Late signals from the PDGF receptors leading to the activation of the p70S6-kinase are necessary for the transition from G1 to S phase in AKR-2B cells.

Platelet-derived growth factor AB (PDGF-AB) has to be permanently present in the culture medium to achieve full proliferation (>90%) of AKR-2B fibroblasts. Upon removal after 1 h incubation time, only a small number of cells (<20%) entered the cell cycle. Concomitantly there was no increase in RNA- and protein-synthesis. The PDGF-receptor autophosphorylation reached a maximum after 30 min incubation with PDGF-AB. Tyrosine phosphorylation was no longer detectable after 2-4 h. The clustering of receptors into coated pits, analyzed by indirect immunofluorescence using a specific antibody against PDGF-beta-receptor, showed in contrast to autophosphorylation a biphasic kinetic. A first maximum was reached after 30 min, followed by a complete disappearance of coated pits, which regenerated in a second phase after 3 h and were long lasting. If PDGF-AB was removed after 1 h, the second phase was obliterated. 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 caused a fast (10 min) activation of MAP-kinase, which returned to background level after 1 h without any further activation later on. In contrast 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-2B cells, induced only a shortlasting p70(S6)-kinase activation. These observations add further evidence for the involvement of the p70(S6)-kinase pathway in the proliferation control of AKR-2B fibroblasts in the late G1 phase (4-8 h after growth factor addition). On the other hand, 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 inhibited by 400 nM wortmannin. It is suggested that a regulatory element exists upstream of the p70(S6)-kinase and the PI-3-kinase. This regulatory 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 must be stimulated within a second later phase of PDGF activation.