Vascular Endothelial Growth Factor Receptor KDR Tyrosine Kinase Activity Is Increased by Autophosphorylation of Two Activation Loop Tyrosine Residues

Bruton's tyrosine kinase (BTK) is a member of the Tec nonreceptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr 551 within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr 551 causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH 2 ) revealed that BTK employs a ternary complex mechanism with K mATP ‫؍‬ 84 ؎ 20 M and K mS1 ‫؍‬ 37 ؎ 8 M. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mM, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate K d is weaker than K m . This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase. Bruton's tyrosine kinase (BTK)2 is a non-receptor tyrosine kinase that plays an essential role in B cells and other hematopoietic cell types (1-4). The importance of BTK is evident from the fact that mutations in BTK cause X-linked agammaglobulinemia, a human disease that causes defects in both B cell maturation and mature B cell function (5, 6). A BTK point mutation in mice results in a similar, albeit milder, B cell deficiency known as X-linked immunodeficiency (7). The indispensable requirement of BTK for B cell function in humans makes BTK a promising target for treatment of inflammatory diseases that involve inappropriate B cell activation.The BTK-mediated signaling events that link B cell receptor ligation to downstream signaling have been well characterized (8, 9). Upon ligation of the B cell receptor, Src family kinases associated with the receptor, such as Lyn, are the first enzymes activated. This activation results in phosphorylation of tyrosine residues that become docking sites for other kinases including phosphatidylinositol 3-kinase. Once recruited, phosphatidylinositol 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate to form phosphatidylinositol 3,4,5-bisphosphate. Formation of phosphatidylinositol 3,4,5-bisphosphate then recruits BTK to the plasma membrane via interaction with its pleckstrin homology domain. Upon recruitment to the membrane, BTK is activated via phosphorylation. BTK proceeds to phosphorylat...

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confidence: 99%

Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase

Dinh¹,

Grünberger

Ho³

et al. 2007

“…The slight, but not significant, reduction in NO release by the 1175 and 1214 mutants compared with wild type receptor may point toward a secondary role for these residues in eNOS signaling, in sharp contrast to Tyr 801 , which is shown to be essential. In addition, we cotransfected with eNOS a Y1059F-VEGFR-2 mutant that has been previously shown to have altered overall tyrosine kinase activity (results not shown) (29,30), and the amounts of NO released from these transfected cells were similar to the kinase-dead K868R and Y801F mutants (results not shown).…”

Section: Figure 1 Stimulation Of No Release By the Vegfr-2 Mutantsmentioning

confidence: 75%

“…Although some slight discrepancies are present in the literature on the implication of certain residues in VEGFR-2 signaling, a consensus emerged on the role of some. Tyrosine residues 1054 and 1059 in the kinase insert domain seem to be needed for maximal intrinsic VEGFR-2 kinase activity (29,30). Other phosphorylated residues have been linked to the association and activation of Src homology 2-containing adaptors, as is the case for Tyr 951 and the VRAP/TSad adaptor and Tyr…”

mentioning

confidence: 99%

Phosphorylation of Tyrosine 801 of Vascular Endothelial Growth Factor Receptor-2 Is Necessary for Akt-dependent Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

Blanes¹,

Oubaha

Rautureau

et al. 2007

Vascular endothelial growth factor (VEGF)-stimulated nitric oxide (NO) release from endothelial cells is mediated through the activation of VEGF receptor-2 (VEGFR-2). Herein, we have attempted to determine which autophosphorylated tyrosine residue on the VEGFR-2 is essential for VEGF-mediated endothelial nitric-oxide synthase (eNOS) activation and NO production from endothelial cells. Tyrosine residues 801, 1175, and 1214 of the VEGFR-2 were mutated to phenylalanine, and the mutated receptors were analyzed for their ability to stimulate NO production. We show, both in COS-7 cells cotransfected with the VEGFR-2 mutants and eNOS and in bovine aortic endothelial cells, that the Y801F-VEGFR-2 mutant is unable to stimulate NO synthesis and eNOS activation in contrast to the wild type, Y1175F-VEGFR-2, and Y1214F-VEGFR-2. However, the Y801F mutant retains the capacity to activate phospholipase C-␥ in contrast to the Y1175F-VEGFR-2. Interestingly, the Y801F-VEGFR-2, in contrast to the wild type receptor, does not fully activate phosphatidylinositol 3-kinase or recruit the p85 subunit upon receptor activation. This results in a complete incapacity of the Y801F-VEGFR-2 to stimulate Akt activation and eNOS phosphorylation on serine 1179 in endothelial cells. In addition, constitutive activation of Akt or a phosphomimetic mutant of eNOS (S1179D) fully rescues the inability of the Y801F-VEGFR-2 to induce NO release. Finally, we generated an antibody that specifically recognizes the phosphorylated form of tyrosine 801 of the VEGFR-2 and demonstrate that this residue is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of tyrosine residue 801 of the VEGFR-2 is essential for VEGFstimulated NO production from endothelial cells, and this is primarily accomplished via the activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS.

“…2B). Tyrosines 1052 and 1057 are activation loop tyrosines and are involved in VEGFR-2 activation (23,30,31). To test which amino acid among the 14 amino acid residues is involved in regulation of autophosphorylation of VEGFR-2, we initially mutated Asn-1160, Gln-1163, Asn-1165, Gln-1167, and Gln-1168 to alanine (A).…”

Section: Regulation Of Ligand-dependent Tyrosine Phosphorylation Of Vmentioning

confidence: 99%

Leucine Motif-dependent Tyrosine Autophosphorylation of Type III Receptor Tyrosine Kinases

Meyer

Qian

Guo

2006

Activation loop tyrosine autophosphorylation is an essential requirement for full kinase activation of receptor tyrosine kinases (RTKs). However, mechanisms involved are not fully understood. In general, kinase domains of RTKs are folded into two main lobes, NH 2 -and COOH-terminal lobes. The COOH-terminal lobe of vascular endothelial growth factor receptor-2 (VEGFR-2) is folded into seven ␣-helices (␣D-␣I). In the studies presented here we demonstrate that leucine residues of helix I (␣I) regulate tyrosine autophosphorylation and phosphotransferase activity of VEGFR-2. The presence of leucines 1158, 1161, and 1162 are essential for tyrosine autophosphorylation and kinase activation of VEGFR-2 and are involved in helix-helix packing via hydrophobic interactions. The presence of leucine 1158 is critical for kinase activation of VEGFR-2 and appears to interact with ␣E, ␣F, ␣H, and ␤7. The analogous residue, leucine 957 on platelet-derived growth factor receptor-␤ and leucine 910 on colony stimulating factor-1R are also found to be critical for tyrosine autophosphorylation of these receptors. Leucines 1161 and 1162 are also involved in helix-helix packing but they play a less critical role in VEGFR-2 activation. Thus, we conclude that leucine motif-mediated helix-helix interactions are critical for kinase regulation of type III RTKs. This mechanism is likely to be shared with other kinases and might provide a basis for the design of a novel class of tyrosine kinase inhibitors.Receptor tyrosine kinases (RTKs) 2 are a large family of enzymes, many of which mediate vital cellular functions of living organisms. The fine-tuning of RTKs function is essential for their normal physiological roles and their aberrant function contributes to human diseases ranging from cancer to diabetes (1-4). RTKs consist of an extracellular region that serves a ligand binding site, a transmembrane domain, and a cytoplasmic region, which possesses intrinsic tyrosine kinase activity. In the inactive state, the activation loop is thought to occupy the active site preventing substrate access and ATP binding (5, 6). Ligand-mediated RTK activation leads to RTK dimerization. Dimerization is believed to facilitate transphosphorylation of one or two tyrosines within the activation loop. Based on the crystal structure of the insulin receptor (7), fibroblast growth factor receptor (8), it is proposed that activation loop tyrosine autophosphorylation removes the activation loop away from the active site and creates appropriate conformation for optimal substrate and ATP binding (6 -8). The catalytic kinase domain of RTKs ranges from 250 to 300 amino acid residues and contains highly conserved amino acid sequences. The typical kinase domain of RTKs is folded into two main lobes, NH 2 -and COOH-terminal lobes. Catalysis occurs in a cleft between the two domains. Residues in the NH 2 -terminal lobe are mainly in ␤-sheets and are involved in ATP binding. Residues in the COOH-terminal lobe, however, are primarily in ␣-helical conformation and are importan...