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.
Maiyar
Anita C
AC
Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, 94720-3200, USA.
Leong
Meredith L L
ML
Firestone
Gary L
GL
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins; D022022:Nuclear Pore / Q000378:metabolism; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*; D000099184:Serum-Glucocorticoid Regulated Kinases
D000255:Adenosine Triphosphate; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D000099184:Serum-Glucocorticoid Regulated Kinases; C087978:karyopherin alpha 2