CA125 phosphorylation is associated with its secretion from the WISH human amnion cell line.
Evidence is presented suggesting that CA125 is a serine and/or threonine phosphoprotein and that its secretion from the human amnion WISH cell line is closely linked to its phosphorylation. It is also indicated that regulation of CA125 secretion requires protein(s) tyrosine phosphorylation. WISH cells treated with a tyrosine phosphatase inhibitor, vanadate/ H2O2, resulted in increased levels of CA125 secretion. Exposure of vanadate-treated cells to epidermal growth factor further enhanced this secretory activity. Immunohistochemistry of vanadate-treated cells resulted in a substantial increase in not only cytoplasmic tyrosine phosphoproteins but also in membrane-associated CA125 when stained with the PY20 anti-phosphotyrosine and M11 anti-CA125 monoclonal antibodies, respectively. M11 immunoprecipitation of CA125 from cells labelled with [32P]-orthophosphate was analyzed by SDS-PAGE and autoradiography. Immunoprecipitates from cell lysates demonstrated that a phosphoprotein of > 200 kD was isolated and immunoreacted with both the OC125 and M11 anti-CA125 monoclonal antibodies by Western blotting. CA125 immunoprecipitated from vanadate-treated cells showed a marked increase in cell-associated CA125 phosphorylation. Although CA125 could be immunoprecipitated from WISH cell media incubated with [32P]-orthophosphate in the presence or absence of vanadate as detected by Western blotting, autoradiographic analysis of the Western blots revealed no [32P]-labelled CA125 co-migrating with the 200-kD plus molecule detected by M11. When the PY20 anti-phosphotyrosine monoclonal antibody was used as the probe, no tyrosine-phosphorylated CA125 was detected in cell lysates.
Fendrick
J L
JL
Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205-7199, USA.
Konishi
I
I
Geary
S M
SM
Parmley
T H
TH
Quirk
J G
JG
O'Brien
T J
TJ
D000650:Amnion / Q000187:drug effects / Q000378:metabolism*; D007611:Aprotinin / Q000494:pharmacology; D018394:CA-125 Antigen / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D015398:Signal Transduction; D014638:Vanadates / Q000494:pharmacology
D018394:CA-125 Antigen; D019000:Phosphotyrosine; D014638:Vanadates; D004815:Epidermal Growth Factor; D007611:Aprotinin