The nuclear localization of glycogen synthase kinase 3β is required its putative PY-nuclear localization sequences

Abstract

Glycogen synthase kinase-3β(GSK-3β), which is a member of the serine/threonine kinase family, has been shown to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that GSK-3β is associated with the karyopherin β2 (Kap β2) (102-kDa), which functions as a substrate for transportation into the nucleus. A potential PY-NLS motif ((109)IVRLRYFFY(117)) was observed, which is similar with the consensus PY NLS motif (R/K/H)X(2-5)PY in the GSK-3β catalytic domain. Using a pull down approach, we observed that GSK-3β physically interacts with Kap β2 both in vivo and in vitro. Secondly, GSK-3β and Kap β2 were shown to be co-localized by confocal microscopy. The localization of GSK-3β to the nuclear region was disrupted by putative Kap β2 binding site mutation. Furthermore, in transient transfection assays, the Kap β2 binding site mutant induced a substantial reduction in the in vivo serine/threonine phosphorylation of GSK-3β, where- as the GSK-3β wild type did not. Thus, our observations indicated that Kap β2 imports GSK-3β through its putative PY NLS motif from the cytoplasm to the nucleus and increases its kinase activity.

Fig. 1.

The putative PY NLS in GSK-3β and interaction between exogenous GSK-3β and Kap β2. (A) GSK-3β contains the putative-conserved Kap β2 binding motif (¹⁰⁹IVRLRYFFY¹¹⁷) within its binding domain (yellow). Two point mutants were prepared in order to define the binding region. GSK-3β mutants in the Kap β2 putative binding motifs, were also prepared, and the mutated sequences were indicated as Y117A (¹⁰⁹IVRLRYFFY ¹¹⁷ changes to ¹⁰⁹IVRLRYFFA ¹¹⁷) or R111A (¹⁰⁹IVRLRYFFY ¹¹⁷ changes to ¹⁰⁹IVALRYFFY ¹¹⁷). For these mutants control, we used the unrelated GSK-3β K292R mutant. (B) Following immunoprecipitation (IP) using an anti-Kap β2 antibody, an immunoblot (IB) was performed using an antibody against GSK-3β (left). The immunoprecipitated GSK-3β complexes were applied to the immunoblot, using an anti-Kap β2 antibody (right). For the negative control, normal mouse serum was used for immunoprecipitation. (C) Confocal fluorescence micrographs showing the endogenous GSK-3β and Kap β2 in HEK293 cells. Kap β2 was visualized by immunofluorescence in fixed and permeablized cells using polyclonal antibodies to human Kap β2 or GSK-3β and Alexa Fluor 568 conjugated donkey anti-rabbit IgG or Alexa Fluor 488 conjugated mouse anti-rabbit IgG. The yellow pattern resulting from the merging of red and green colors indicates the co-localization of both proteins at a specific region of the nuclear membrane and nuclear. (D) HEK293 cells were transiently transfected with expression vectors, HA-GSK-3β WT, R111A, Y117A. Following immunoprecipitation (IP) using an anti-HA antibody, either Kap β2 (upper lane) or GSK-3β (down lane) was detected with the immunoblot (IB) using an antibody against Kap β2 or GSK-3β. (E) In vitro pull down assay with the fusion protein of GSK-3β (WT, R111A, Y117A, K292R). Whole cell lysates of HEK293 cells was incubated with 1 μg of each glutathione agarose tagged recombinant GSK-3β (WT, R111A, Y117A, K292R). The immunoblot was performed to detect Kap β2 with its antibody (upper lane). The recombinant GSK-3β (WT, R111A, Y117A, K292R) protein amount were monitored with the coomasaie blue staining (bottom lane).

Fig. 2.

The subcellular localization of exogenous GSK-3β PY mutants. Confocal fluorescence micrographs of HA-GSK-3β WT, R111A, Y117A, or K292R in HEK293 cells. Kap β2 was visualized by immunofluorescence in fixed and permeablized cells using a polyclonal antibody against human a Kap β2 and Alexa Fluor 568 conjugated donkey anti-rabbit IgG. The yellow pattern resulting from the merging of red and green colors indicates colocalization of both proteins at a specific region of the plasma membrane or cytoplasm, similar to the results obtained for endogenous GSK-3β shown in Fig. 1C. All constructs were shown as green color and performed to determine whether it merged with Kap β2. The transfected HA- GSK-3β wt (detected in both the cytoplasm and the nucleus) was merged (yellow) with GSK- 3β nuclear speckles around nuclear pore (A). The transfected HA-GSK-3β PY mutant (R111A, Y117A) was not detected in nuclear speckles around nuclear pore, and was not merged with Kap β2 in the nuclear (B and C). To control the specificity of GSK-3β PY mutant (R111A, Y117A) subcellular localization, that of GSK-3β K292R mutant was also visualized in (D).

Fig. 3.

Comparison the phosphorylation at ⁹Ser and ²¹⁶Tyr residue of GSK-3β with its PY mutant. HA-GSK-3β WT or its mutant (R111A, Y117A, or K292R) was transfected and immunoprecitated with HA antibody, as described in “Material and Methods”. The phosphorylation of GSK-3β was detected with IB using an anti-phospho ²¹⁶Tyr (upper lane) or ⁹Ser (middle lane) GSK-3β antibody. The untansfectied HEK293 cells was immunoprecitated with HA antibody as the negative control. The amount of GSK-3β protein in the experiment was monitored by GSK-3β antibody (under lane).

Fig. 4.

The protein stability of GSK-3β and its PY mutant. HEK293 cells (2.5 × 10⁵ cells per well) in 100 mm plates were transfected with 8.0 μg of expression vector with HA-GSK 3β wt or its PY mutant plasmid. The medium was replaced with medium containing 200 μg/ml cycloheximide 36 h after transfection (0-hr time point). Cell lysates were harvested at 0, 8, 16, and 24 h then analyzed by immunoprecipitation and Western blotting using anti-HA antibodies, and assayed in five time repeats. The relative optical density (OD) was measured by image analysis of the dried SDS-PAGE gel with the Fuji Image Quant software (Fujifilm, Japan), according to the manufacturer’s instructions.