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Comparative Study
. 2009 Nov;76(5):1123-9.
doi: 10.1124/mol.109.060129. Epub 2009 Aug 31.

Protein kinase A and B-Raf mediate extracellular signal-regulated kinase activation by thyrotropin

Lisa A Vuchak  1 , Oxana M Tsygankova, Gregory V Prendergast, Judy L Meinkoth
Affiliations
Comparative Study

Protein kinase A and B-Raf mediate extracellular signal-regulated kinase activation by thyrotropin

Lisa A Vuchak et al. Mol Pharmacol. 2009 Nov.

Abstract

Thyrotropin (TSH) regulates thyroid cell proliferation and function through cAMP-mediated signaling pathways that activate protein kinase A (PKA) and Epac/Rap1. The respective roles of PKA versus Epac/Rap1 in TSH signaling remain unclear. We set out to determine whether PKA and/or Rap1 mediate extracellular signal-regulated kinase (ERK) activation by TSH. Neither blocking Rap1 activity nor silencing the expression of Rap1 impaired TSH or forskolin-induced ERK activation in Wistar rat thyroid cells. Direct activation of Epac1 failed to stimulate ERK activity in starved cells, suggesting that Epac-induced Rap1 activity is not coupled to ERK activation in rat thyroid cells. By contrast, PKA activity was required for cAMP-stimulated ERK phosphorylation and was sufficient to increase ERK phosphorylation in starved cells. Expression of dominant-negative Ras inhibited ERK activation by TSH, forskolin, and N(6)-monobutyryl (6MB)-cAMP, a selective activator of PKA. Silencing the expression of B-Raf also inhibited ERK activation by TSH, forskolin, and 6MB-cAMP, but not that stimulated by insulin or serum. Depletion of B-Raf impaired TSH-induced DNA synthesis, indicating a functional role for B-Raf in TSH-regulated proliferation. Collectively, these results position PKA, Ras, and B-Raf as upstream regulators of ERK activation and identify B-Raf as a selective target of cAMP-elevating agents in thyroid cells. These data provide the first evidence for a functional role for B-Raf in TSH signaling.

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Figures

Fig. 1.
Fig. 1.
TSH stimulates cAMP-dependent MEK and ERK activation. Starved WRT cells were stimulated with TSH (A) or forskolin (fsk) (B) for the times indicated (in minutes). C, cells were pretreated with U0126 (10 μM) for 30 min before stimulation. Total cell lysates were analyzed for ERK and MEK activation by Western blotting for phospho-ERK (ERK-P) and phospho-MEK1/2 (MEK-P). Equal protein loading was confirmed by Western blotting for Rac1 or ERK2.
Fig. 2.
Fig. 2.
cAMP stimulates Rap-independent ERK activation. A, WRT cells infected with Rap1GAP adenovirus (AdRap1GAP, 500 particles/cell) were fixed and stained for HA-Rap1GAP at days 1 and 2 after infection. Nuclei were stained with 4′,6-diamidino-2-phenylindole. B, cells infected with AdRap1GAP were starved for 24 h, stimulated with forskolin (F) or TSH (T) for 2 min (on day 2 after infection), subjected to RalGDS-RBD pull-down assay, and subsequently blotted for activated Rap1 (Rap1GTP). Total cell lysates were subjected to Western blotting for Rap1 and Rap1GAP. C, cells infected with AdRap1GAP were starved and stimulated with TSH (top) or forskolin (bottom), and total cell lysates were analyzed for ERK phosphorylation and Rap1GAP expression. Equal protein loading was confirmed by Western blotting for ERK2. D, the results from three experiments are summarized.
Fig. 3.
Fig. 3.
Rap1 is not coupled to ERK activation. A, WRT cells transfected with Rap1 siRNAs (Rap#1 and Rap#2) were starved, stimulated with forskolin for 2 min, and ERK phosphorylation, Rap1, and Akt expression (as a loading control) were analyzed. Rap1 expression was reduced by more than 80% after transfection with Rap1-directed siRNAs. B, the results from three experiments are summarized. C, starved cells were stimulated with EcAMP or TSH for the indicated times (in minutes). Lysates were subjected to RalGDS-RBD pull-down assay and blotted for Rap1GTP. Total cell lysates were analyzed for Rap1 expression. D, starved cells were stimulated with EcAMP for the indicated times (in minutes) and analyzed for ERK phosphorylation. Western blotting for actin was used to ensure equal protein loading.
Fig. 4.
Fig. 4.
ERK activation in FRTL5 and PCCL3 cells does not require Rap activity. FRTL-5 (A) and PCCL3 cells (B) infected with Rap1GAP adenovirus (500 particles/cell) were starved for 24 h, stimulated with forskolin for the indicated times (in minutes), and subjected to Western blotting for ERK phosphorylation, Rap1GAP, and Akt expression, the latter to confirm equal protein loading. Similar results were obtained in two experiments in each cell line.
Fig. 5.
Fig. 5.
ERK activation is PKA-dependent. A, WRT cells infected with LacZ or PKI adenoviruses were starved overnight and stimulated with forskolin for the indicated times (in minutes). ERK and PKA substrate phosphorylation were analyzed by Western blotting. ERK2 expression documented equal protein loading. B, PKI significantly reduced forskolin-stimulated ERK activation (*, p < 0.05 at 2 and 5 min). C, starved cells were stimulated with 6MB-cAMP for the indicated times (in minutes) and analyzed by Western blotting for phospho-ERK, phospho-MEK, and Rac1 as a loading control. MEK1 activation by 6-MB-cAMP was analyzed in a single experiment.
Fig. 6.
Fig. 6.
Ras activity is required for ERK activation. A, WRT cells transfected with HA-ERK and FLAG-RasN17 or empty vector were starved, stimulated with forskolin for 2 min, and HA-ERK immunoprecipitated (IP) using an HA antibody and subjected to Western blotting for phospho-ERK and HA-ERK expression. Whole-cell extracts (CE) were analyzed for FLAG-RasN17 expression. B, cells transfected as described in A were stimulated with 6MB-cAMP for 2 min. Two experiments using forskolin and 6-MB-cAMP and a single experiment using TSH were performed with similar results.
Fig. 7.
Fig. 7.
B-Raf is required for cAMP-stimulated ERK activation. WRT cells transfected with scrambled- (Scr) versus B-Raf-directed siRNAs were starved, stimulated with forskolin (2 min), insulin (5 min) (A) or TSH (2 min) (B) and subjected to Western blotting for phospho-ERK and B-Raf. Equal protein loading was confirmed by blotting for ERK2 or Akt. Depletion of B-Raf using an independent set of siRNAs (B-Raf#2) inhibited ERK activation by forskolin and TSH. C, siRNA-transfected cells were stimulated with 6MB-cAMP for the times indicated (in minutes) and analyzed for phospho-ERK, B-Raf, and Akt as a loading control. D, the decrease in ERK activation in B-Raf-depleted cells was statistically significant (**, p < 0.01).
Fig. 8.
Fig. 8.
B-Raf is required for TSH-dependent DNA synthesis. Cells pretreated with U0126 (10 μM) for 1 h or depleted of B-Raf were stimulated with TSH, insulin (A), or 3H growth medium (B), and DNA synthesis was analyzed. The percentage of BrdU-positive nuclei (13.2% for TSH, 19.1% for insulin, 52.6% for 3H versus 2.0% for starved cells) in mitogen-stimulated cells was set to 1. The decrease in DNA synthesis in U0126-treated (**, p < 0.01 for TSH; **, p < 0.01 for 3H) and B-Raf-depleted (***, p < 0.001 for TSH; *, p < 0.05 for 3H) cells was statistically significant.
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