THE NOVEL CYCLIC DINUCLEOTIDE 3'-5'-CYCLIC-DIGUANYLIC ACID BINDS TO P21(RAS) 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 [H-3]thymidine incorporation, up to 200-fold at 50 mu M c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher th;ln untreated cells. Stimulation of [H-3]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 [P-32]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 p21(ras) 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 p21(ras) 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.