Characterization and activation of cyclic adenosine 3':5'-monophosphate-dependent protein kinase in C1300 murine neuroblastoma clones growing in vivo.
Cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases from three clones of C1300 neuroblastoma, established as growing tumors in A/jax mice, were identified and characterized. Mean (+/- SD) intratumor concentrations of cAMP ranged from 5.0 +/- 2.4 to 6.8 +/- 1.9 pmol/mg protein; cytosolic protein kinase activity ratios, calculated from the -cAMP/+cAMP value, ranged from 0.12 +/- 0.02 to 0.18 +/- 0.02. The total amount of cytosolic cAMP-binding activity was highest in the NBA2 clone and nearly equal in N-18 and NBP2. By DEAE chromatography, two peaks of cAMP-binding activity eluting at ionic strengths of 0.09 and 0.15 M of NaCl were resolved from each clone. The latter peak was associated with catalytic activity (type II cAMP-dependent protein kinase), whereas the former was not (free type I cAMP-binding protein). Photoaffinity labeling with 8-azido (N3)-[32P]cAMP, followed by sodium-dodecyl-sulfate:polyacrylamide gel electrophoresis, resolved four major cAMP-binding proteins with molecular weights ranging from 39,000 to 56,000 in each clone. The mol. wt 47,000 protein was judged to be a free regulatory subunit of type I kinase, and the mol. wt 39,000 protein a cleavage product. The mol. wt 54,000 and 56,000 proteins were both endogenously phosphorylated and both had a lower affinity for 8-N3-[32P]cAMP, characteristic of type II kinase. Selected properties of the cAMP-binding proteins (kinetic and autophosphorylating features, response to tryptic hydrolysis, specificity for cAMP and temperature sensitivity) did not differ appreciably among clones. Despite initially low intratumor concentrations of cAMP, it was possible to activate the protein kinase system by simultaneous injection of N6, O2'-dibutyryl-cAMP and papaverine. This indicates that mouse C1300 neuroblastoma can be used profitably to study cAMP-induced neuroblast differentiation.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002999:Clone Cells; D000242:Cyclic AMP / Q000494:pharmacology; D002373:Cyclic AMP Receptor Protein; D004789:Enzyme Activation; D051379:Mice; D009374:Neoplasms, Experimental / Q000201:enzymology; D009447:Neuroblastoma / Q000201:enzymology*; D010208:Papaverine / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D010208:Papaverine; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin