PROLONGED VS TRANSIENT ROLES FOR EARLY CELL-CYCLE SIGNALING COMPONENTS

Both p21ras and phosphatidylinositol 3-kinase (PI 3-k) are critical el ements in signaling pathways mediating insulin/IGF-I induced cell cycl e progression. For example, microinjection of antibodies, peptides, or recombinant proteins which block the interaction of the SH2 domains o f the PI 3-k p85 alpha subunit with tyrosine phosphorylated intracellu lar targets blocks insulin mediated DNA synthesis. We report here that this inhibitory phenotype is observed whether the injections are made into quiescent cells (the standard approach), or at any time point du ring G(1) phase subsequent to stimulation. This observation is not tru e, however, for the major substrate of the insulin/IGF-I receptor (IRS -1) despite the well known interaction of p85 with IRS-1, Antibodies t o IRS-1 are inhibitory only when injected during the first 15 min of G (1) phase, as are antibodies to another major IRS-1 binding protein, t he tyrosine phosphatase SHP2. We also have microinjected reagents whic h target proteins involved in the formation of rasGTP and which mediat e some of the downstream effects of ras activation. Reagents which tar get the formation of rasGTP (Shc and dominant negative ras protein) in hibit DNA synthesis only at points early in G(1), as do reagents which target components of the MAP kinase pathway. Injection of antibodies to p21ras itself, or a recombinant Raf-l protein domain which binds to the effector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G(1) phase. The result s point to a continuous requirement for both PI 3-k and ras activity u ntil cellular commitment to DNA synthesis, although some of the molecu les which are both upstream and downstream of these activities are onl y required transiently. Our results are also consistent with a Raf-l i ndependent ras activity late in G(1), as well as IRS-1 independent eff ects of PI 3-kinase.