PMID- 10102632
OWN - NLM
STAT- MEDLINE
DCOM- 19990422
LR  - 20121115
IS  - 0950-9232 (Print)
IS  - 0950-9232 (Linking)
VI  - 18
IP  - 8
DP  - 1999 Feb 25
TI  - Autophosphorylation of KDR in the kinase domain is required for maximal
      VEGF-stimulated kinase activity and receptor internalization.
PG  - 1619-27
AB  - 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.
FAU - Dougher, M
AU  - Dougher M
AD  - Wyeth-Ayerst Oncology Research, Pearl River, New York 10965, USA.
FAU - Terman, B I
AU  - Terman BI
LA  - eng
PT  - Journal Article
PL  - England
TA  - Oncogene
JT  - Oncogene
JID - 8711562
RN  - 0 (Cell Adhesion Molecules)
RN  - 0 (Codon)
RN  - 0 (Cytoskeletal Proteins)
RN  - 0 (Endothelial Growth Factors)
RN  - 0 (Isoenzymes)
RN  - 0 (Lymphokines)
RN  - 0 (PXN protein, human)
RN  - 0 (Paxillin)
RN  - 0 (Phosphoproteins)
RN  - 0 (Receptors, Growth Factor)
RN  - 0 (Vascular Endothelial Growth Factor A)
RN  - 0 (Vascular Endothelial Growth Factors)
RN  - 21820-51-9 (Phosphotyrosine)
RN  - EC 2.7.10.1 (Protein-Tyrosine Kinases)
RN  - EC 2.7.10.1 (Receptor Protein-Tyrosine Kinases)
RN  - EC 2.7.10.1 (Receptors, Vascular Endothelial Growth Factor)
RN  - EC 2.7.10.2 (Focal Adhesion Kinase 1)
RN  - EC 2.7.10.2 (Focal Adhesion Protein-Tyrosine Kinases)
RN  - EC 2.7.10.2 (PTK2 protein, human)
RN  - EC 3.1.4.- (Type C Phospholipases)
RN  - EC 3.1.4.3 (Phospholipase C gamma)
SB  - IM
MH  - Amino Acid Substitution
MH  - Catalysis
MH  - Catalytic Domain
MH  - Cell Adhesion Molecules/metabolism
MH  - Cell Line, Transformed
MH  - Codon/genetics
MH  - Cytoskeletal Proteins/metabolism
MH  - Endocytosis/*physiology
MH  - Endothelial Growth Factors/*pharmacology
MH  - Focal Adhesion Kinase 1
MH  - Focal Adhesion Protein-Tyrosine Kinases
MH  - Humans
MH  - Isoenzymes/metabolism
MH  - Kidney
MH  - Lymphokines/*pharmacology
MH  - Mutagenesis, Site-Directed
MH  - Neovascularization, Physiologic/physiology
MH  - Paxillin
MH  - Phospholipase C gamma
MH  - Phosphoproteins/metabolism
MH  - Phosphorylation
MH  - Phosphotyrosine/physiology
MH  - *Protein Processing, Post-Translational
MH  - Protein-Tyrosine Kinases/metabolism
MH  - Receptor Protein-Tyrosine Kinases/chemistry/*metabolism
MH  - Receptors, Growth Factor/chemistry/*metabolism
MH  - Receptors, Vascular Endothelial Growth Factor
MH  - Structure-Activity Relationship
MH  - Type C Phospholipases/metabolism
MH  - Vascular Endothelial Growth Factor A
MH  - Vascular Endothelial Growth Factors
MH  - src Homology Domains
EDAT- 1999/04/02 00:00
MHDA- 1999/04/02 00:01
CRDT- 1999/04/02 00:00
PHST- 1999/04/02 00:00 [pubmed]
PHST- 1999/04/02 00:01 [medline]
PHST- 1999/04/02 00:00 [entrez]
AID - 10.1038/sj.onc.1202478 [doi]
PST - ppublish
SO  - Oncogene. 1999 Feb 25;18(8):1619-27. doi: 10.1038/sj.onc.1202478.