The novel cyclic dinucleotide 3'-5' cyclic diguanylic acid binds to p21ras and enhances DNA synthesis but not cell replication in the Molt 4 cell line.
1. The effect of the novel, naturally occurring nucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid Molt 4 cell line was studied. When exposed to this guanine nucleotide. Molt 4 cells exhibited a marked increase in [3H]thymidine incorporation, up to 200-fold at 50 microM c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher than untreated cells. Stimulation of [3H]thymidine incorporation into the cells was time- and concentration-dependent. This effect was specific and was not observed with GMP or cyclic GMP, nor with the unhydrolysable GTP analogues, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate. C-di-GMP entrance into the cells was experimentally verified and occurred without using any means of cell permeabilization. SDS/PAGE analysis of cells exposed to [32P]c-di-GMP, followed by autoradiography, revealed the labelling of three low-molecular-mass proteins at 18-27 kDa. The labelling is highly specific to c-di-GMP and its extent was not affected by other guanine nucleotides. 2. One of the c-di-GMP-binding proteins was found to be the p21ras protein, by immunoprecipitation with the anti-Ras monoclonal antibody Y13-259. The effects described appear to be unique for c-di-GMP and, taken together, raise the possibility that an irreversible binding of this guanine nucleotide to the growth-promoting p21ras protein results in a fixed active conformation of this protein affecting DNA synthesis. Strikingly, although at 48 h of growth markedly high DNA levels were found in Molt 4 cells treated with c-di-GMP, this guanine nucleotide had no effect on cell replication during this period. Thus Molt 4 cells exposed to c-di-GMP enter the S phase uncoordinated with their overall replication rate.
D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002463:Cell Membrane Permeability; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects; D012680:Sensitivity and Specificity; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D011506:Proteins; D014316:Tritium; C062025:bis(3',5')-cyclic diguanylic acid; D006160:Guanosine Triphosphate; D004247:DNA; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D006152:Cyclic GMP; D013936:Thymidine