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==Crystal structure of cytoplasmic kinase domain of Tie2 in complex with decipera compound DP1919== | ==Crystal structure of cytoplasmic kinase domain of Tie2 in complex with decipera compound DP1919== | ||
<StructureSection load=' | <StructureSection load='6mwe' size='340' side='right' caption='Caption for this structure' scene=''> | ||
The protein we are focusing one is a protein kinase receptor to a family of ligands called angiopoietins. This receptor is a Tyrosine Kinase TIE2. We are going to analyze the kinase domain of this protein | |||
The protein we are focusing one is a protein kinase receptor to a family of ligands called angiopoietins. This receptor is a Tyrosine Kinase TIE2. We are going to analyze the <scene name='80/802664/Entire_molecule/1'>kinase domain</scene> of this protein. | |||
It acts as cell-surface receptor for the ligands ANGPT1, ANGPT2 and ANGPT4 and regulates among others angiogenesis, endothelial cell survival and maintenance of vascular quiescence. It is important in the regulation of both normal physiologic and pathologic angiogenesis. The later is a fundamental step in the transition of tumors from a benign state to a malignant one. | It acts as cell-surface receptor for the ligands ANGPT1, ANGPT2 and ANGPT4 and regulates among others angiogenesis, endothelial cell survival and maintenance of vascular quiescence. It is important in the regulation of both normal physiologic and pathologic angiogenesis. The later is a fundamental step in the transition of tumors from a benign state to a malignant one. | ||
Angiogenesis is the process in which new blood vessels are formed from pre-existing blood vessels. The growth of these new blood vessels requires migration and proliferation of endothelial cells (ECs). It is an event controlled by angiogenic growth factors such as vascular endothelial growth factor (VEGF). | |||
While ANGPT1 is a TIE2 agonist and has a higher binding affinity to it than ANGPT2, ANGPT2 can act as a context-dependent agonist. Thus, the ANGPT/TIE2 kinase signaling pathway is an attractive anti-vascular target. | While ANGPT1 is a TIE2 agonist and has a higher binding affinity to it than ANGPT2, ANGPT2 can act as a context-dependent agonist. Thus, the ANGPT/TIE2 kinase signaling pathway is an attractive anti-vascular target. | ||
== Function == | == Function == | ||
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TIE2 maintains the vascular integrity of mature vessels by enhancing endothelial barrier function and inhibiting apoptosis of endothelial cells.<ref>PMID:18425120</ref> | TIE2 maintains the vascular integrity of mature vessels by enhancing endothelial barrier function and inhibiting apoptosis of endothelial cells.<ref>PMID:18425120</ref> | ||
ANGPT1 is a TIE2 agonist : in vitro, it binds to TIE2 and induces its activation via tyrosine phosphorylation. In vivo, it was proven that inactivation of ANGPT1 or over expression of ANGPT2 produce similar effects.<ref>PMID:19223473</ref> | ANGPT1 is a TIE2 agonist : in vitro, it binds to TIE2 and induces its activation via tyrosine phosphorylation. In vivo, it was proven that inactivation of ANGPT1 or over expression of ANGPT2 produce similar effects.<ref name="Angiopoietin 2">PMID: 19223473</ref> | ||
ANGPT2 is a competitive antagonist of TIE2 or a partial agonist of TIE2 depending on the context. In stressed ECs, one recent report suggests that ANGPT2 may activate TIE2 signaling in the absence of ANGPT1 and in high concentrations. | ANGPT2 is a competitive antagonist of TIE2 or a partial agonist of TIE2 depending on the context. In stressed ECs, one recent report suggests that ANGPT2 may activate TIE2 signaling in the absence of ANGPT1 and in high concentrations. | ||
[[Image:controlANG.png]] | [[Image:controlANG.png]] | ||
''Fig 1. These cells express Tie2 and are marked with GFP. They were incubated with vehicle (control; left column) and COMP–Ang1 (right column). The scale bars represent 10 μm.''<ref | |||
''Fig 1. These cells express Tie2 and are marked with GFP. They were incubated with vehicle (control; left column) and COMP–Ang1 (right column). The scale bars represent 10 μm.''<ref name="Angiopoietin 2"/> | |||
===• Signal transduction and kinase activity=== | ===• Signal transduction and kinase activity=== | ||
Receptor tyrosine kinases are transmembrane proteins with a [[2gy5|ligand-binding extracellular domain]], a single membrane-spanning domain, a juxtamembrane region, a catalytic domain, and a C-terminal tail.<ref>PMID:11513602</ref> | Receptor tyrosine kinases are transmembrane proteins with a [[2gy5|ligand-binding extracellular domain]], a single membrane-spanning domain, a juxtamembrane region, a catalytic domain, and a C-terminal tail.<ref name="Mechanistic effects of autophosphorylation">PMID: 11513602</ref> In cell culture, ANGPT1 induces phosphorylation of TIE2 and stimulates endothelial cell migration and survival. | ||
The activation of the receptor is due to a ligand-induced dimerization : the extracellular receptor domain dimerization brings the cytosolic kinase domains next to each other for intermolecular autophosphorylation. The latter occurs when one subunit of the dimeric receptor phosphorylates tyrosine residues on the other subunit. It happens in a sequential manner : Tyr-992 in the kinase activation loop is phosphorylated first, followed by autophosphorylation at Tyr-1108 and at additional tyrosine residues. Autophosphorylation also has multiple functions including recruitment of downstream signaling molecules.<ref | The activation of the receptor is due to a ligand-induced dimerization : the extracellular receptor domain dimerization brings the cytosolic kinase domains next to each other for intermolecular autophosphorylation. The latter occurs when one subunit of the dimeric receptor phosphorylates tyrosine residues on the other subunit. It happens in a sequential manner : Tyr-992 in the kinase activation loop is phosphorylated first, followed by autophosphorylation at Tyr-1108 and at additional tyrosine residues. Autophosphorylation also has multiple functions including recruitment of downstream signaling molecules.<ref name="Mechanistic effects of autophosphorylation" /> | ||
Looking more closely at the TIE2 intracellular domain, 1106 is found at the base of a loop formed between the C-terminus tail and the C-terminus lobe of the kinase. The OH group of Tyr-1106 is thus directly into the solvent and accessible to phosphorylation. However, Tyr-1100 is not solvent exposed : thereby implying that the carboxy-terminal tail must undergo a conformational change upon activation of the receptor to expose this tyrosine residue for phosphorylation.<ref>PMID:12665569</ref> | Looking more closely at the TIE2 intracellular domain, 1106 is found at the base of a loop formed between the C-terminus tail and the C-terminus lobe of the kinase. The OH group of Tyr-1106 is thus directly into the solvent and accessible to phosphorylation. However, Tyr-1100 is not solvent exposed : thereby implying that the carboxy-terminal tail must undergo a conformational change upon activation of the receptor to expose this tyrosine residue for phosphorylation.<ref name="A unique autophosphorylation">PMID: 12665569</ref> | ||
Consequent to ANGPT1 stimulation, the SH2 domain-containing p85 subunit of [[2pna|phosphatidylinositol (PI) 3-kinase]] is recruited to TIE via tyrosine residue 1100 in the C-end tail of the receptor, leading to activation of the enzyme.<ref | Consequent to ANGPT1 stimulation, the SH2 domain-containing p85 subunit of [[2pna|phosphatidylinositol (PI) 3-kinase]] is recruited to TIE via tyrosine residue 1100 in the C-end tail of the receptor, leading to activation of the enzyme.<ref name="A unique autophosphorylation"/> | ||
Interestingly, inhibition of PI 3′ kinase activity can only partially inhibit the chemotactic effect of ANGPT1 on endothelial cells, thereby implying that additional TIE2 binding partners may also contribute to ANGPT1-mediated endothelial cell migration. Phosphorylation of TIE2 further results in its association with a docking protein related to downstream of kinase (Dok), known as Dok-R, it allows Dok-R to serve as a substrate of TIE2 and thereby become tyrosine phosphorylated.<ref | Interestingly, inhibition of PI 3′ kinase activity can only partially inhibit the chemotactic effect of ANGPT1 on endothelial cells, thereby implying that additional TIE2 binding partners may also contribute to ANGPT1-mediated endothelial cell migration. Phosphorylation of TIE2 further results in its association with a docking protein related to downstream of kinase (Dok), known as Dok-R, it allows Dok-R to serve as a substrate of TIE2 and thereby become tyrosine phosphorylated.<ref name="A unique autophosphorylation"/> | ||
== Structural highlights == | == Structural highlights == | ||
The kinase domain of the TIE2 receptor is composed of two chains : A & B. | |||
===• Catalytic activation === | ===• Catalytic activation === | ||
ATP + L-tyrosyl-[protein] = ADP + H+ + O-phospho-L-tyrosyl-[protein] | ATP + L-tyrosyl-[protein] = ADP + H+ + O-phospho-L-tyrosyl-[protein] | ||
Angiopoietin binding leads to receptor dimerization and activation by autophosphorylation at Tyr-992 on the kinase activation loop. | Angiopoietin binding leads to receptor dimerization and activation by autophosphorylation at Tyr-992 on the kinase activation loop. | ||
[[Image:Réaction Phosphorylation.jpg]] | [[Image:Réaction Phosphorylation.jpg]] | ||
''Fig 2. Phosphorylation reaction'' | ''Fig 2. Phosphorylation reaction'' | ||
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===• Description of total protein=== | ===• Description of total protein=== | ||
[[Image:TIE2 Schema.jpg]] | [[Image:TIE2 Schema.jpg]] | ||
''Fig 3. Scheme of the whole TIE2 receptor'' | ''Fig 3. Scheme of the whole TIE2 receptor'' | ||
====Important sites ==== | ====Important sites ==== | ||
AA= Amino Acid | AA= Amino Acid | ||
– Binding site : AA 855 | – Binding site : <scene name='80/802664/Amino_acid_855/1'>AA 855</scene> | ||
– Active site : AA 964 | – Active site :<scene name='80/802664/Asp964/2'> AA 964</scene> | ||
====Regions ==== | ====Regions ==== | ||
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– Transmembrane region : AA 749 to 769 | – Transmembrane region : AA 749 to 769 | ||
– Cytoplasmic region : AA 770 to 1124 (already found 3D structures: [[1fvr]], [[2oo8]], [[2osc]], [[2wqb]], [[3bea]], [[3l8p]], [[4x3j]] | – Cytoplasmic region : AA 770 to 1124 (already found 3D structures: [[1fvr]], [[2oo8]], [[2osc]], [[2wqb]], [[3bea]], [[3l8p]], [[4x3j]]) | ||
====Domains ==== | ====Domains ==== | ||
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– '''Mutation in position 849 : Arginine → Tryptophane:''' Change from large size and basic (R) to large size and aromatic (W). Increased autophosphorylation and kinase activation; no effect on location at membrane. | – '''Mutation in position 849 : Arginine → Tryptophane:''' Change from large size and basic (R) to large size and aromatic (W). Increased autophosphorylation and kinase activation; no effect on location at membrane. | ||
Arginine at position 849 is found in six residues upstream of the invariant lysine K855 in the kinase domain (sequence preserved among the human, bovine, murine and rat TIE2 sequences). This seems to prove that a basic amino acid is essential for this position. In addition, arginine located a few amino acids before invariant lysine is involved in stabilizing the kinase domain (hydrogen binding of arginine with a proline downstream). It is therefore possible that R849 may also be involved in the stabilization of the kinase domain. Thus, the substitution of R849 by a W could modify the conformation of the kinase domain, leading to a decrease in inhibitory mechanisms and involving autophosphorylation.<ref>PMID:8980225</ref> | Arginine at position 849 is found in six residues upstream of the invariant lysine K855 in the kinase domain (sequence preserved among the human, bovine, murine and rat TIE2 sequences). This seems to prove that a basic amino acid is essential for this position. In addition, arginine located a few amino acids before invariant lysine is involved in stabilizing the kinase domain (hydrogen binding of arginine with a proline downstream). It is therefore possible that R849 may also be involved in the stabilization of the kinase domain. Thus, the substitution of R849 by a W could modify the conformation of the kinase domain, leading to a decrease in inhibitory mechanisms and involving autophosphorylation.<ref name="Vascular dysmorphogenesis">PMID: 8980225</ref> | ||
[[Image:Venous Malformations Diagram.jpg]] | [[Image:Venous Malformations Diagram.jpg]] | ||
''Fig 4. Diagram : Comparison of the Kinase Activities of Normal and Mutant TIE2 Receptors. (B) Cells infected with wild-type baculovirus (wt) or virus expressing normal TIE2 (R2) or mutant TIE2 (W2). Cells expressing the mutation at position 849 (Arginine → Tryptophan) have an autophosphorylation activity 6 to 10 times higher than wild cells.''<ref | |||
''Fig 4. Diagram : Comparison of the Kinase Activities of Normal and Mutant TIE2 Receptors. (B) Cells infected with wild-type baculovirus (wt) or virus expressing normal TIE2 (R2) or mutant TIE2 (W2). Cells expressing the mutation at position 849 (Arginine → Tryptophan) have an autophosphorylation activity 6 to 10 times higher than wild cells.''<ref name="Vascular dysmorphogenesis"/> | |||
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[[Image:Venous Malformations Immunohistochemistry.jpg]] | [[Image:Venous Malformations Immunohistochemistry.jpg]] | ||
''Fig 5. Pictures of immunohistochemistry of VMs with Antibodies against Smooth Muscle Cells 𝛂-Actin <ref | |||
''Fig 5. Pictures of immunohistochemistry of VMs with Antibodies against Smooth Muscle Cells 𝛂-Actin <ref name="Vascular dysmorphogenesis"/>'' | |||
''B = Abnormal channels'' | ''B = Abnormal channels'' | ||
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== Medical relevance == | == Medical relevance == | ||
===•Venous Malformations=== | ===•Venous Malformations=== | ||
