MULTIPLE SITE PHOSPHORYLATION OF TYROSINE-HYDROXYLASE - DIFFERENTIAL REGULATION INSITU BY 8-BROMO-CAMP AND ACETYLCHOLINE

Abstract

Suspension cultures of purified bovine adrenal chromaffin cells incorporated 32P from exogenous 32Pi into a protein of MW = .apprx. 60,000 (isolated by discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis). Phosphorylated tyrosine hydroxylase, purified from chromaffin cell supernatants by immunoprecipitation, comigrated with the MW .simeq. 60,000 band. Tryptic fragments prepared from either the MW .simeq. 60,000 band or the immunoprecipitated tyrosine hydroxylase band were analyzed after separation with 2-dimensional electrophoresis/chromatography. Two distinct 32P-peptides were present in either sample. After a 2- to 3-min lag period, 32P incorporation into both peptides was relatively linear with time for at least 20 min. In the presence of Ca, exogenous acetylcholine (100 .mu.M) increased 32P incorporation into both of the 32P-labeled tryptic peptides, whereas 8-bromo-cAMP (1 mM) increased 32P incorporation into only 1 of the 2. Ethyleneglycol-bis(.beta.-aminoethylether)-N,N,N'',N''-tetraacetic acid and MnCl2 inhibited the acetylcholine-induced phosphorylation of both tryptic peptides. Thus, tyrosine hydroxylase is phosphorylated in situ at > 1 site, and the phosphorylation of these sites is affected differently by acetylcholine and 8-bromo-cAMP. Kinase activity other than (or in addition to) cAMP-dependent protein kinase activity probably attends tyrosine hydroxylase in the intact chromaffin cells, and multiple kinase activities may be involved in the short-term regulation of catecholamine biosynthesis by afferent activity.