Antiapoptotic effect of heterozygously expressed mutant RI (Ala336-->Asp) subunit of cAMP kinase I in a rat leukemia cell line.
cAMP induced rapid apoptosis (> 90% cell death in 6 h) of non-growth-arrested rat leukemia IPC-81 cells. A cell clone selected for cAMP resistance had a normally functioning apoptotic machinery whose triggering required about 30-fold higher cellular cAMP than in the parent cells. The cAMP subresponsiveness was due to a heterozygous point mutation (Ala336-->Asp) in the RI subunit of cAMP-dependent protein kinase I. In fact, apoptosis correlated with intracellular cAMP binding to the subresponsive RI. The mutated alanine is invariantly present in cyclic nucleotide kinases, but of unknown function. The mutation decreased the cAMP affinity to site B by increasing the cAMP dissociation rate 500x. The ability of site B to discriminate adenine-modified cAMP analogues was affected, suggesting that Ala336 faced the adenine moiety of cAMP. That the heterozygously expressed RID336 was a dominant suppressor of apoptosis was explained by a higher expression of R than C subunits in the mutant cells by preferential expression of the mutant form of RI, and by the ability of mutant RI to exert dominant negative control of activation of wild type cAMP kinase at moderate cAMP levels. Apoptosis was induced at a similar cAMP level in cells treated with cholera toxin or other cAMP elevating agents, indicating that cAMP kinase was essential for toxin action.
Institut National de la Santé et de la Recherche Médicale U301, Institute of Hematology, Centre Hayem, Hôpital St. Louis, Paris, France.
E
E
Duprez
B T
BT
Gjertsen
O
O
Bernard
M
M
Lanotte
S O
SO
Døskeland
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000378:metabolism; D002999:Clone Cells; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004351:Drug Resistance / Q000502:physiology; D004789:Enzyme Activation; D006579:Heterozygote; D007700:Kinetics; D007942:Leukemia, Experimental; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012334:RNA, Neoplasm; D001224:Aspartic Acid; D000242:Cyclic AMP; D011494:Protein Kinases; D000409:Alanine