Abstract 21431: Identification of the Receptor Tyrosine Kinase Tie1 as a Novel Akt Substrate: Requirement for Tie1 Phospho-T794 in Vascular Development
The Tie family of receptors, Tie1 and Tie2, are highly homologous receptor tyrosine kinases (RTKs). In contrast to Tie2 signal transduction, Tie1 signaling is still poorly understood. To investigate the potential structural differences between Tie1 and −2 that mediate their distinct biological properties, we used a bioinformatics and biochemical approach to identify Tie1 as a potential substrate for the serine/threonine kinase Akt. Examination of the receptors' juxtamembrane (JM) domains revealed a high probability Akt consensus phosphorylation site (RXRXXS/T) at Threonine 794 in Tie1 but not Tie2 that is conserved across orthologs, including zebrafish. Akt regulates a myriad of cellular processes including growth, proliferation, survival, and metabolism. We hypothesized that Akt-mediated phosphorylation of Tie1-T794 plays an important role in endothelial cell (EC) biology. Using recombinant GST-Tie1 kinase fusion proteins and cultured ECs expressing Tie1, Tie1 T794 was found to be phosphorylated upon addition of membrane targeted myristoylated-Akt. Similarly, activation of endogenous Akt in response to the Tie receptor ligand Angiopoietin-1 induced phosphorylation of Tie1 T794. The non-phosphorylatable Tie1 mutant T794A abrogated Akt-mediated Tie1 phosphorylation in vitro. To determine functional consequences of Tie1 phosphorylation at T794, loss-of-function experiments were performed in a zebrafish model of vascular development. Vascular development was disrupted by morpholino knockdown of Tie1, with impaired sprouting of intersomitic vessels, edema, and vascular hemorrhage. The mutant phenotype was rescued by concomitant expression of wild-type (WT) murine Tie1 but not Tie1-T794A. Furthermore, when expressed in the absence of morpholino knockdown, the T794A mutant induced a similar mutant phenotype, acting as a dominant negative inhibitor. These findings suggest a novel mechanism by which Tie1 signaling and function are regulated by Akt-mediated phosphorylation. Studies are underway to determine the precise role of Akt-mediated Tie1 phosphorylation in vascular growth and remodeling.
- © 2010 by American Heart Association, Inc.