Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization

We have previously reported the identification of four autophosphorylation sites on the KDR VEGF receptor. Two of these sites (tyrosines 951 and 996) are located in the receptor's kinase insert domain, and two (tyrosines 1054 and 1059) are located in the catalytic domain. In order to clarify the functional significance of these sites, we made DNA constructs in which tyrosine codons were replaced with those for phenylalanine, and expressed the DNA constructs in 293 cells. VEGF binding to cells expressing the native receptor led to a rapid increase in receptor and PLC gamma phosphorylation, and a slower increase in the phosphorylation of p(125)FAK and paxillin. VEGF binding to KDR(Y951F) and KDR(Y996F) expressing cells resulted in phosphorylation of all cellular substrates tested, although the level of PLC gamma phosphorylation was decreased for KDR(Y996F). The decreased level of PLC gamma phosphorylation was not because PLC gamma-containing SH2 domains bind to the Y996 autophosphorylation site. We conclude that there exists receptor autophosphorylation sites not previously identified which allow for signaling via PLC gamma, as well as p(125)FAK and paxillin. VEGF binding to cells expressing KDR mutated at both tyrosine's 1054 and 1059 activated receptor autophosphorylation but at a level which was only 10% of that seen for cells expressing native receptor. Tyrosine phosphorylation of cell signaling proteins was not observed in KDR(Y1054,1059) expressing cells. Utilizing an in vitro assay which directly measures receptor catalytic activity allowed us to determine that the tyrosine kinase activity of the native receptor was significantly greater than that for the double mutant. We conclude from this result that VEGF-induced autophosphorylation at tyrosines 1054 and 1059 is a required step for allowing maximal KDR kinase activity. Maximal rates of receptor kinase activity is required for VEGF-induced receptor internalization, as internalization was delayed in the KDR(Y1054,1059F) expressing cells when compared to cells expressing native receptor.