Role of WW Domain-containing Oxidoreductase WWOX in Driving T Cell Acute Lymphoblastic Leukemia Maturation*

Whether tumor suppressor WWOX (WW domain-containing oxidoreductase) stimulates immune cell maturation is largely unknown. Here, we determined that Tyr-33-phosphorylated WWOX physically binds non-phosphorylated ERK and IκBα in immature acute lymphoblastic leukemia MOLT-4 T cells and in the naïve mouse spleen. The IκBα·ERK·WWOX complex was shown to localize, in part, in the mitochondria. WWOX prevents IκBα from proteasomal degradation. Upon stimulating MOLT-4 with ionophore A23187/phorbol myristate acetate, endogenous IκBα and ERK undergo rapid phosphorylation in <5 min, and subsequently WWOX is Tyr-33 and Tyr-287 de-phosphorylated and Ser-14 phosphorylated. Three hours later, IκBα starts to degrade, and ERK returns to basal or non-phosphorylation, and this lasts for the next 12 h. Finally, expression of CD3 and CD8 occurs in MOLT-4 along with reappearance of the IκBα·ERK·WWOX complex near 24 h. Inhibition of ERK phosphorylation by U0126 or IκBα degradation by MG132 prevents MOLT-4 maturation. By time-lapse FRET microscopy, IκBα·ERK·WWOX complex exhibits an increased binding strength by 1–2-fold after exposure to ionophore A23187/phorbol myristate acetate for 15–24 h. Meanwhile, a portion of ERK and WWOX relocates to the nucleus, suggesting their role in the induction of CD3 and CD8 expression in MOLT-4.

The pathway by which T cells differentiate via various stages of selection has been well documented (1). Briefly, bone marrow pluripotent hematopoietic stem cell differentiates into precursor T cell and then migrates to the thymus. In mouse thymus, precursor T cell (CD44 low ) differentiates into CD4-CD8 double-negative cell and then goes through four stages, namely stage I CD44ϩCD25-, stage II CD44ϩ CD25ϩ, stage III CD44-CD25ϩ with pre-TCR 2 (pre-T cell receptor) expression, and stage IV CD44-CD25Ϫ (1). After stage IV, double-negative cells differentiate into CD4/CD8 double-positive cells with TCR (T cell receptor) expression. The CD4 and CD8 single-positive cells, regulatory T cells (Treg), and natural killer-like T cells (NKT) are generated after further positive and negative selection (2,3). In comparison, human hematopoietic progenitor cells, possessing CD34, CD7, and CD5 markers, undergo similar developmental stages to become mature T cells (4,5).
T cell receptor-␤ and -␣ (TCR-␤/␣) subunits form a complex on the cell membrane in the double-negative stage. The surface CD3 complex expression level is elevated during double-positive to single-positive transition (1). TCR-␤/␣ and CD3 complexes on the cell surface receive stimulatory signals from major histocompatibility complex (MHC) for triggering the downstream activation cascade for cell maturation and activation (6). MAPK pathway is implicated in the positive/negative selection involving TCR␣, CD3␥, CD3␦, and PLC␥1. Positive selection results from a low, sustained level of ERK activation, whereas negative selection occurs in response to a large burst of ERK activation concomitant with JNK and p38 activation (1).
Substantial evidence shows that nuclear factor B (NF-B) and its inhibitors (IB) are involved in the T cell maturation (7). IB␣ induces a limited number of double-positive CD4 ϩ CD8 ϩ cells (8). Transgenic mice expressing a superinhibitory form of IB␣ (IB␣ ⌬32/36 ) have an increased number of CD4 ϩ CD8 ϩ double-positive cells but decreased number of CD4 ϩ or CD8 ϩ single-positive cells (9). A low level of calcium ionophore A23187 and 12-myristate 13-acetate (PMA), designated IoP hereafter, is known to induce the differentiation of mononuclear cells, human T-lymphoblastic cell lines HPB-ALL and MOLT-3, and normal lymphocytes (10). Indeed, IoP replaces TCR signals to induce positive selection of CD4 ϩ T cells (11). In contrast, PMA induces apoptosis of T cells at micromolar concentrations (12).
The upstream signal preceding the aforementioned MAPK/ NF-B pathway is largely unknown during T cell differentia-tion. Here, we investigated whether tumor suppressor WW domain-containing oxidoreductase, known as WWOX, FOR, or WOX1, participates in the early signaling of T cell acute lymphoblastic leukemia (T-ALL) maturation. WWOX plays a crucial role in tumor suppression, metabolism, ataxia, epilepsy, neural disorders, neuronal damages and degeneration, and anti-viral immune responses (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Here, we showed that WWOX binds IB␣ and ERK and the complex localizes, in part, in the mitochondria of immature T lymphoid cells. IoP rapidly induces IB␣ and ERK phosphorylation to cause dissociation of the IB␣⅐ERK⅐WWOX complex that ultimately leads to nuclear localization of WWOX and ERK.

