cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1.
The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [gamma-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization.
Csar
X F
XF
University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Victoria, Australia.
Ward
A C
AC
Hoffmann
B W
BW
Guy
G G
GG
Hamilton
J A
JA
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism; D004247:DNA / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology; D007951:Leukemia, Myeloid; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010761:Phosphorus Radioisotopes; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D006131:Growth Inhibitors; D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins; D016179:Granulocyte Colony-Stimulating Factor; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras)