Sandbox Reserved 1490

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. 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.

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Function

Structural highlights

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• Catalytic activation

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.

Disease

• Dominantly inherited venous malformations (VMCM)

Disease description: A vascular morphogenesis error characterized by dilated serpiginous channels. All mutations occur in the protein kinase domain.

– 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.

With this mutation, Venous Malformations (VMs) contain a Disproportionately high ratio of Endothelial Cells (ECs) to Smooth Muscle Cells (SMCs)

Fig 1 : 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.

Fig 2 : Immunohistochemistry of VMs with Antibodies against Smooth Muscle Cells 𝛂-Actin

B = Abnormal channels

C = Normal veins (v) and arteries (a)

Scale bars, 200 𝛍m

Antibodies directed against SMCs 𝛂-Actin from cells with VMs show that the vessels have a specific and abnormal staining (B) compared to normal vessels (C)

– Mutation in position 897 : Tyrosine → Cystéine. Change from large size and aromatic (Y) to medium size and polar (C). An error of vascular morphogenesis characterized by dilated, serpiginous channels

– Mutation in position 897 : Tyrosine → Sérine. Change from large size and aromatic (Y) to small size and polar (S). An error of vascular morphogenesis characterized by dilated, serpiginous channels.

– Mutation in position 915 : Arginine → Histidine. Change from large size and basic (R) to medium size and polar (H). An error of vascular morphogenesis characterized by dilated, serpiginous channels.

– Mutation in position 918 : Arginine → Cystéine. Change from large size and basic (R) to medium size and polar (C). An error of vascular morphogenesis characterized by dilated, serpiginous channels.

– Mutation in position 919 : Valine → Leucine. Similar physico-chemical property. Both residues are medium size and hydrophobic. An error of vascular morphogenesis characterized by dilated, serpiginous channels.

– Mutation in position 925 : Alanine → Sérine. Change from small size and hydrophobic (A) to small size and polar (S). Increased ligand-independent autophosphorylation and kinase activation.

– Mutation in position 1100 : Lysine → Asparagine. Change from large size and basic (K) to medium size and polar (N). Increased ligand-independent autophosphorylation and kinase activation.

• Mutagénèse

– Transfer in position 855 : Loss of kinase activity – Transfer in position 1102 : Deletes interaction with CHS1

Relevance