LATE SIGNALS FROM THE PDGF RECEPTORS LEADING TO THE ACTIVATION OF THEP70(S6)-KINASE ARE NECESSARY FOR THE TRANSITION FROM G1 TO S-PHASE INAKR-2B CELLS

Platelet-derived growth factor AB (PDGF-AB) has to be permanently pres ent in the culture medium to achieve full proliferation (>90%) of AKR- BB fibroblasts. Upon removal after I h incubation time, only a small n umber of cells (<20%) entered the cell cycle. Concomitantly there was no increase in RNA- and protein-synthesis. The PDGF-receptor autophosp horylation reached a maximum after 30 min incubation with PDGF-AB. Tyr osine phosphorylation was no longer detectable after 2-4 h. The cluste ring of receptors into coated pits, analyzed by indirect immunofluores cence using a specific antibody against PDGF-beta-receptor, showed in contrast to autophosphorylation a biphasic kinetic. A first maximum wa s reached after 30 min, followed by a complete disappearance of coated pits, which regenerated in a second phase after 3 h and were long las ting. If PDGF-AB was removed after I h, the second phase was obliterat ed. The involvement of two different signalling pathways in these two phases was investigated in detail: (1) The ras-raf-MAP-kinase pathway and (2) the PI-3-kinase/p70(S6)-kinase pathway. PDGF-AB addition cause d a fast (10 min) activation of MAP-kinase, which returned to backgrou nd level after 1 h without any further activation later on. In contras t PDGF-AB led to a rapid (15-30 min) activation of the p70(S6)-kinase that persisted for 8-12 h just prior to the entry of the cells into S- phase. If PDGF-AB was removed after 1 h, the activation of this kinase ceased 3 h later. PDGF-AA, which is unable to promote division of AKR -SB cells, induced only a shortlasting p70(S6)-kinase activation. Thes e observations add further evidence for the involvement of the p70(S6) -kinase pathway in the proliferation control of AKR-BB fibroblasts in the late G1 phase (4-8 h after growth factor addition). On the other h and, if the p70(S6)-kinase activation was prevented by the addition of 10 nM rapamycin, the cell division was not inhibited but only delayed by 4 h. Similar kinetics were observed when the PI-3-kinase was inhib ited by 400 nM wortmannin. It is suggested that a regulatory element e xists upstream of the p70(S6)-kinase and the PI-3-kinase. This regulat ory element should be responsible for the transmission of late signals required for the progression through the cell cycle. This element is not involved in the immediate responses after PDGF-AB addition but mus t be stimulated within a second later phase of PDGF activation. (C) 19 98 Academic Press.