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
Xf. Csar et al., 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, Biochemical journal, 322, 1997, pp. 79-87
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.