Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative PI3K p85 subunit. Consistently, Gas6 activates the PI3K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

No abstract

The involvement of p53 in regulating diverse cellular processes dictates that it must respond to multiple signaling mechanisms, thus coordinating the response to various ''stress conditions.'' Genotoxic stress has served as a paradigm to dissect the transactivation-dependent branch of the pathway by which p53 can induce growth arrest. Alternate mechanisms have been invoked to explain transactivationindependent effects of p53, especially in the context of apoptosis. We have identified a p53-dependent pathway initiated by the gas1 product, a plasma membrane protein highly expressed during G0, which activates a transactivation-independent p53 growth arrest function. Through a detailed deletional analysis and site-specific mutagenesis of p53 we show that the Gas1-dependent signal transduction relies on a proline-rich region (amino acids 63-85) of murine p53. In vivo competition experiments using combinations of such mutants implicate this functional domain of p53 as a docking site in the transmission of antiproliferative signals.

In normal cells, induction of quiescence is accompanied by the increased expression of growth arrest-specific genes (gas). One of them, gas1, is regulated at the transcriptional level and codes for a membrane-associated protein (Gas1) which is down regulated during the G 0 -to-S phase transition in serum-stimulated cells. Gas1 is not expressed in growing or transformed cells, and when overexpressed in normal fibroblasts, it blocks the G 0 -to-S phase transition. Moreover, Gas1 blocks cell proliferation in several transformed cells with the exception of simian virus 40-or adenovirus-transformed cell lines. In this paper, we demonstrate that overexpression of Gas1 blocks cell proliferation in a p53-dependent manner and that the N-terminal domaindependent transactivating function of p53 is dispensable for Gas1-induced growth arrest. These data therefore indicate that the other intrinsic transactivation-independent functions of p53, possibly related to regulation of apoptosis, should be involved in mediating Gas1-induced growth arrest.