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Prolonged vs transient roles for early cell cycle signaling components
1University of California at San Diego Department of Medicine, La Jolla 92093, USA.
Abstract:
Both p21ras and phosphatidylinositol 3-kinase (PI 3-k) 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 p85alpha 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 G1 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 first 15 min of G1 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 effects of ras activation. Reagents which target the formation of rasGTP (Shc and dominant negative ras protein) inhibit DNA synthesis only at points early in G1, 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 effector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G1 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 G1, as well as IRS-1 independent effects of PI 3-kinase.
Insights
Both phosphatidylinositol 3-kinase (PI 3-k) and p21ras signaling are crucial for cell cycle progression. Their continuous activity is required until DNA synthesis commitment, with some upstream and downstream molecules only needed transiently.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Insulin/IGF-I signaling pathways regulate cell cycle progression.
- p21ras and phosphatidylinositol 3-kinase (PI 3-k) are key mediators in these pathways.
- Understanding the temporal requirements of signaling molecules is crucial for cell cycle control.
Purpose of the Study:
- To investigate the temporal requirements of PI 3-k and p21ras signaling during the G1 phase of the cell cycle.
- To determine the roles of specific molecules like IRS-1 and SHP2 in insulin/IGF-I induced cell cycle progression.
- To elucidate the relationship between PI 3-k, p21ras, and downstream effectors like MAP kinase.
Main Methods:
- Microinjection of inhibitory antibodies, peptides, and recombinant proteins into quiescent and stimulated cells.
- Targeting of specific signaling molecules including PI 3-k, IRS-1, SHP2, Shc, dominant-negative ras, MAP kinase components, p21ras, and Raf-1.
- Assessment of inhibition of DNA synthesis and cell cycle progression.
Main Results:
- Inhibition of PI 3-k activity blocked DNA synthesis at all time points in G1 phase.
- Inhibition of IRS-1 and SHP2 was only effective when applied early in G1 phase.
- Inhibition of rasGTP formation, MAP kinase pathway components, p21ras, and Raf-1 showed varying temporal requirements, with p21ras and Raf-1 inhibition affecting the entire G1 phase.
- Evidence suggests Raf-1 independent ras activity and IRS-1 independent PI 3-kinase effects late in G1.
Conclusions:
- Both PI 3-k and p21ras signaling exhibit a continuous requirement throughout G1 phase until commitment to DNA synthesis.
- Certain upstream and downstream signaling molecules have transient roles.
- The study reveals distinct temporal dependencies for different components of the insulin/IGF-I signaling network, including potential Raf-1 and IRS-1 independent pathways.