Role of STRAP in regulating GSK3β function and Notch3 stabilization

Abstract

Glycogen synthase kinase 3β (GSK3β) can regulate a broad range of cellular processes in a variety of cell types and tissues through its ability to phosphorylate its substrates in a cell- and time-specific manner. Although it is known that Axin and presenilin help to recruit β-catenin/Smad3 and tau protein to GSK3β, respectively, it is not clear how many of the other GSK3β substrates are recruited to it. Here, we have established the binding of GSK3β with a novel scaffold protein, STRAP, through its WD40 domains. In a new finding, we have observed that STRAP, GSK3β and Axin form a ternary complex together. We show for the first time that intracellular fragment of Notch3 (ICN3) binds with GSK3β through the ankyrin repeat domain. This binding between STRAP and GSK3β is reduced by small-molecule inhibitors of GSK3β. Further studies revealed that STRAP also binds ICN3 through the ankyrin repeat region, and this binding is enhanced in a proteasomal inhibition-dependent manner. In vivo ubiquitination studies indicate that STRAP reduces ubiquitination of ICN3, suggesting a role of STRAP in stabilizing ICN3. This is supported by the fact that STRAP and Notch3 are co-upregulated and co-localized in 59% of non-small cell lung cancers, as observed in an immunohistochemical staining of tissue microarrays. These results provide a potential mechanism by which STRAP regulates GSK3β function and Notch3 stabilization and further support the oncogenic functions of STRAP.

Figure 1

GSK3β and STRAP physically interact with each other. (A) STRAP-HA and GSK3β-myc constructs were transiently transfected into 293T cells. Cells were subjected to lysis 48 hours after tranfection, immunoprecipitation using 1 µg of pre-immuneanti-rabbit IgG or 1 µg anti-HA antibody and immunoblotted with anti-myc antibody as indicated. Bottom parts show comparable expression of GSK3β-myc and STRAP-HA in the lysates. (B) Same as above except immunoprecipitations were done with anti-mouse IgG and anti-myc antibodies and immunoblotting was done with anti-HA antibody. All antibodies are from Santa Cruz Biotechnology. Bottom parts show comparable expression of GSK3β and STRAP in the lysates. (C) GSK3β interacts with the WD40-domain region of STRAP. STRAP-HA, CT1-STRAP-HA and GSK3β-myc constructs were transiently transfected into 293T cells. Immunoprecipitation was done with anti-myc and immunoblotting with anti-HA antibody as indicated. Light chain of the myc antibody used for immunoprecipitation is visible just below the CT1-STRAP-HA band. Bottom parts show comparable expression of STRAP-HA, CT1-STRAP-HA and GSK3β-myc in the lysates.

Figure 2

STRAP and GSK3β phosphorylation status. (A) Effect of lithium chloride and small-molecule inhibitors of GSK3β on STRAP and GSK3β binding. STRAP-HA and GSK3β-myc constructs were transiently transfected into 293T cells. 35 hours after transfection, cells were treated with SB415286 (20 µM), SB216763 (25 µM) and AR-A014418 (20 µM) as shown in figure. 48 hours after tranfection, cells were subjected to lysis, immunoprecipitation using 1 µg anti-HA antibody and immunoblotted with anti-myc antibody as indicated. Bottom parts show comparable expression of GSK3β-myc and STRAP-HA in the lysates. (B) STRAP has no effect of the phosphorylation/activation status of GSK3β in a part of cell lines. MEFs from wild-type and STRAP-null mice were used, and STRAP was also knocked down in NmuMG, HeLa and HT29 cells using a lentiviral shRNA construct (Open Biosystems). Lysates were prepared, and total proteins (30 µg) were analyzed for phospho-Ser9-GSK3β, total GSK3β (Cell Signaling) and also β-actin as a loading control. (P: parental cells; V: vector control cells; S1 and S2: two STRAP knockdown clones; +/+: wild-type MEFs and −/−: STRAP-null MEFs).

Figure 3

STRAP and GSK3β form a ternary complex together with Axin. (A) 293T cells were co-transfected with STRAP-FLAG, GSK3β-HA and Axin-myc in different combinations as indicated. Cell lysates were prepared and co-immunoprecipitated with 2.5 µg of anti-Flag antibody. After five washes with the wash buffer, bound proteins were eluted using the FLAG peptide (Sigma). The eluate was diluted in the lysis buffer and subjected to a second immunoprecipitation with the anti-HA antibody. After washes, the bound proteins were eluted and analyzed by western blotting with anti-myc antibody. Bottom parts show comparable expression of STRAP-FLAG, GSK3β-HA and Axin-myc in the lysates. (B) Same as above except the second immunoprecipitation was done using anti-myc antibody and western analysis was done using anti-HA antibody.

Figure 4

GSK3β binds specifically with ICN3. (A) ICN3-HA and GSK3β-myc constructs were transiently transfected into 293T cells. Cells were subjected to lysis 48 hours after tranfection, immunoprecipitation using 1 µg pre-immune mouse IgG or 1 µg anti-myc antibody and immunoblotted with anti-HA antibody as indicated. Bottom parts show comparable expression of GSK3β-myc and ICN3-HA in the lysates. (B) Same as above except immunoprecipitations were done with pre-immune rabbit IgG and anti-HA antibodies, and immunoblotting was done using anti-myc antibody. The band above the GSK3β band is the heavy chain. All antibodies are from Santa Cruz Biotechnology. Bottom parts show comparable expression of GSK3β-myc and ICN3-HA in the lysates. (C) The ANK domain 1,863–2,000 aa region of Notch3-IC physically interacts with GSK3β. HEK-293T cells transfected with 1 µg of HA-GSK3β and various deletion constructs of ICN3 as indicated. The lysates from these cells were incubated with anti-HA antibodies for 3 hours and then with G-sepharose beads for 1 hour. Complexes were precipitated anti-HA antibody and analyzed by western blot with anti-myc antibody to detect GSK3β-myc. Bottom parts show equal expression of the ICN3 fragments and GSK3β.

