Dibutyryl cyclic AMP-induced phosphorylation of specific proteins in adenohypophysial cells

Brattin, W.J.; Portanova, R.

Molecular and Cellular Endocrinology 23(1): 77-90

1981


ISSN/ISBN: 0303-7207
PMID: 6266900
DOI: 10.1016/0303-7207(81)90118-0
Accession: 005140114

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Abstract
An attempt was made to identify the natural substrates of cAMP-dependent protein kinase in pituitary cells. Studies were performed using 2 systems: intact rat pituitary cells stimulated with dibutyryl cAMP (DBC) after preincubation with 32Pi, and pituitary-cell extracts stimulated with cAMP in the presence of [.gamma.-32P]ATP. Phosphorylation of proteins was analyzed by 2-dimensional gel electrophoresis, followed by autoradiography. In intact cells, the only clear and reproducible effect of DBC stimulation is increased phosphorylation of 3 proteins (termed A, B and C), each with a MW of .apprx. 20,000 daltons. The time-course and dose-dependence of phosphorylation of A, B and C are generally similar to that for DBC-induced hormone secretion, which is consistent with a role for these proteins in the secretory mechanism. When [.gamma.-32P]ATP is added to cell extracts, proteins A, B and C are not measurably phosphorylated, either in the absence or presence of cAMP. Proteins A, B and C may not be directly phosphorylated by cAMP-dependent protein kinase, but may be phosphorylated indirectly by a 2nd kinase. Growth hormone and prolactin are readily phosphorylated in cell extracts by cAMP-dependent protein kinase (although they are not phosphorylated in vivo). This finding makes clear the need for caution in interpreting results from broken cell systems.