Importin-alpha mediates the regulated nuclear targeting of serum- and glucocorticoid-inducible protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain.
The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-alpha nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of ... importin-alpha with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-alpha, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-alpha to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131-141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-alpha and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-alpha as well as the PI3-kinase-dependent phosphorylation of Sgk. Our results define a new role importin-alpha in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.
Mesh Terms:
1-Phosphatidylinositol 3-Kinase, Active Transport, Cell Nucleus, Adenosine Triphosphate, Animals, Catalytic Domain, Cell Line, Culture Media, Serum-Free, Enzyme Inhibitors, Humans, Immediate-Early Proteins, Nuclear Pore, Nuclear Proteins, Protein Binding, Protein Sorting Signals, Protein-Serine-Threonine Kinases, Receptors, Cytoplasmic and Nuclear, Recombinant Fusion Proteins, Signal Transduction, Two-Hybrid System Techniques, alpha Karyopherins
1-Phosphatidylinositol 3-Kinase, Active Transport, Cell Nucleus, Adenosine Triphosphate, Animals, Catalytic Domain, Cell Line, Culture Media, Serum-Free, Enzyme Inhibitors, Humans, Immediate-Early Proteins, Nuclear Pore, Nuclear Proteins, Protein Binding, Protein Sorting Signals, Protein-Serine-Threonine Kinases, Receptors, Cytoplasmic and Nuclear, Recombinant Fusion Proteins, Signal Transduction, Two-Hybrid System Techniques, alpha Karyopherins
Mol. Biol. Cell
Date: Mar. 01, 2003
PubMed ID: 12631736
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