Figure 5

STRAP binds ICN3, and this binding is significantly upregulated in presence of MG132. (A) 1 µg of STRAP-FLAG and ICN3-HA constructs were transiently transfected into HEK-293T cells. Where indicated, cells were treated with 40 µM of the proteasomal inhibitor MG132 (Sigma Biotechnology) for 5 hours before lysis. Forty-eight hours after transfection, cells were subjected to lysis, immunoprecipitation using 1 µg anti-FLAG antibody and immunoblotted with anti-HA antibody. (B) Same as above except immunoprecipitation was done using anti-HA antibody and western analysis with anti-FLAG antibody. For both (A and B), bottom parts show comparable expression of STRAP-FLAG and ICN3 in the lystaes. (C) Homology between mouse ICN3 and ICN2. Protein sequences of mouse ICN3 and ICN2 were compared, and homology is indicated by the common sequence placed between the Notch2 and Notch3 sequences. (D) STRAP binds weakly with ICN1. One µg of STRAP-HA, GSK3β-HA and ICN1-myc constructs were transiently transfected into HEK-293T cells as indicated. Forty-eight hours after transfection, cells were subjected to lysis, immunoprecipitation using 1 µg anti-HA antibody and immunoblotted with anti-myc antibody. Heavy chain band is visible just above the GSK3β-myc band. Bottom parts show comparable expression of STRAP-HA, GSK3β-HA and ICN3-myc in the lysates.

Figure 6

STRAP also binds ICN3 through the ANK domain region. (A) ICN3 deletion fragments C4 and C5 do not bind with STRAP. HEK-293 cells were transiently transfected with 1 µg of STRAP-myc and the HA-tagged ICN3 deletion constructs in different combinations as indicated. All cells were treated with the proteasomal inhibitor MG132 (40 µM) for 5 hours before cell lysis. Cells were lysed 48 hours after transfection, subjected to immunoprecipitation with 1.5 µg of anti-myc antibody and immunoblotted with anti-HA antibody to detect co-immunoprecipitated ICN3 deletion fragments. The middle part indicates the same western blot as in the top part, after stripping and immunoblotting with anti-myc antibody to reveal equal immunoprecipitation of STRAP-myc. The bottom two parts indicate comparable expressions of the ICN3 deletion constructs and STRAP-myc in the lysates. (B) This is a reverse of the experiment in part (A). HEK-293 cells were transiently transfected with 1 µg of STRAP-myc and HA-tagged ICN3 deletion constructs. Lane 1 is a negative control transfected only with STRAP-myc. Cell in lanes 3, 5, 7, 9, 11, 13, 15, 17 and 19 were treated with 40 µM of MG132 for 5 hours. Cells were lysed 48 after transfection and subjected to immunoprecipitation with 1.5 µg of anti-HA antibody. Western analysis of the bound proteins was done using anti-myc antibody. Lower part indicates comparable expressions of STRAP-myc and ICN3 deletion constructs in the lysates.

Figure 7

Effect of STRAP on ICN3 activity. (A) STRAP decreases ubiquitination of ICN3. HEK-293 cells were transfected with 0.8 µg of ICN3 and His₆-tagged ubiquitin and 1 µg of STRAP-FLAG in combinations as indicated. The cells were lysed in a modified lysis buffer as detailed the Materials and Methods. Proteins tagged with His₆-ubiquitin molecules were pulled down with Nickel-Nitrilo Tri-Acetic Acid (Ni-NTA) agarose beads. Eluted proteins were subjected to electrophoresis and immunoblotting with anti-HA antibody to specifically detect ubiquitinated species of ICN3. Lower parts indicate equal expression of ICN3 in the lysates. (B) STRAP does not alter ubiquitination of ICN3 fragments C4 (1,663–1,878) andC5 (1,663–1,768). HEK-293 cells were transfected with 0.8 µg of ICN3 deletion constructs and His₆-tagged ubiquitin and 1 µg of STRAP-FLAG in combinations as indicated. The rest of the procedure was as described above. Top part shows ubiquitination pattern of the Notch3 fragments in the absence and presence of STRAP-FLAG and lower part shows the expression of STRAP-FLAG and Notch3 deletion constructs in the lysates. (C) STRAP inhibits Notch3 mediated transactivation. HEK-293 cells were plated in 12-well plates, transfected with 0.5 µg of the HES1-promoter luciferase reporter construct and different combinations of ICN3-HA and STRAP-FLAG. All wells were also transfected with 20 ng of beta-galactosidase construct. Cells were lysed, luciferase activity was normalized using beta-galactosidase activity and averaged for triplicates before representing here. The experiment was replicated three times.

Figure 8

Immunohistochemical analysis of Notch3 and STRAP expression in lung cancer TMA. Left hand columns show expression of Notch3 in lung cancer TMA (Novus anti-Notch3 antibody). Right hand column shows expression of STRAP in the serial section of the same lung cancer samples. All of the positively stained pulmonary adenocarcinomas show a predominantly cytoplasmic localization of Notch3 and STRAP (bottom two parts), whereas the squamous carcinomas exhibit a predominantly nuclear localization of both Notch3 and STRAP (top part).