Interaction of the Flt-1 Tyrosine Kinase Receptor with the p85 Subunit of Phosphatidylinositol 3-Kinase

Abstract

We have examined the interactions of the p85 regulatory subunit of phosphatidylinositol 3-kinase with the endothelium-specific Flt-1 receptor tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tail of the Flt-1 receptor in order to identify the site(s) that is responsible for the p85 interactions. A single tyrosine to phenylalanine change at position 1213 inhibits the binding of both p85 SH2 domains. Phosphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophosphorylation. The amino acids following this tyrosine are VNA and thus define a novel binding site for p85.

VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells

“…Some groups reported that PI 3 kinase is activated in response to VEGF (Guo et al, 1995;Xia et al, 1996) and others that it is not (Waltenberger et al, 1994). Recently Flt-1 kinase has been shown to associate with p85 subunit of PI 3 kinase when these molecules are overexpressed in yeast cells (Conningham et al, 1995). However, it is not clear whether this interaction takes place in primary endothelial cells, because the Flt family receptors do not have tyrosine residues of the recognition motif for PI 3 kinase (tyrosine-X-X-methionine) within the kinase insert nor in the carboxyl tail.…”

“…Activation of PI 3 kinase is not essential for VEGF signaling to DNA synthesis Recently Flt-1 kinase has been shown to associate with p85 subunit of PI 3 kinase when these molecules were overexpressed in yeast cells (Conningham et al, 1995). It is not clear yet whether or not this interaction is biologically signi®cant in primary endothelial cells.…”

Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis

“…They are located in the C-terminal tail domain and may serve as docking sites for signaling molecules such as PLCg, Nck, Crk, SHP-1/2, or the p85 subunit of PI3 kinase, as shown by yeast two hybrid assays and by in vitro binding studies (Cunningham et al, 1995;Igarashi et al, 1998a). Activation of PLCg and RasGAP has been reported in VEGFR-1 transfected ®broblasts (Seetharam et al, 1995).…”

The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC-γ pathway and partially induces mitotic signals in NIH3T3 fibroblasts

“…More recently Flt-1 kinase has been shown to associate with p85 subunit of PI 3 kinase when these molecules were overexpressed in yeasts (Conningham et al, 1995). It is not clear yet whether or not this interaction could be seen in primary endothelial cells.…”