Transforming growth factor-alpha increases tyrosine phosphorylation of microtubule-associated protein kinase in a small intestinal crypt cell line (IEC-6).
The small intestinal crypt cell line (IEC-6) is an undifferentiated, untransformed, mitotically active cell used in this study to determine the effect of transforming growth factor-alpha (TGF-alpha) on tyrosine phosphorylation levels of cellular proteins. Thymidine incorporation increased maximally after addition of 2 ng/ml TGF-alpha for 24 h. At the same dose, TGF-alpha induced the tyrosine phosphorylation of proteins with approximate molecular masses of 42, 44, 52, 80, 150 and 175 kDa as shown by Western blots treated with anti-phosphotyrosine antibody. The most intense phosphorylation was seen in the 42 kDa (p42) and 44 kDa (p44) proteins, which were identified as two isoforms of microtubule-associated protein kinase (MAPK). This phosphorylation was seen as early as 5 min post stimulation and was dose dependent. Both p42 and p44 were found in the nucleus after stimulation, although a basal level of unphosphorylated protein was present before stimulation. The observed tyrosine phosphorylation of p42 and p44 was inhibited by genistein, a tyrosine kinase inhibitor, and tyrphostin 23, an epidermal growth factor receptor tyrosine kinase inhibitor. We conclude that MAPK is tyrosine phosphorylated in response to TGF-alpha stimulation of IEC-6 cells.
Department of Physiology, University of Connecticut Health Center, Farmington 06030.
B L
BL
Oliver
R I
RI
Sha'afi
J J
JJ
Hajjar
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects* / Q000378:metabolism; D002396:Catechols / Q000494:pharmacology; D002460:Cell Line; D005591:Chemical Fractionation; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019833:Genistein; D006131:Growth Inhibitors / Q000494:pharmacology; D007421:Intestine, Small / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D007529:Isoflavones / Q000494:pharmacology; D008970:Molecular Weight; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D013936:Thymidine / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*; D020032:Tyrphostins
D000911:Antibodies, Monoclonal; D002396:Catechols; D006131:Growth Inhibitors; D007529:Isoflavones; D009570:Nitriles; D016211:Transforming Growth Factor alpha; D020032:Tyrphostins; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C060641:tyrphostin A23; D013936:Thymidine