulate various metabolic processes.
Binding of the SH2 domains of PLC to tyrosine autophosphorylation sites in activated receptors facilitates tyrosine phosphorylation of PLC as well as membrane translocation; a process medi ated in part by binding of the PH domain to products of PI-3 kinase. Tyrosine phosphorylation is essential for PLC activation leading to hydrolysis of PtdIns(4,5)P2 and the generation of the two second messengers Ins(1,4,5)P3 and diacylglycosol.
Binding of the SH2 domains of p85, the regulatory subunit of PI-3 kinase to tyrosine autophosphorylation sites on activated receptors releases an autoinhibitory constraint that stimulates the catalytic domain (p110). PI-3 kinase catalyzes the phosphorylation of the 3' positions of the inositol ring of PtdIns(4)P and PtdIns(4,5)P2 to generate PtdIns(3,4)P2 and PtdIns(3,4,5)P3 respectively.
Ribbon diagram of the PTK domain of FGFR1. The helices are shown in red, the beta strands in green, the nucleotide binding loop in orange, the catalytic loop in blue, the activation loop in purple, the kinase insert in black, and the side chains of Tyr-653 and Tyr 654 in yellow. The Amino and Carboxy termini are denoted by N and C, respectively.
Molecular surface representation of the PTK domains of FGFR1 (FGFR1K) and Insulin receptor (IRK) showing the difference in the conformation of the activation loops (green). The catalytic l oop is shown in orange, nitrogen atoms are blue and oxygen atoms are red.
Molecular surface representation of the conformation of the activation loops of Insulin receptor PTK domain (IRK) and tyrosine phosphorylated PTK domain of Insulin receptor (IRK3P). The ac tivation loops are colored green, the catalytic loops are in orange and the peptide substrate is pink. Carbon atoms are colored white, nitrogen atoms blue, oxygen atoms red and phosphorous atoms yellow. AMP-PNP is also shown in the IRK3P structure (Courte sy of S.R. Hubbard).
Molecular surface representation of the catalytic domain of FGFR1 with two PTK inhibitors. The inhibitors are bound to the cleft between the two lobes of the PTK domain. The surface is colored purple for atoms of the hinge region, light blue for atoms of the nucleotide binding loop and yellow for atoms of the catalytic loop of the PTK domain.
Oxygen-aromatic interactions of the phenyl ring of Phe489. Carbon atoms are orange, oxygen atoms are red, nitrogen atoms are blue and hydrogen atoms are black. An alignment of the amino acid sequences of residues in the hinge region of FGFR1, PDGFR, Insulin R, EGFR and VEGFR are shown.
Surface of the nucleotide binding region of the catalytic PTK core of FGFR1 in complex with an ATP analogue (AMP-PCP), or with two PTK inhibitors. Carbon atoms are in orange, oxygen atoms are red, nitrogen atoms are blue and phosphorous atoms are purple.
Surface of the nucleotide binding region of the catalytic domain of FGFR1 in complex with two PTK inhibitors. Carbon atoms are in orange, oxygen atoms are red and nitrogen atoms are in blue.