title: Thyrotropin and insulin-like growth factor I regulation of tyrosine phosphorylation in FRTL-5 cells. Interaction between cAMP-dependent and growth factor-dependent signal transduction.
abstract: Pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiated the mitogenic response to insulin-like growth factor I (IGF-I) (Tramontano, D., Moses, A. C., Veneziani, B. M., and Ingbar, S. H. (1988) Endocrinology 122, 127-132; Takahashi, S.-I., Conti, M., and Van Wyk, J. J. (1990) Endocrinology 126, 736-745). The present study was undertaken to determine whether this synergism between TSH and IGF-I in FRTL-5 cells was correlated with changes in tyrosine phosphorylation of intracellular proteins. Tyrosine phosphorylation in intact cells was determined by gel electrophoresis and immunoblotting using monospecific anti-phosphotyrosine antibodies. Cells were preincubated for up to 24 h with TSH, dibutyryl cAMP, forskolin, or cholera toxin and then incubated for an additional 1 min in the absence or presence of IGF-I. As reported by others, IGF-I rapidly increased tyrosine phosphorylation of a 175-kDa protein as well as a less intense band of 90-100 kDa. Pretreatment for 6-12 h with either TSH or other agents that elevate intracellular cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of the 175-kDa substrate by 3-5-fold. Since TSH did not increase IGF receptor number of kinase activity, the effect of TSH is assumed to be exerted at a step distal to IGF receptor tyrosine kinase. Surprisingly, IGF-I-independent tyrosine phosphorylation was also increased by pretreatment with TSH. When intact cells were analyzed TSH produced a time- and concentration-dependent increase in tyrosine phosphorylation of a prominent 120-125-kDa substrate and less prominent 100- and 80-kDa substrates. Assays using Triton X-100-soluble extracts incubated with MgCl2, ATP, and orthovanadate demonstrated that TSH pretreatment increased tyrosine phosphorylation over that observed in untreated cells. In this cell-free assay, TSH pretreatment enhanced the phosphorylation of multiple substrates. These studies suggest that a cAMP stimulus that initiates a trophic effect can be propagated indirectly through multiple pathways including enhancement of tyrosine phosphorylation.
authors.affiliation: Department of Pediatrics, University of North Carolina, Chapel Hill 27599.
authors.forename: S
authors.initials: S
authors.lastname: Takahashi
authors.forename: M
authors.initials: M
authors.lastname: Conti
authors.forename: C
authors.initials: C
authors.lastname: Prokop
authors.forename: J J
authors.initials: JJ
authors.lastname: Van Wyk
authors.forename: H S
authors.initials: HS
authors.lastname: Earp
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D013379:Substrate Specificity; D013501:Surface-Active Agents; D013972:Thyrotropin / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D013501:Surface-Active Agents; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D013972:Thyrotropin; D000242:Cyclic AMP