Phosphorylation of heat shock protein-90 by TSH in FRTL-5 thyroid cells.

Context: Although it is well established that thyrotropin (TSH) initiates signal transduction systems resulting in protein kinase(s) activation, the phosphorylated targets have not been fully characterized.

Objective/design: In FRTL-5 thyroid cells, we used two-dimensional (2D) gel images of silver-stained proteins isolated from FRTL- 5 thyroid cells following TSH stimulation to identify potential phosphorylation targets.

Results: We characterized a 90 kDa protein that had undergone a pH shift and subsequently identified it as heat shock protein-90 (hsp-90) following in-gel trypsin digestion and mass spectroscopy. This was confirmed by Western blot using a monoclonal antibody against hsp-90. Western blot analysis of the 2D gel images using a polyclonal antibody directed at phosphoserine/threonine sites showed that TSH induced the phosphorylation of hsp-90. Western blotting of hsp-90 following stimulators of the signal transduction systems mediated by TSH indicated that TSH-mediated hsp-90 phosphorylation occurs through protein kinases A and C.

Conclusion: In summary, we have demonstrated that TSH action stimulates the phosphorylation of hsp-90 in FRTL-5 thyroid cells. Abnormalities of hsp-90 phosphorylation may be a mediator in the development of thyroid disease.