Abstract
The v-Ki-Ras oncoprotein dedifferentiates thyroid cells and inhibits nuclear accumulation of the catalytic subunit of cAMP-dependent protein kinase. After activation of v-Ras or protein kinase C, the regulatory subunit of type II protein kinase A, RIIbeta, translocates from the membranes to the cytosol. RIIbeta mRNA and protein were eventually depleted. These effects were mimicked by expressing AKAP45, a truncated version of the RII anchor protein, AKAP75. Because AKAP45 lacks membrane targeting domains, it induces the translocation of PKAII to the cytoplasm. Expression of AKAP45 markedly decreased thyroglobulin mRNA levels and inhibited accumulation of C-PKA in the nucleus. Our results suggest that: 1) The localization of PKAII influences cAMP signaling to the nucleus; 2) Ras alters the localization and the expression of PKAII; 3) Translocation of PKAII to the cytoplasm reduces nuclear C-PKA accumulation, resulting in decreased expression of cAMP-dependent genes, including RIIbeta, TSH receptor, and thyroglobulin. The loss of RIIbeta permanently down-regulates thyroid-specific gene expression.
Highlights
Ras is a small GTP binding protein that serves as a central molecular switch
We demonstrate that the intracellular location of RII subunits profoundly affects the nuclear accumulation of C-PKA and, cAMP-regulated thyroglobulin mRNA levels
Ras and cAMP Signals in the Thyroid Cell—Thyroid cells exposed to v-Ras dedifferentiate
Summary
DNA Plasmids, and Transfections—The TL cell line is derived from the FRTL-5 thyroid cell line, which has been extensively characterized with respect to thyroglobulin expression. Assays (final volume, 25 l) were performed at 30 °C for 10 min in a solution containing 100 M ATP, [␥-32P]ATP (Amersham Corp.) (125–150 cpm/pmol) at a final concentration of 10 Ci/100 l of reaction mixture, 10 mM MgCl2, 20 mM Hepes, pH 7.4, 100 M kemptide (Sigma). Immunoblot Analysis—Nuclear, cytosolic, or membrane proteins were resolved by SDS-PAGE (see above), transferred to nitrocellulose, rinsed in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20), incubated with 10% nonfat dry milk in TBST, and incubated with anti-C-PKA antibodies (see above) in 5% nonfat dry milk in TBST for 1 h. Anti-PKA catalytic or regulatory subunit immunoreactivity was detected using the specific polyclonal antibody (see above) in PBS containing 0.2% gelatin for 45 min at 22 °C. Covalent incorporation was accomplished by exposure of the reactions at 20 °C to UV light (254 nm) at a distance of 5 cm for 15 min
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