CAMP SUPPRESSES P21(RAS) 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-s timulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 c ells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulat ed extracellular signal-related protein kinase 1 (Erk-1), p21(ras) and Raf-1 activation. The Erk-1 activity was not down-regulated by the in crease in intracellular cAMP levels, whereas p21(ras) and Raf-1 activi ties were, suggesting that Erk-1 activity might not be dependent on up stream p21(ras) and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstr eam substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylat ion patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [gamma-P-32]ATP. The two phosphoryla tion patterns were found to have many differences. To gain further ins ights into the possible relevance of these phosphorylation patterns an d as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytoso lic extracts of P-32-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated p roteins 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 com mon to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The r esults are consistent with a Raf-1-independent pathway for Erk-1 activ ation in G-CSF-treated myeloid cells, and a number of potential downst ream substrates of these kinases have been identified for further char acterization.