Protein tyrosine phosphorylation induced by epidermal growth factor and insulin-like growth factor-I in a rat clonal dental pulp-cell line.
Both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) produce a dose-dependent stimulation in the rate of cell division in a rat clonal dental pulp-cell line (RDP 4-1). To elucidate the initial mitogen-induced cellular events that may mediate mitogenic action, the effects of EGF and IGF-I on cellular protein tyrosine phosphorylation were examined. In a dose-dependent manner, EGF (1-100 ng/ml) transiently stimulated tyrosine phosphorylation in four major proteins with apparent molecular weights of 220, 180, 140 and 120 kDa, and in five other more minor proteins (90, 80, 65, 55 and 44 kDa). IGF-I (1-100 ng/ml) dose-dependently stimulated the tyrosine phosphorylation of 160- and 140-kDa proteins, and had a smaller effect on the 80-, 65- and 44 kDa proteins. In contrast to the action of EGF, IGF-I-induced tyrosine phosphorylation was sustained for more than 60 min, particularly that of the 160-kDa phosphoprotein. From the results of specific immunoprecipitation/Western-blot analyses, the 180-kDa EGF-sensitive protein could be identified as the EGF receptor (EGF-R). Among the IGF-I-sensitive pulp cell proteins, the 160-kDa protein was identified as insulin-receptor substrate-1. Both mitogenic treatments stimulated the tyrosine phosphorylation of a weak, 44-kDa protein, which we have identified as the extracellular signal-regulated kinase-1. Despite the presence of phosphoproteins of the correct size, neither the IGF-I receptor (IGF-I-R) nor the phospholipase C gamma-isoform could be identified as tyrosine kinase substrates in either treatment. Pretreatment with the tyrosine kinase inhibitor genistein (20 micrograms/ml) significantly inhibited EGF- and IGF-I-induced tyrosine phosphorylation in permeabilized RDP 4-1 cells, and the tyrosine phosphatase inhibitor orthovanadate (1 mM) significantly prolonged the duration of the mitogen-stimulated tyrosine phosphorylation in both intact or permeabilized cells. Phorbol 12-myristate 13-acetate (100 nM), which activates protein kinase C (PKC), inhibited the tyrosine phosphorylation induced by either growth factor. This action was blocked by pretreatment with staurosporine (200 nM, 15 min), a selective PKC inhibitor. However, neither removing external Ca2+ with EGTA (1 mM) nor inducing Ca2+ influx with A23187 ionophore (2 microM) significantly altered EGF- or IGF-I-induced phosphorylation. These findings strongly suggest that authentic EGF-R and IGF-I-R on RDP 4-1 cells are coupled to complex, tyrosine kinase-mediated, intracellular signalling systems that are sensitive to a PKC-dependent mechanism. EGF- and IGF-I-induced tyrosine phosphorylation cascades may have important roles in vivo in the regulation of dental pulp-cell proliferation and ultimately may affect dentine formation.
Kawase
T
T
Department of Pharmacology, Niigata University School of Dentistry, Japan.
Orikasa
M
M
Ogata
S
S
Burns
D M
DM
D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002999:Clone Cells; D003782:Dental Pulp / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology* / Q000502:physiology; D066246:ErbB Receptors / Q000378:metabolism; D019833:Genistein; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology* / Q000502:physiology; D007529:Isoflavones / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000096:biosynthesis; D011493:Protein Kinase C / Q000819:agonists / Q000378:metabolism; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D018970:Insulin-Like Growth Factor Binding Protein 1; D007529:Isoflavones; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D019833:Genistein; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D013755:Tetradecanoylphorbol Acetate