Prolonged vs transient roles for early cell cycle signaling components

Abstract

Both p21ras and phosphatidylinositol 3-kinase are critical elements in signaling pathways mediating insulin/IGF-I induced cell cycle progression. For example, microinjection of antibodies, peptides, or recombinant proteins which block the interaction of the SH2 domains of the PI 3-k p85a subunit with tyrosine phosphorylated intracellular 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 during G 1 phase subsequent to stimulation. This observation is not true, however, for the major substrate of the insulin/ IGF-I receptor (IRS-1) despite the well known interaction of p85 with IRS-1. Antibodies to IRS-1 are inhibitory only when injected during the ®rst 15 min of G 1 phase, as are antibodies to another major IRS-1 binding protein, the tyrosine phosphatase SHP2. We also have microinjected reagents which target proteins involved in the formation of rasGTP and which mediate some of the downstream e ects of ras activation. Reagents which target the formation of rasGTP (Shc and dominant negative ras protein) inhibit 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-1 protein domain which binds to the e ector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G 1 phase. The results point to a continuous requirement for both PI 3-k and ras activity until cellular commitment to DNA synthesis, although some of the molecules which are both upstream and downstream of these activities are only required transiently. Our results are also consistent with a Raf-1 independent ras activity late in G 1 , as well as IRS-1 independent e ects of PI 3-kinase.

Raf-1 activation suppresses neuroendocrine marker and hormone levels in human gastrointestinal carcinoid cells

“…A baseline level of nonactive, unphosphorylated ERK1/2 was seen in all cells. several studies (2,13,24,29) have recently observed raf-1-mediated effects in the absence of MEK activation through induction of other target signaling factors including MEK kinase 1, cell cycle proteins such as retinoblastoma protein, p53, cdc25, and apoptosis signal-regulated kinase 1. Moreover, studies using raf-1 knockout mice have shown that MEK kinase activity is not necessary for raf-1 function (18).…”

Connecting signaling and cell cycle progression in growth factor-stimulated cells

“…By injecting the antibody after exposing cells to the growth factor it has been possible to assess when the signaling enzyme is contributing to mitogenic signaling. Blocking Ras, SHP-2 or PI3K hours after the initial wave of growth factor-dependent signaling had occurred prevented growth factor-dependent entry into S phase (Bennett et al, 1996;Dobrowolski et al, 1994;Roche et al, 1994Roche et al, , 1996Rose et al, 1998). These studies strongly suggested that signaling enzymes are important for mitogenic signaling at times beyond the initial burst of signaling.…”

A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation

“…For instance, timed-microinjection experiments with dominant-negative proteins or neutralizing antibodies demonstrated that Ras activity is necessary throughout the whole G 1 phase in order for DNA synthesis to occur in fibroblasts. The blockade of individual downstream Ras effectors revealed a transient requirement for Raf-1 early in G 1 , whereas PI-3 kinase activity was required throughout G 1 (37). It has also been reported that Raf-mediated transformation requires the activation of NF-B (45).…”