Abstract
PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).
Highlights
The physiological functions of PDK1 in living organisms have been investigated by engineering of the corresponding gene
To examine the role of PDK1-dependent signaling in cell proliferation and control of the cell cycle, we have investigated the effects of PDK1 deficiency in mouse embryonic fibroblasts (MEFs)
Whereas insulin-like growth factor-1 (IGF-1) induced a marked increase in Akt activity in Pdk1lox/lox MEFs, no such effect was apparent in Pdk1-KO MEFs (Fig. 1B)
Summary
Antibodies—Rabbit polyclonal antibodies to PDK1 were kindly provided by F. Cell Cycle Synchronization and Cell Cycle Analysis by Flow Cytometry—Cells were cultured overnight in serum-free medium and subjected to cell cycle arrest by incubation for 16 or 24 h in culture medium containing aphidicolin (1 g/ml) (Sigma) or nocodazole (200 ng/ml) (Sigma), respectively. The fixed cells were washed with PBS, incubated for 30 min at 37 °C with RNase A (100 g/ml), stained with propidium iodide (50 g/ml), and analyzed by flow cytometry (FACSCalibur; BD Biosciences). Immunocytofluorescence Analysis—Cells were seeded on glass coverslips before infection with adenoviral vectors They were subsequently fixed with 3% paraformaldehyde, washed, and incubated consecutively with monoclonal antibodies to p27Kip and with fluorescein isothiocyanate-conjugated goat antibodies to mouse immunoglobulin G (Jackson ImmunoResearch, West Grove, PA) together with propidium iodide. Images were acquired with a confocal laser-scanning microscope (LSM5 PASCAL, Carl Zeiss)
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