Dougher M, Terman B I
Wyeth-Ayerst Oncology Research, Pearl River, New York 10965, USA.
Oncogene. 1999 Feb 25;18(8):1619-27. doi: 10.1038/sj.onc.1202478.
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 p125FAK and paxillin. VEGF binding to KDR(Y951F) and KDR(Y996F) expressing cells resulted in phosphorylation of all cellular substrates tested, although the level of PLCgamma phosphorylation was decreased for KDR(Y996F). The decreased level of PLCgamma phosphorylation was not because PLCgamma-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 PLCgamma, as well as p125FAK 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.
我们之前报道过在KDR血管内皮生长因子（VEGF）受体上鉴定出四个自身磷酸化位点。其中两个位点（酪氨酸951和996）位于受体的激酶插入结构域，另外两个（酪氨酸1054和1059）位于催化结构域。为了阐明这些位点的功能意义，我们构建了DNA构建体，其中酪氨酸密码子被苯丙氨酸密码子取代，并在293细胞中表达这些DNA构建体。VEGF与表达天然受体的细胞结合导致受体和磷脂酶C-γ（PLC-γ）磷酸化迅速增加，以及粘着斑激酶（p125FAK）和桩蛋白磷酸化缓慢增加。VEGF与表达KDR（Y951F）和KDR（Y996F）的细胞结合导致所有测试的细胞底物磷酸化，尽管KDR（Y996F）的PLCγ磷酸化水平有所降低。PLCγ磷酸化水平降低并非因为含PLCγ的Src同源2（SH2）结构域与Y996自身磷酸化位点结合。我们得出结论，存在先前未鉴定的受体自身磷酸化位点，这些位点允许通过PLCγ以及p125FAK和桩蛋白进行信号传导。VEGF与在酪氨酸1054和1059处均发生突变的KDR表达细胞结合可激活受体自身磷酸化，但激活水平仅为表达天然受体细胞的10%。在表达KDR（Y1054,1059）的细胞中未观察到细胞信号蛋白的酪氨酸磷酸化。利用直接测量受体催化活性的体外试验，我们能够确定天然受体的酪氨酸激酶活性明显高于双突变体。从该结果我们得出结论，VEGF诱导的酪氨酸1054和1059处的自身磷酸化是实现最大KDR激酶活性的必要步骤。VEGF诱导的受体内化需要最大速率的受体激酶活性，因为与表达天然受体的细胞相比，表达KDR（Y1054,1059F）的细胞中的内化延迟。
