In order to understand the mechanism by which cyclic 3':5' adenosine monophosphate (cAMP) regulates insulin secretion, cAMP dependent protein phosphorylation was studied in a transplantable hamster islet cell tumor. Single cell suspensions prepared by enzymatic digestion of the tumors released insulin into the incubation media. Glucagon (3 nM to 3 μM) stimulated cellular cAMP accumulation and insulin release in a dose dependent manner and these effects were enhanced by 1 mM theophylline. 8 Bromoadenosine 3':5' monophosphate (8Br cAMP) (1 mM) increased insulin release. Somatostatin (10 μg/ml) inhibited basal and glucagon or 8Br cAMP stimulated insulin release without significantly lowering cellular cAMP in glucagon stimulated cells. For analysis of phosphoproteins, cells were incubated with carrier free 32P1 following which lysates were prepared and analyzed by sodium dodecyl sulfate slab gel electrophoresis and autoradiography. Of the numerous 32P labeled protein bands found, only one (P1, Mr = 28,000) displayed a significant increase in 32P incorporation when cells were incubated under conditions that raise the concentration of cellular cAMP. Somatostatin did not affect 32P incorporation into P1 or any other protein band. When cells were incubated with glucagon, an increase in cellular cAMP was evident after 1 min, enhanced 32P incorporation into P1 after 1 to 5 min, and stimulation of insulin release after 5 to 10 min. Analysis of subcellular fractions led to the designation of P1 as a 40 S ribosomal protein. Two dimensional electrophoresis of 32P labeled basic ribosomal proteins showed two labeled proteins, P1 and P2, both of which were localized to the 40 S ribosomal subunit. Only phosphorylation of P1 was stimulated by cAMP. The cAMP dependent ribosomal phosphoprotein, P1, may be identical with a ribosomal phosphoprotein demonstrated in a variety of tissues and species. Its physiological role remains to be established.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - 1977|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology