Raf-1 and ERK2 kinases are required for phorbol 12,13-dibutyrate-stimulated proliferation of rat lymphoblasts. ERK2 activation precedes Raf-1 hyperphosphorylation

Rat lymphoblasts are arrested in the G₁ phase of the cell cycle and can be promoted to proceed up to the S phase, when they are stimulated by phorbol ester. In this work, we have studied some details of the phorbol 12,13-dibutyrate (PBu₂)-stimulated proliferation. We show that in response to PBu₂ at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC ζ antibody recognizes two bands of 75 and 82 kDa. These two activities are separated using a Mono Q chromatography and we show that p75 is the classical PKC ζ isoform, while p82 might be a related isoform which is PBu₂ sensitive. Our data show that there is a correlation between the ability of PBu₂ to promote mitogenesis and to activate ERK2 kinase, suggesting that ERK2 kinase might be the limiting step of the process. We also show that ERK kinase activation precedes Raf-1 kinase hyperphosphorylation, suggesting that Raf-1 kinase activation is not required for ERK kinase activation. This idea was checked using a Raf-1 kinase antisense (AS) oligonucleotide. The results obtained with the Raf-1 AS oligonucleotide indicate that this serine/threonine kinase is dispensable for ERK kinase activation, but needed for the PBu₂ mitogenic signaling even as late as 7 h after the delivery of the signal.