Tyrosine hydroxylase in bovine adrenal chromaffin cells: angiotensin II-stimulated activity and phosphorylation of Ser19, Ser31, and Ser40.
The aim of this study was to determine the effect of angiotensin II (AII) on tyrosine hydroxylase (TOH) activity and phosphorylation in bovine adrenal chromaffin cells (BACCs). We report here that stimulation of BACCs with AII (100 nM) produced a significant increase in both TOH activity and phosphorylation over a period of 10 min. The increase in TOH activity was receptor-mediated. Tryptic phosphopeptide analysis by HPLC revealed that AII stimulated an increase in phosphorylation of three sites on TOH, Ser19, Ser31, and Ser40, with the largest increase being observed for Ser31 phosphorylation. Pretreatment of the cells with the protein kinase C inhibitor Ro 31-8220 (10 microM, 15 min) did not affect TOH activity or phosphorylation produced by AII. The inhibitor also did not affect the TOH activity or Ser40 phosphorylation produced by forskolin (10 microM, 10 min). In contrast, Ro 31-8220 fully inhibited the TOH activation as well as Ser31 and Ser40 phosphorylation of TOH produced by phorbol 12,13-dibutyrate (500 nM, 10 min). Removal of extracellular Ca2+ from the incubation medium inhibited the AII-induced TOH activity by 50% and significantly blocked Ser19 and Ser31 phosphorylation but did not affect Ser40 phosphorylation in response to AII. These results indicate that AII activates a complex and perhaps novel signaling pathway leading to the phosphorylation and activation of TOH. The TOH activation by AII appears to be partially independent of Ser40 phosphorylation, suggesting a potentially important role for Ser31 phosphorylation.
Bobrovskaya
L
L
Discipline of Medical Biochemistry, Faculty of Medicine and Health Sciences, University of Newcastle, New South Wales, Australia.
Cheah
T B
TB
Bunn
S J
SJ
Dunkley
P R
PR
D000311:Adrenal Glands / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D009434:Neural Pathways / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011945:Receptors, Angiotensin / Q000502:physiology; D012694:Serine / Q000378:metabolism*; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007211:Indoles; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C; D002118:Calcium; C064758:Ro 31-8220