Differential activation of p44mapk (ERK1) by α-thrombin and thrombin-receptor peptide agonist

alpha-Thrombin (thrombin), a potent mitogen for CCL39 hamster lung fibroblasts, stimulates phosphoinositide-specific phospholipase C (PI-PLC) and inhibits adenylate cyclase via cleavage of a specific G-protein-coupled receptor (TH-R), recently cloned from human and hamster cells. This action can be entirely mimicked by the synthetic peptide SFFLRNP, referred to here as TMP (thrombin-mimicking peptide). TMP corresponds to the first seven amino acids of the new N-terminus generated by thrombin cleavage of the hamster TH-R. Although thrombin and TMP apparently generate identical early transmembrane signals, only thrombin is mitogenic on its own. TMP needs to be associated with fibroblast growth factor (FGF), a tyrosine kinase-activating growth factor, to induce cell-cycle re-entry. Here, we have examined the early and late phase of p44 MAP kinase (p44mapk) activation in G0-arrested CCL39 cells after stimulation by thrombin, TMP, FGF or TMP+FGF. We found that: (i) both thrombin and TMP rapidly activate p44mapk in a dose-dependent manner with maximum activation at around 5 min, (ii) after the initial burst of activation, a second and long-lasting wave of activation is observed in response to thrombin (10-100 nM) but not to TMP (up to 300 microM), (iii) FGF alone (25 ng/ml), like thrombin, rapidly and persistently activates p44mapk (20-fold at 5 min and about 3-fold after 2 h), (iv) TMP added together with FGF strongly potentiates the second and sustained phase of p44mapk activation. From these results we propose that: (1) thrombin-induced mitogenesis is mediated only in part by the TH-R recently cloned and (2) activation of p44mapk, in particular the long-lasting phase that correlates with DNA synthesis, is an obligatory event for cell-cycle re-entry.