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

Biochem J. 1995 Nov 1;311 ( Pt 3)(Pt 3):921-7. doi: 10.1042/bj3110921.

Abstract

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.

MeSH terms

  • Cell Cycle / drug effects
  • Cell Cycle / physiology
  • Cell Division / drug effects
  • Cell Line
  • Cell Membrane Permeability
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / metabolism
  • Cyclic GMP / pharmacology
  • DNA / biosynthesis*
  • Guanosine Triphosphate / metabolism
  • Humans
  • Lymphocytes / cytology*
  • Lymphocytes / drug effects
  • Lymphocytes / metabolism
  • Proteins / metabolism
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • S Phase / drug effects
  • Sensitivity and Specificity
  • Thymidine / metabolism
  • Tritium

Substances

  • Proteins
  • Tritium
  • bis(3',5')-cyclic diguanylic acid
  • Guanosine Triphosphate
  • DNA
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • Cyclic GMP
  • Thymidine