Protein kinase A and B-Raf mediate extracellular signal-regulated kinase activation by thyrotropin.
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.
Vuchak
Lisa A
LA
Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6061, USA.
Tsygankova
Oxana M
OM
Prendergast
Gregory V
GV
Meinkoth
Judy L
JL
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D048493:Proto-Oncogene Proteins B-raf / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013972:Thyrotropin / Q000502:physiology*
D013972:Thyrotropin; D000242:Cyclic AMP; C482123:Braf protein, rat; D048493:Proto-Oncogene Proteins B-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases