cSrc is a major cytosolic tyrosine kinase in vascular tissue.
We are interested in identifying, in vascular tissue, nonreceptor tyrosine kinases that may be responsible for the contractile actions of G-protein-coupled agonists such as angiotensin II. By using a series of chromatographic steps, including ion exchange, hydrophobic, and affinity chromatography, we have isolated a major fraction of tyrosine kinase activity from the cytosolic fraction of porcine aorta tissue. According to (i) its immunologic cross-reactivity with the monoclonal anti-cSrc antibody, m327, and with the N-terminally directed monoclonal cSrc2-17 antibody, (ii) its inhibition by the C-terminal cSrc kinase, CSK, and (iii) its specificity for phosphorylating tyrosine 15 in the cdc2(6-20) peptide kinase substrate, we conclude that the kinase we have isolated represents porcine cSrc. A substantial proportion of the enzyme (>70%) was recovered in the cytoplasmic fraction from aorta tissue. The profile of inhibition of the human and porcine cSrc enzymes by a spectrum of tyrosine kinase inhibitors (PP1 >> AG82 > AG490 approximately/= genistein > AG10) was compared with the profile of inhibition of angiotensin II mediated contraction in a porcine coronary vascular preparation (AG10 >> genistein > or = AG82 > or = AG490; PP1 inactive). The different inhibitory profiles indicated that cSrc does not represent the vascular tyrosine kinase responsible for the contractile actions of angiotensin II. We suggest, nonetheless, that cSrc plays a key role for other actions of angiotensin II in intact vascular tissue, such as the regulation of mitogen-activated protein kinase activity and gene transcription.
Oda
Y
Y
Endocrine Research Group, Faculty of Medicine, The University of Calgary, AB, Canada.
Renaux
B
B
Bjorge
J
J
Saifeddine
M
M
Fujita
D J
DJ
Hollenberg
M D
MD
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000502:physiology; D000818:Animals; D001011:Aorta; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016391:Genes, src; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013552:Swine; D014018:Tissue Distribution; D014661:Vasoconstriction / Q000187:drug effects
D004791:Enzyme Inhibitors; D000804:Angiotensin II; D016392:Proto-Oncogene Proteins pp60(c-src)