Venous malformations can cause significant morbidity due to pain, disfigurement and organ dysfunction. Before understanding a lot better the mechanisms leading to this disease, therapies were limited to compression therapy and ablation of malformed veins by sclerotherapy and surgery.<ref>PMID:29668117</ref> | Venous malformations can cause significant morbidity due to pain, disfigurement and organ dysfunction. Before understanding a lot better the mechanisms leading to this disease, therapies were limited to compression therapy and ablation of malformed veins by sclerotherapy and surgery.<ref name="Therapies for Venous Malformations">PMID: 29668117</ref> | ||
A gene test for TIE2 and PIK3CA mutations is the most definite biomarker for VMs. The mutations in the sequence of this proteins cover a large proportion of the causes (about 80%) of all VMs. | A gene test for TIE2 and PIK3CA mutations is the most definite biomarker for VMs. The mutations in the sequence of this proteins cover a large proportion of the causes (about 80%) of all VMs. | ||
In a blood coagulation reaction, fibrinogen is transformed to fibrin that is cleaved by plasmin in fibrinolysis, resulting in the formation of D‐dimers as a fibrin degradation product. | In a blood coagulation reaction, fibrinogen is transformed to fibrin that is cleaved by plasmin in fibrinolysis, resulting in the formation of D‐dimers as a fibrin degradation product. | ||
Unlike other vascular malformations, VMs patients often have elevated D‐dimers. D‐dimer testing has shown to be useful to separate VMs from other vascular or lymphatic malformations which usually present with normal D‐dimers. Interestingly, VM patients with identified TIE2 or PIK3CA mutations had high D‐dimers when compared to patients with no detectable mutation in these genes. A high serum level of D‐dimers is not solely due to static blood flow in the lesions, but also to an intrinsic signalling defect in ECs due to constantly high TIE2/PIK3CA activity.<ref | Unlike other vascular malformations, VMs patients often have elevated D‐dimers. D‐dimer testing has shown to be useful to separate VMs from other vascular or lymphatic malformations which usually present with normal D‐dimers. Interestingly, VM patients with identified TIE2 or PIK3CA mutations had high D‐dimers when compared to patients with no detectable mutation in these genes. A high serum level of D‐dimers is not solely due to static blood flow in the lesions, but also to an intrinsic signalling defect in ECs due to constantly high TIE2/PIK3CA activity.<ref name="Therapies for Venous Malformations"/> | ||
[[Image:electro.png]] | [[Image:electro.png]] | ||
''Fig 6. Western blot of p-TIE2 in Human Endothelial Cells transfected with TIE2-WT (Wild type) or with mutant TIE2 (L914F). Tubulin served as loading control. The hyperphosphorylation is clearly visible.''<ref>PMID:26258417</ref> | |||
''Fig 6. Western blot of p-TIE2 in Human Endothelial Cells transfected with TIE2-WT (Wild type) or with mutant TIE2 (L914F). Tubulin served as loading control. The hyperphosphorylation is clearly visible.''<ref name="Molecular Therapies">PMID: 26258417</ref> | |||
Thus, genetic and transplantation‐based models offer versatile tools to study the pathology of VMs, as well as the efficacy and safety of potential molecular therapies. | Thus, genetic and transplantation‐based models offer versatile tools to study the pathology of VMs, as well as the efficacy and safety of potential molecular therapies. | ||
Rapamycin is the first molecular therapy for VMs. It is currently being tested in a multicenter clinical trial on lymphatico-vascular malformations.<ref | Rapamycin is the first molecular therapy for VMs. It is currently being tested in a multicenter clinical trial on lymphatico-vascular malformations.<ref name="Molecular Therapies"/> | ||
[[Image:lesion area.png]] | [[Image:lesion area.png]] | ||
''Fig 7. (C) HUVECs lesional area measured every 2 days for 16 days. (D) Vascular volume at day 15 measured by analysis of color Doppler 3D image stacks. When compared with the vehicle-treated group, the lesional area was significantly smaller in the rapamycin-treated group from day 4 to day 16 and in the TIE2-TKI–treated group from day 8 to day 14.''<ref | |||
''Fig 7. (C) HUVECs lesional area measured every 2 days for 16 days. (D) Vascular volume at day 15 measured by analysis of color Doppler 3D image stacks. When compared with the vehicle-treated group, the lesional area was significantly smaller in the rapamycin-treated group from day 4 to day 16 and in the TIE2-TKI–treated group from day 8 to day 14.''<ref name="Molecular Therapies"/> | |||
===•Cancers=== | ===•Cancers=== | ||