Protein kinase A: regulation and receptor-mediated delivery of antisense oligonucleotides and cytotoxic drugs.
Protein kinases help regulate eukaryotic cell division. We investigated the regulation of cAMP-dependent protein kinase A (PKA) and casein kinase (CK) type I activity in normal cells and in cancer. To assess this activity in biopsies we suggest a new parameter--the ratio of CK activity and total PKA activity divided by cAMP concentration: CK/PKA/cAMP. In 98 samples of colon mucosa in normal, inflamed, polyp, and adenocarcinoma cells, we found this parameter to be fairly constant in normal conditions and increased 10-fold in colon cancer; the ratio does not depend on the place of biopsy or the patient's age or sex. Experiments with model systems of concanavalin A-stimulated lymphocytes and regenerating rat liver showed that in normal cell proliferation the parameter increases 2-3-fold, as compared to a 30-fold increase in cancer. Unlike normal cells, malignant cells show CK activation and decrease of cAMP; therefore, PKA activity decreases. This suggests a correlation of CK and PKA activity and significant damage to their regulation at pathological changes of tissue proliferation. To further study concerted CK and PKA regulation we used monoclonal antibodies (mAbs) against cAMP-dependent protein kinase regulatory subunit RKII beta. We produced 11 antibodies in three groups: inhibiting, which block cAMP binding with RII beta and inhibit holoenzyme formation (RS6); activating, which enhance cAMP binding and do not affect holoenzyme formation (RS28); and neutral (RS17). To investigate mAb influence on protein kinase regulation in live cells we permeabilized pheochromocytoma PC12 by digitonin. When used at 5-microM concentration for 5 min, digitonin allowed us to deliver mAb into PC12 cells at 30-34-nM concentration, leaving 68-75% viable cells. Protein kinase activity was measured within 0.5 and 4 h after incorporation of mAbs into cells. After 30 min incorporation, mAb RS6 blocked PKA activation in PC12 cells under the influence of cAMP; other mAbs showed no effect. mAb RS6 caused a 4-fold increase of free C subunit activity 4 h after incorporation. mAb RS38 decreased R2C2 activity and did not influence C subunit activity. The change of free C subunit activity caused by mAb incorporation was followed by a synchronized, well-balanced change of CK type I activity, which suggests a correlation between the two phosphorylation systems of cell proteins.
Russian Research Center for Molecular Diagnostics and Therapy, 113149 Moscow, Russia.
P G
PG
Sveshnikov
I D
ID
Grozdova
M V
MV
Nesterova
E S
ES
Severin
D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D047388:Casein Kinases; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes; D008214:Lymphocytes / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D047388:Casein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases