Prolonged vs transient roles for early cell cycle signaling components.
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.
University of California at San Diego Department of Medicine, La Jolla 92093, USA.
D W
DW
Rose
S
S
Xiao
T S
TS
Pillay
W
W
Kolch
J M
JM
Olefsky
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D016193:G1 Phase; D007328:Insulin / Q000494:pharmacology / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000502:physiology; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000502:physiology*; D011972:Receptor, Insulin / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D000906:Antibodies; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C516727:Ptpn11 protein, rat; C509248:Ptpn6 protein, mouse; C509250:Ptpn6 protein, rat; D016283:Proto-Oncogene Proteins p21(ras)