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. 2003 Nov 25;100(24):14006-11.
doi: 10.1073/pnas.2335486100. Epub 2003 Nov 17.

Nuclear translocation of 3'-phosphoinositide-dependent protein kinase 1 (PDK-1): a potential regulatory mechanism for PDK-1 function

Mei A Lim  1 , Chintan K Kikani, Michael J Wick, Lily Q Dong
Affiliations

Nuclear translocation of 3'-phosphoinositide-dependent protein kinase 1 (PDK-1): a potential regulatory mechanism for PDK-1 function

Mei A Lim et al. Proc Natl Acad Sci U S A. .

Abstract

3'-Phosphoinositide-dependent protein kinase 1 (PDK-1) phosphorylates and activates members of the AGC protein kinase family and plays an important role in the regulation of cell survival, differentiation, and proliferation. However, how PDK-1 is regulated in cells remains elusive. In this study, we demonstrated that PDK-1 can shuttle between the cytoplasm and nucleus. Treatment of cells with leptomycin B, a nuclear export inhibitor, results in a nuclear accumulation of PDK-1. PDK-1 nuclear localization is increased by insulin, and this process is inhibited by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Consistent with the idea that PDK-1 nuclear translocation is regulated by the PI3-kinase signaling pathway, PDK-1 nuclear localization is increased in cells deficient of PTEN (phosphatase and tensin homologue deleted on chromosome 10). Deletion mapping and mutagenesis studies unveiled that presence of a functional nuclear export signal (NES) in mouse PDK-1 located at amino acid residues 382 to 391. Overexpression of constitutively nuclear PDK-1, which retained autophosphorylation at Ser-244 in the activation loop in cells and its kinase activity in vitro, led to increased phosphorylation of the predominantly nuclear PDK-1 substrate p70 S6KbetaI. However, the ability of constitutively nuclear PDK-1 to induce anchorage-independent growth and to protect against UV-induced apoptosis is greatly diminished compared with the wild-type enzyme. Taken together, these findings suggest that nuclear translocation may be a mechanism to sequestrate PDK-1 from activation of the cytosolic signaling pathways and that this process may play an important role in regulating PDK-1-mediated cell signaling and function.

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Figures

Fig. 1.
Fig. 1.
PDK-1 is a cytoplasmic nuclear-shuttling protein. (A) Localization of HA-Grb10γ or Myc-mPDK-1 in CHO/IR cells treated with vehicle (70% methanol) or 5 nM LMB for 2.5 h. Fixed cells were stained with an antibody to the tag, followed by staining with anti-mouse Alexa Fluor 488 and 4′,6-diamidino-2-phenylindole (DAPI). Images were captured with an inverted fluorescent microscope (IX70). (B) Confocal images depicting the localization of endogenous PDK-1 in HeLa cells treated with vehicle or 20 nM LMB for the indicated time points. (Upper) Endogenous PDK-1 was visualized by staining with a monoclonal anti-PDK-1 antibody. (Lower) Confocal differential image contrast. (Bars = 20 μm.) (C) Schematic representation of the C-terminal truncations of mPDK-1 and summary of their localization in CHO/IR cells treated with either vehicle or 5 nM LMB. (D Top) Amino acid alignment of mouse and human PDK-1. (Middle and Bottom) Confocal images showing the localization of Myc-tagged mPDK-1, mPDK-1Δ382–391, or mPDK-1L383S/F386S in CHO/IR cells. DIC, Differential image contrast.
Fig. 2.
Fig. 2.
PDK-1 nuclear localization depends on the PI3-kinase pathway. (A) Confocal images showing localization of Myc-mPDK-1α in PTEN+/+ and PTEN–/– cells. (Bar = 20 μm.) (B) Graphical representation of PDK-1α and β localization in PTEN+/+ and PTEN–/– cells; bars represent mean ± SEM from at least three independent experiments. P value was determined by using Student's t test. (C) Subconfluent PTEN+/+ and Pten–/– cells were lysed and fractionated into cytoplasmic (C) and nuclear (N) fractions. (Top) Anti-PDK-1 blot. (Middle) Anti-β-tubulin blot. (Bottom) Anti-histone blot. (D) CHO/IR transiently expressing Myc-mPDK-1 were serum-starved in the presence/absence of DMSO, 50 nM wortmannin (Wort), or 50 μM LY 294002 (LY) for 1 h before stimulation with 20 nM insulin (Ins) for 20 min. Localization of PDK-1 was visualized by staining with an antibody to the tag. Data are mean ± SEM from three independent experiments.
Fig. 3.
Fig. 3.
Nuclear PDK-1 is phosphorylated in its activation loop and is kinaseactive. (A) Confocal images showing localization of Myc-mPDK-1, mPDK-1Δ382–391, or mPDK-1S244A in murine hepatocytes treated with vehicle or 10 nM LMB for 2.5 h. (Bars = 20 μm.) (B) In vitro kinase activities of immunopurified Myc-tagged mPDK-1, mPDK-1K114G, mPDK-1Δ382–391, or mPDK-1K114G/Δ382–391 against a PKB-based peptide substrate. (C) Phosphorylation of immunopurified FLAG-p70 S6KβI by PDK-1 proteins from HeLa cells treated with 10–6 M insulin for 10 mins. p70 S6KβI phosphorylation (Top), p70 S6KβI expression (Middle), and PDK-1 expression (Bottom) levels were detected by blotting with anti-phospho-Thr PDK-1 substrate, anti-FLAG, and anti-Myc antibodies, respectively. (D) Graphical representation of phosphorylation of p70 S6KβI by PDK-1. Bars represent the mean ± SEM from three independent experiments.
Fig. 4.
Fig. 4.
Effect of nuclear PDK-1 localization on cellular transformation and UV-induced apoptosis. (A Left) Western blot of whole cell lysates from Comma-1D cell lines showing expression levels of eYFP-mPDK-1 and eYFP-mPDK-+Δ382–391. (Upper Left) Anti-PDK-1 blot. (Lower Left) β-Tubulin blot (loading level control). (Right) Pictures of colony formation on soft agar (Upper Right) and the graphical representation of colony formation assay (Lower Right). Bars represent the mean ± SEM from three independent experiments. (B Left) Western blot of whole cell lysates from NMuMg cell lines expressing eYFP, eYFP-mPDK-1, and eYFP-mPDK-1Δ382–391 showing expression of mPDK-1. (Upper Left) Anti-PDK-1 blot. (Lower Left) β-Tubulin blot. (Right) Inverted contrast image of an ethidium bromide-stained gel showing DNA fragmentation of nuclear DNA purified from NMuMg cells exposed to 70 J/m2 UV light. Data are representative of three independent experiments.
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