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 G(1) 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 (PBu(2))-stimulated proliferation. We show that in response to PBu(2) at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC zeta 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 zeta isoform, while p82 might be a related isoform which is PBu(2) sensitive. Our data show that there is a correlation between the ability of PBu(2) 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 PBu(2) mitogenic signaling even as late as 7 h after the delivery of the signal.