Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Serine/threonine phosphorylation of adenylyl cyclase (AC) has been shown to regulate enymatic activity; however, the role of tyrosine kinase-mediated phosphorylation is unclear. We have previously demonstrated that insulin enhances adenylyl cyclase activity (ACA). Further, the insulinomimetic vanadate (a tyrosine phosphatase inhibitor) enhances stimulation of ACA. The cellular mechanisms responsible for this enhancement remain unclear. To examine the effect of tyrosine kinase activation on ACA, an epitope-tagged AC isoform VI (AC6) was constructed and transiently transfected in HEK 293 cells (HEK-AC6). Vanadate mediated a dose-dependent enhancement of forskolin-stimulated ACA. The tyrosine kinase inhibitor Tyrphostin B46 attenuated the vanadate-mediated enhancement of forskolin-stimulated ACA whereas the inactive tyrosine kinase inhibitor Tyrphostin A1 had no effect. To determine whether the vanadate-mediated enhancement of ACA was related to phosphorylation of AC, HEK-AC6 were treated with or without vanadate (300 µmol/L) in the presence of [32P]orthophosphate. Following vanadate treatment, [32P]phosphate incorporation into AC6 was significantly enhanced (316 + 56 % of control); Tyrphostin B46 blunted this effect (to 120 + 16% of control). Phosphoamino acid analysis demonstrated that vanadate treatment resulted in a selective increase in phosphoserine, but not phospho-tyrosine nor phosphothreonine content. Anti-AC6 immunoprecipitation/anti-phosphotyrosine immunoblotting demonstrated that AC6 was not directly tyrosine phosphorylated. Thus, vanadate enhances ACA in a tyrosine kinase-dependent manner. This effect parallels a selective enhancement in serine phosphorylation of AC. Phosphorylation of AC may represent an important mechanism by which tyrosine kinase receptor systems modify AC-mediated responses.

Back to the top.
Poster Number PAtan0766
Keywords: adenylyl cyclase, tyrosine kinase, phosphorylation, vascular