Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

Abstract

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.Tie2 (also called Tek) is a member of a novel family of receptor tyrosine kinases (RTKs) (16,17,37,42,72) that are expressed predominantly on endothelial cells or their embryonic precursors (14,16,17,37,42) and that are required for normal vascular development (15,52,55). Functional disruption of Tie2 in transgenic mice results in embryonic lethality by day E9.5 to 10.5 with effects on the microvasculature resulting in reduced numbers of endothelial cells and abnormalities of vascular morphogenesis (15,55). Knockout of the activating Tie2 ligand, angiopoietin-1 (Ang1), or overexpression of a related, inhibitory ligand, angiopoietin-2 (Ang2), resulted in phenotypes similar to the Tie2 knockout (43, 64). Taken together, these findings suggest a role for Tie2 in endothelial cell maintenance, survival, and/or vascular morphogenesis (24).In addition to a role in embryonic vascular development, data from our laboratory suggest that Tie2 plays an important role in the adult vasculature. For example, Tie2 expression is increased in the vasculature of malignant breast tumors (49), and a soluble extracellular domain of Tie2 inhibits tumor angiogenesis and growth (39). Tie2 is also broadly expressed and tyrosine phosphorylated in a variety of adult tissues in which the endothelium is normally quiescent (69). These findings are consistent with a dual role for Tie2 in both the growth and the maintenance of the adult vasculature.To better understand the role of Tie2 in vascular growth and maintenance, it will be important to identify the signal transduction pathways responsible for these functions. Currently, little is known about the specific signaling proteins and pathways utilized by Tie2. We demonstrated previously that Tie2 a...

Angiogenic Actions of Angiopoietin-1 Require Endothelium-Derived Nitric Oxide

“…Enhanced EC survival in response to Ang1 has been shown to be dependent on activation of Akt by PI3-kinase (also known as PKB). 10,17,20,41 Akt promotes cell survival through its ability to phosphorylate Bad and procaspase-9. 42,43 In addition, a recent report has demonstrated that Ang1 increased EC survival by the induction of the apoptosis inhibitor, survivin, again through the PI3-kinase/Akt pathway.…”

“…15 In addition, the p85 subunit of phosphatidylinositol (PI) 3-kinase can interact specifically with the phosphorylated Tie2 through its SH2 domain. Although an initial report suggested that activated Tie2 does not associate with PI3-kinase, 16 subsequent studies have confirmed that activation of Tie2 leads to PI3-kinase activation, 17 the lipid products of which participate in the regulation of cell survival by activation of the serine/ threonine kinase, Akt. 18 Indeed, the PI3-kinase/Akt pathway has been shown to transduce the antiapoptotic effect of both vascular endothelial growth factor (VEGF) 19 and Ang1.…”

“…18 Indeed, the PI3-kinase/Akt pathway has been shown to transduce the antiapoptotic effect of both vascular endothelial growth factor (VEGF) 19 and Ang1. 17,20 Recently, Akt has also been demonstrated to directly phosphorylate endothelial nitric oxide synthase (eNOS), leading to calcium/calmodulin-independent enzyme activation. 21,22 Endothelium-derived nitric oxide (NO) is a crucial mediator in the regulation of EC growth, survival, and angiogenesis.…”

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

“…Subcellular fractionation and immunofluorescence imaging of endogenous Akt suggest that it is strongly recruited to the PM in response to growth factor stimulation (Carvalho et al, 2000;Currie et al, 1999;. Furthermore, live-cell imaging of fluorescent Akt reporter constructs have revealed important insights such as isoform specificity, chemotaxis, phospholipid binding, conformational changes in Akt, nuclear activation and membrane diffusion rates, across a range of cell lines (Calleja et al, 2003;Gonzalez and McGraw, 2009;Kontos et al, 1998;Lasserre et al, 2008;Servant et al, 2000;Wang and Brattain, 2006). Numerous groups have assessed Akt recruitment to the PM in response to various stimuli in live cells by using GFP-tagged full-length Akt and Akt PH domain constructs.…”

E7080, a novel inhibitor that targets multiple kinases, has potent antitumor activities against stem cell factor producing human small cell lung cancer H146, based on angiogenesis inhibition

“…6 However, the role of SCF/KIT signaling in tumor angiogenesis is still unclear 7 to data, except for the role of SCF in the recruitment of bone marrow-derived progenitor cells, 6,[8][9][10] together with angiopoietin-1 and stromal derived factor 1 a.…”