Importin-α 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
| Autor: | Meredith L. Leong, Anita C. Maiyar, Gary L. Firestone |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2003 |
| Předmět: |
alpha Karyopherins
Recombinant Fusion Proteins Active Transport Cell Nucleus Receptors Cytoplasmic and Nuclear Importin Biology Protein Serine-Threonine Kinases Protein Sorting Signals environment and public health Article Culture Media Serum-Free Cell Line Immediate-Early Proteins Phosphatidylinositol 3-Kinases Adenosine Triphosphate Catalytic Domain Two-Hybrid System Techniques Animals Humans Nuclear pore Nuclear protein Enzyme Inhibitors Protein kinase A Molecular Biology Nuclear Proteins Alpha Karyopherins Cell Biology Biochemistry Nuclear Pore Signal transduction Nuclear transport Nuclear localization sequence Protein Binding Signal Transduction |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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