The carboxyl terminus of VEGFR-2 is required for PKC-mediated down-regulation.
Vascular endothelial growth factor receptor-2 (VEGFR-2/Flk-1) is a receptor tyrosine kinase (RTK) whose activation regulates angiogenesis. The regulatory mechanisms that attenuate VEGFR-2 signal relay are largely unknown. Our study shows that VEGFR-2 promotes phosphorylation of c-Cbl, but activation, ubiquitylation, and down-regulation of VEGFR-2 are not influenced by c-Cbl activity. A structure-function analysis of VEGFR-2 and pharmacological approach revealed that down-regulation of VEGFR-2 is mediated by a distinct mechanism involving PKC. A tyrosine mutant VEGFR-2, defective in PLC-gamma1 activation underwent down-regulation efficiently in response to ligand stimulation, suggesting that activation of classical PKCs are not involved in VEGFR-2 down-regulation. Further studies showed that the ectodomain of VEGFR-2 is dispensable for PKC-dependent down-regulation. Progressive deletion of the carboxyl-terminal domain showed that at least 39 amino acids within the carboxyl-terminal domain, immediately C-terminal to the kinase domain, is required for efficient PKC-mediated down-regulation of VEGFR-2. Mutation of serine sites at 1188 and 1191, within this 39 amino acid region, compromised the ability of VEGFR-2 to undergo efficient ligand-dependent down-regulation. Altogether the results show that the regulatory mechanisms involved in the attenuation of VEGFR-2 activation is mediated by nonclassical PKCs and the presence of serine sites in the carboxyl terminal of VEGFR-2.
Singh
Amrik J
AJ
Department of Ophthalmology, Boston University School of Medicine, MA 02118, USA.
Meyer
Rosana D
RD
Band
Hamid
H
Rahimi
Nader
N
D000818:Animals; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051966:Phospholipase C gamma; D011493:Protein Kinase C / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D011518:Proto-Oncogene Proteins; D012694:Serine; C016658:phorbolol myristate acetate; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D040301:Vascular Endothelial Growth Factor Receptor-2; D011493:Protein Kinase C; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D013755:Tetradecanoylphorbol Acetate