Annexin VII and annexin XI are tyrosine phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells.
The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases.
Furge
L L
LL
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Chen
K
K
Cohen
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017310:Annexin A7 / Q000378:metabolism*; D017302:Annexins / Q000378:metabolism*; D004285:Dogs; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000008:administration & dosage* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000008:administration & dosage* / Q000494:pharmacology
D017310:Annexin A7; D017302:Annexins; D010982:Platelet-Derived Growth Factor; C069276:peroxovanadate; D014638:Vanadates; D014443:Tyrosine