A Rac GTPase-activating protein, MgcRacGAP, is a nuclear localizing signal-containing nuclear chaperone in the activation of STAT transcription factors

Abstract

In addition to their pleiotropic functions under physiological conditions, transcription factors STAT3 and STAT5 also have oncogenic activities, but how activated STATs are transported to the nucleus has not been fully understood. Here we show that an MgcRacGAP mutant lacking its nuclear localizing signal (NLS) blocks nuclear translocation of p-STATs both in vitro and in vivo. Unlike wild-type MgcRacGAP, this mutant did not promote complex formation of phosphorylated STATs (p-STATs) with importin alpha in the presence of GTP-bound Rac1, suggesting that MgcRacGAP functions as an NLS-containing nuclear chaperone. We also demonstrate that mutants of STATs lacking the MgcRacGAP binding site (the strand betab) are hardly tyrosine phosphorylated after cytokine stimulation. Intriguingly, mutants harboring small deletions in the C'-adjacent region (betab-betac loop region) of the strand betab became constitutively active with the enhanced binding to MgcRacGAP. The molecular basis of this phenomenon will be discussed, based on the computer-assisted tertiary structure models of STAT3. Thus, MgcRacGAP functions as both a critical mediator of STAT's tyrosine phosphorylation and an NLS-containing nuclear chaperone of p-STATs.

FIG. 1.

Identification of the nuclear localization signal of MgcRacGAP. (A) Schematic diagram of various Flag-tagged deletion mutants in the INT domain of MgcRacGAP and summary of the localizations of these mutants. (B) Expression of various deletion mutants and their potential to interact with a known partner, MKLP-1. Cells were transfected with expression vectors carrying Flag-tagged WT or various deletion mutants in the INT domain. After 36 h, cells were immunoprecipitated with the anti-Flag Ab and blotted with the anti-Flag Ab (upper panel) or anti-MKLP-1 Ab (lower panel). (C) Localization of Flag-tagged WT and various deletion mutants in the INT domain of MgcRacGAP in HeLa cells. Cells were transfected with expression vectors carrying Flag-tagged WT or various deletion mutants in the INT domain. After 36 h, cells were immunostained with the anti-Flag Ab (green) and DAPI (blue) and viewed with a fluorescence microscope IX70 (Olympus). Bar, 10 μm. (D) Localization of GFP-fused NLS of MgcRacGAP. GFP-fused aa182-200 of MgcRacGAP, aa182-200(182AAA/199AA), and large T antigen-NLS were expressed in HeLa cells. After 36 h, living cells were viewed with a fluorescence IX70 microscope (Olympus). Bar, 10 μm.

FIG. 2.

Nuclear translocation of p-STAT5A requires the NLS of MgcRacGAP. (A) The recombinant proteins used in the experiments are shown, after CBB staining of purified proteins (a) or Western blot analysis of the STAT5A-Flag protein purified from Sf-9 cells with or without coexpression with the kinase domain of JAK, using the anti-p-STAT5 Ab (b). (B) Nuclear transport assay of STAT5A. HeLa cells were permeabilized using 40 μg/ml digitonin and were incubated at 37°C for 30 min with 50 μl IM. The IM contained TB, ERS, and a single protein or combinations of the following purified proteins, as indicated: 1 μM STAT5A, p-STAT5A, V12Rac1, MgcRacGAP, 199AA-MgcRacGAP, importin α1, importin β1, Ran, or NTF2. After the import reaction, the cells were fixed. STAT5A protein was detected using the anti-STAT5A Ab. Cells were examined using a FLUOVIEW FV300 confocal microscope (Olympus). A representative result of three independent experiments is shown. Bar, 10 μm. (C) The ternary protein complex composed of p-STAT5A, GTP-bound Rac1, and 199AA-MgcRacGAP did not bind importin α1 in the transport buffer. Purified STAT5A and p-STAT5A were incubated with importin α1 in the absence or presence of the indicated combinations of V12Rac1, N17Rac1, MgcRacGAP, or 199AA-MgcRacGAP in the transport buffer containing 5% bovine serum albumin for blocking nonspecific binding. One microgram of each purified protein was used for each sample. After the incubation, STAT5A was immunoprecipitated (IP) with anti-STAT5A Ab and washed three times with transport buffer. The immunoprecipitates were subjected to Western blot analysis with the anti-importin α1, anti-Rac1, anti-MgcRacGAP, anti-STAT5A Ab, or anti-p-STAT5A Ab. (D) GTP-bound Ran (L69Ran) dissociates Rac1, importin α1, and importin β1 from the import complex composed of p-STAT5A. Purified p-STAT5A was incubated with purified V12Rac1, MgcRacGAP, and importin α1 in the presence of the indicated combinations of purified importin β1 alone, importin β1 plus Q69Ran (GTP-bound Ran), or importin β1 plus N24Ran (GDP-bound Ran) in the transport buffer containing 5% bovine serum albumin for blocking nonspecific binding. One microgram of each purified protein was used for each sample. After the incubation, STAT5A was immunoprecipitated with anti-STAT5A Ab and washed three times with transport buffer. The immunoprecipitates were subjected to Western blot analysis with the anti-importin α1, anti-Rac1, anti-MgcRacGAP, anti-importin β1, or anti-STAT5A Ab.

FIG. 3.

The NLS of MgcRacGAP is required for the transcriptional activation of p-STAT3 in 5C cells. (A) Suppression of MgcRacGAP by TET in 5C cells. The 5C cells were treated with TET for the time indicated and lysed. Cell lysates were separated on SDS-PAGE and immunoblotted with the anti-chicken MgcRacGAP Ab (upper panel) or anti-α-tubulin Ab (lower panel). (B) Flag-tagged WT MgcRacGAP rescued 5C cells from becoming multinucleated after addition of TET. The 5C cells transduced with mock or Flag-tagged WT were stained with rhodamine-conjugated phalloidin (red) and DAPI (blue) 12 h after the addition of TET and viewed using a FLUOVIEW FV300 confocal microscope (Olympus). Bar, 10 μm. (C) Subcellular localization of p-STAT3 in 5C cells after addition of TET in the absence or presence of G-CSF. Cell fractionation was performed using 5C cells transiently transfected with the expression vector for the G-CSF receptor (G-CSFR). Twenty-four hours after transfection, live cells were isolated using Ficoll-Paque Plus (Amersham) and used for further analysis. Cells were treated or untreated with TET for 4 h and were incubated with 100 ng/ml of G-CSF for 15 min before cell fractionations. Fractionated samples were then subjected to Western blotting with anti-p-STAT3, anti-Flag, anti-RhoA, or anti-HDAC Ab (upper panels). The total amount of p-STAT3 was also examined using whole-cell lysates of 5C cells by Western blotting with anti-p-STAT3 (lower panel). C, cytosol; N, nuclear. (D) Effect of NLS mutants of MgcRacGAP on cell proliferation. Flag-tagged WT or various MgcRacGAP mutants (182AAA, 199AA, and 182AAA/199AA) were transduced into 5C cells by using a retrovirus vector, pMXs-IG. GFP-positive cells were selected by addition of TET. The number of transfectants was counted at the indicated time points after selection. GFP-positive mock-transduced cells, which were analyzed using fluorescence-activated cell sorting, were used as a control. (E) Expression levels of the Flag-tagged WT or mutant MgcRacGAPs in 5C transfectants. Cell lysates from 5C cells expressing mock, WT, or mutant MgcRacGAPs (1 × 10⁷/lane) were examined by Western blotting using the anti-Flag M2 monoclonal antibody (upper panel) or anti-α-tubulin Ab (lower panel). (F) G-CSF-induced phosphorylation of STAT3 in 5C cells expressing Flag-tagged WT or mutant MgcRacGAPs. The 5C cells expressing WT or mutant MgcRacGAPs cotransfected with the expression vector for G-CSFR were stimulated with 100 ng/ml of G-CSF for 15 min in the presence of TET, followed by Western blotting (5 × 10⁶ cells/lane) using the anti-p-STAT3 antibody (upper panel) or anti-STAT3 Ab (lower panel). (G) Subcellular localization of p-STAT3 in 5C cells expressing Flag-tagged WT, 182AAA, 199AA, or 182AAA/199AA with or without G-CSF stimulation in the presence of TET. Cell fractionation was performed using 5C transfectants cotransfected with the expression vector for G-CSFR. Twenty-four hours after transfection, live cells were isolated using Ficoll-Paque Plus (Amersham) and used for further analysis. Cells were incubated with 100 ng/ml of G-CSF for 15 min before cell fractionations. Fractionated samples were then subjected to Western blotting with anti-p-STAT3, anti-Flag, anti-RhoA, or anti-HDAC Ab. (H) G-CSF-induced transcriptional activation of STAT3 was suppressed by depletion of MgcRacGAP. Expression of Bcl-xL or GAPDH mRNA was examined in the 5C transfectants expressing WT, 182AAA, 199AA, or 182AAA/199AA with or without G-CSF stimulation. Cells transiently transfected with G-CSFR were serum starved with or without G-CSF stimulation for 7 h in the presence of TET, followed by semiquantitative RT-PCR.

FIG. 4.

The NLS of MgcRacGAP is not required for activation of NF-κB p65 in 5C cells. (A) The NLS of MgcRacGAP was required for transcriptional activities of STAT3. Luciferase activities were examined in the lysates of 5C transfectants cotransfected with the STAT3 reporter plasmid, internal control plasmid, expression vector for the G-CSF receptor, or expression vector for the WT-STAT3 (pME/STAT3). After the transfection, cells were incubated with 100 ng/ml of G-CSF for the last 12 h before cell lysates were prepared. Cell lysates were then subjected to a dual luciferase reporter system (Promega). The results shown are the averages ± standard deviations of three independent experiments. (B) The NLS of MgcRacGAP was required for transcriptional activities of STAT5. This experiment was identical to that in panel A, except that 5C transfectants were cotransfected with the STAT5 reporter plasmid, internal control plasmid, or expression vector for the WT STAT5A (pME/STAT5A), together with either the mock or expression vector for ITD-Flt3. (C) The NLS of MgcRacGAP is dispensable for the nuclear translocation of NF-κB p65 in 5C cells. Immunostaining was performed using the 5C transfectants cotransfected with the expression vector for NF-κB p65. After the transfection, cells were serum starved for 3 h, incubated with 30 nM PMA and 1 μM ionomycin for 30 min, and stained with the anti-NF-κB p65 and DAPI. Cells were viewed with a FLUOVIEW FV300 confocal microscope (Olympus). Bar, 10 μm. (D) The NLS of MgcRacGAP was dispensable for transcriptional activities of NF-κB. Luciferase activities were examined in the lysates of 5C transfectants cotransfected with the NF-κB reporter plasmid (k9) carrying a firefly luciferase gene driven by the IL-6 promoter together with the internal control plasmid. After the transfection, cells were incubated with 30 nM PMA and 1 μM ionomycin for 12 h before cell lysates were prepared. Cell lysates were then subjected to a dual luciferase reporter system (Promega). The results shown are the averages ± standard deviations of three independent experiments.

FIG. 5.

Correlation between binding abilities of STAT3 to MgcRacGAP and activities of STAT3. (A) Schematic diagrams showing a series of the deletion sites of STAT3 mutants. (B) Transcriptional activities of STAT3 mutants harboring deletions in DB2. Luciferase activity was examined as described in Materials and Methods. As a control, a reported constitutively active mutant of STAT3C was used. The results shown are the averages ± standard deviations of three independent experiments. (C) MgcRacGAP binding abilities of the STAT3 mutants. Tyrosine phosphorylation and binding affinity to MgcRacGAP of Flag-tagged deletion mutants of DB2-STAT3 in the absence or presence of IL-6-stimulation were determined by immunoprecipitation using the anti-Flag Ab followed by Western blotting with the anti-p-STAT3, anti-MgcRacGAP, or anti-Flag Ab. Expression, tyrosine phosphorylation, and interaction with MgcRacGAP of the Flag-tagged deletion mutants of DB2-STAT3 (lower panel, middle panel, and upper panel, respectively) were examined by immunoprecipitation using 293T cells transfected with each of the STAT3 mutants in the absence (upper three panels) or presence (lower three panels) of IL-6-stimulation for 30 min.

FIG. 6.

The series of deletion mutants of STAT5A in DB2 showed similar phenotypes to those of STAT3. (A) Schematic diagrams showing a series of the deletion mutants of STAT5A. (B) The mutants lacking D1 to -5 and D10 (STAT5A-dD1 to -5 and STAT5A-dD10) as well as the STAT5A-dDB2 lacked their transcriptional activities even under EPO stimulation. Luciferase activity was examined in the lysates of unstimulated or EPO (18 ng/ml)-stimulated 293T cells cotransfected with the expression vector for the EPO receptor (EPOR) and STAT5 reporter plasmid together with internal control reporter plasmids and either the mock vector (pME), the expression vector for the Flag-tagged WT STAT5A, or a series of STAT5A mutants harboring deletions in DB2. As a control, the constitutively active STAT5A1*6 mutant was used. The results shown are the averages ± standard deviations of three independent experiments. (C) The mutants of STAT5A harboring deletions in the two strands (βaprime] and βb) lost binding affinities to MgcRacGAP or did not undergo tyrosine phosphorylation, while the mutants harboring deletions in the region following the strand βb showed enhanced binding affinities to MgcRacGAP and underwent enhanced tyrosine phosphorylation. Expression, tyrosine phosphorylation, and interaction with MgcRacGAP of the Flag-tagged deletion mutants of DB2-STAT5A (lower panel, middle panel, and upper panel, respectively) were examined by immunoprecipitation using 293T cells cotransfected with EPOR and each of the STAT5A mutants in the absence (upper three panels) or presence (lower three panels) of EPO stimulation for 30 min.

FIG. 7.

A constitutively active STAT3 mutant, STAT3-d358L, preferentially bound MgcRacGAP and Rac1 and accumulated to the nucleus. (A) STAT3-d358L supports proliferation of BaF-BO3-G133F3 cells in the presence of G-CSF. BaF-BO3-G133F3 cells expressing mock vector or the Flag-tagged WT STAT3, STAT3C, STAT3-d358L, or H-RasV12 were cultured in the presence of G-CSF, and the cell numbers were determined at the indicated times. BaF-BO3-G133 cells were used as a control. (B) Similar expression levels of WT STAT3, STAT3C, and STAT3-d358L were confirmed by Western blotting with the anti-Flag Ab (upper panel). The expression and activation of H-RasV12 were examined by Western blotting with the anti-Flag Ab (lower panel; input lane) and by pull-down assay using GST-Raf-RBD (lower panel; other lanes), respectively. (C) The STAT3-d358L mutant preferentially accumulated to the nucleus. 293T cells were transfected with pME/STAT3-d358L-Flag (upper panels) or pME/WT-STAT3-Flag (lower panels). After 24 h, the cells were stimulated with IL-6 for the time indicated and fixed, followed by immunostaining with the anti-p-STAT3 or anti-Flag Ab (data not shown). Bar, 10 μm. (D) STAT3-d358L constitutively bound MgcRacGAP and Rac1. Interaction of MgcRacGAP or Rac1 with WT STAT3 or STAT3-d358L was examined by coimmunoprecipitation (IP) using 293T cells transfected with either WT-STAT3 or STAT3-d358L in the absence or presence of IL-6 stimulation (upper two panels). Expression and tyrosine phosphorylation of Flag-tagged WT STAT3 or STAT3-d358L (lower two panels) were also examined.

FIG. 8.

A current model of nuclear import of p-STATs and a working hypothesis for membrane targeting and phosphorylation of STATs. In the present work, we demonstrated that the NLS of MgcRacGAP accompanied by GTP-bound Rac1 is essential for nuclear translocation of p-STATs via importin α/β. We also propose that binding of MgcRacGAP to STATs is required for their tyrosine phosphorylation after cytokine stimulation. Interestingly, the mutants that preferentially bind MgcRacGAP become constitutively active. Altogether, we conclude that MgcRacGAP critically functions both as a mediator of STAT's tyrosine phosphorylation and as an NLS-containing nuclear chaperone of p-STATs.

FIG. 9.

Structure of the wild-type STAT3 and homology models of the hydrophobic core surrounding Leu358. The crystal structure of the β-barrel domain of the wild-type STAT3β (A) and its hydrophobic core region (B). In panel A, the B-factors are color coded, with lower values in blue and higher values in red, to show that the loop bearing L358 is well-ordered and rigid. (C to E) The hydrophobic core regions of the dD7, dL358, and L358A mutants, respectively. In panels B to E, only the residues corresponding to the boxed region of panel A are shown. The key residue, Leu358, is highlighted in orange, and the surrounding hydrophobic residues that form the core together with Leu358 are shown in yellow. The figures are in stereo view (wall eye) and were produced using MOLMOL (20). It should be noted that all of these modeled core structures are packed less tightly than the wild-type structure.