Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway.
In dog thyroid epithelial cells (thyrocytes) in primary culture, thyrotropin (TSH) acting through cAMP induces proliferation and differentiation expression, whereas epidermal growth factor (EGF) and tumor-promoting phorbol esters induce proliferation and dedifferentiation. In these cells we have demonstrated mitogen-activated protein (MAP) kinase phosphorylation by 32P labeling and two-dimensional gel electrophoresis and by immunodetection with anti-MAP kinase and anti-phosphotyrosine antibodies after one- or two-dimensional gel electrophoresis. MAP kinase localization was demonstrated by immunochemical staining. We show the following results. (i) As in other systems, EGF and phorbol esters induced p42 and p44 MAP kinases phosphorylation on tyrosine, serine, and threonine. This effect was rapid, peaking after 5 and 15 min, respectively, followed by a slow decline thereafter. It preceded a translocation of MAP kinase immunoreactivity from cytoplasm to nucleus. (ii) Carbamylcholine, a potent stimulator of the Ca(2+)-phosphatidylinositol cascade which is unable to induce DNA synthesis, stimulated MAP kinases phosphorylation and nuclear staining with kinetics similar to those observed after EGF action, indicating that MAP kinase phosphorylation was not sufficient for mitogenesis. (iii) The cAMP-dependent mitogenic cascade elicited by TSH and forskolin did not involve the phosphorylation and nuclear translocation of p42 and p44 MAP kinases at any time during the entire prereplicative phase. Activation of MAP kinases by phosphorylation is therefore not a necessary step in the G0-G1 transition in this mitogenic cascade.
Institut de Recherche Interdisciplinaire, Université Libre de Bruxelles, Belgium.
F
F
Lamy
F
F
Wilkin
M
M
Baptist
J
J
Posada
P P
PP
Roger
J E
JE
Dumont
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002455:Cell Division; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D010703:Phorbol Esters / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology* / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism*
D010703:Phorbol Esters; D004815:Epidermal Growth Factor; D013972:Thyrotropin; D000242:Cyclic AMP; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases