Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium.
Epidermal growth factor (EGF) rapidly stimulates the formation of inositol 1,4,5-trisphosphate in a variety of cell types. Previously we have found that in intact cells stimulation of phospholipase C (PLC) activity by EGF is correlated with the retention of increased amounts of PLC activity by a phosphotyrosine immunoaffinity matrix, suggesting that the EGF-receptor tyrosine kinase phosphorylates PLC. We now define parameters of the mechanism by which EGF addition to A-431 cells stimulates phosphotyrosine immunoisolation of PLC activity and demonstrate that EGF addition to A-431 cells increases tyrosine phosphorylation of PLC. EGF rapidly and reversibly stimulated the anti-phosphotyrosine recovery of increased PLC activity when cells were treated with growth factor at 3 degrees C, indicating that receptor internalization is not required and that the phosphorylation event occurs prior to formation of inositol 1,4,5-trisphosphate. Also, the EGF stimulation of anti-phosphotyrosine recovery of PLC activity occurred in the absence of extracellular Ca2+. Stimulation of PLC activity in intact cells by other agonists, such as bradykinin or ATP, did not result in increased anti-phosphotyrosine recovery of PLC activity, suggesting two separate mechanisms exist in A-431 cells for hormone-stimulated formation of inositol phosphates. Finally, using monoclonal antibodies that specifically recognize three distinct PLC isozymes, we show that an approximately 145-kDa PLC isozyme (PLC-II) is present in A-431 cells and that EGF treatment of A-431 cells stimulates phosphorylation of PLC-II on both tyrosine and serine residues.
Wahl
M I
MI
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146.
Nishibe
S
S
Suh
P G
PG
Rhee
S G
SG
Carpenter
G
G
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000502:physiology*; D007118:Immunoassay; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000096:biosynthesis; D007700:Kinetics; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007295:Inositol Phosphates; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D001920:Bradykinin; D002118:Calcium