Experimental Procedures
Cell Lines-Human T cell acute lymphoblastic leukemia MOLT-4 and Jurkat cells, monocytic U937 and THP-1 cells, breast MDA-MB-231 cancer cells, and embryonic HEK293 fibroblasts were from American Type Culture Collections (ATCC, Manassas, VA) and cultured according to the provider's instructions.
DNA Constructs-Murine full-length WWOX and dominant-negative (dn) cDNA constructs were made as described (21,22). Expression constructs for murine IB␣ (1-301), IB␣295N-(1-295), and IB␣243C-(244 -295) were made in pECFP-C1 vector (cloning site, EcoRI) (27). Additional IB␣ clones were made in pECFP-C1 using the following primer sets: Förster (Fluorescence) Resonance Energy Transfer (FRET)-FRET microscopy was carried out as previously described (25,26,28,29). MOLT-4 cells were transiently overexpressed with ECFP-IB␣ (or other indicated constructs) and EYFP-WWOX expression constructs and cultured for 24 -36 h. FRET analysis was performed using an inverted fluorescence microscope (Nikon Eclipse TE-2000U). Cells were stimulated with an excitation wavelength 440 nm. FRET signals were detected at an emission wavelength 535 nm. ECFP and EYFP were used as donor and acceptor fluorescent molecules, respectively. The FRET images were corrected for background fluorescence from an area without cells and spectral bleed-through. , where fret ϭ fret image, bk ϭ background, cf ϭ correction factor, don ϭ donor image, and acc ϭ acceptor image. The equation normalizes the FRET signals to the expression levels of the proteins. Where indicated, time-lapse microscopy for FRET was carried out to determine IoP-regulated binding interactions among ECFP-IB␣, EGFP-ERK, and DsRed-WWOX, as described (29). An inverted Olympus IX81 fluorescence microscope and the internal software for FRET analysis were used. As a negative control, a dominant-negative EGFP-ERK(K71R/K72R) (or dn-EGFP-ERK) was generated and used to replace the wild type EGFP-ERK.
Co-immunoprecipitation, Western Blotting, and Immunofluorescence Microscopy-MOLT-4 and Jurkat T cell were exposed to IoP for indicated times, lysed with a lysis buffer (0.5% Nonidet P-40, 10% glycerol in PBS), and then cleared with protein-G-Sepharose beads. Aliquots of an indicated IgG antibody were added to the lysates for immunoprecipitation, as described (23,24,26). Additionally, cytoplasmic and nuclear extractions were prepared for immunoprecipitation and immunostaining (23,24,26). Similarly, cells were treated with IoP for the indicated times followed by immunostaining processing with specific antibodies. Primary antibodies were against ERK1, ERK2, IB␣, and WWOX (Santa Cruz Biotechnology). Fluorescent secondary antibodies, including donkey anti-rabbit Alexa Fluor 488 IgG and donkey anti-goat or -mouse Alexa Fluor 594 IgG (eBioscience) were used for visualization by fluorescent microscopy and confocal microscopy (24 -29). Where indicated, mitochondria were stained with MitoTracker Red (Thermo Fisher Scientific) (22,26).
Naïve BALB/c Mice-Whole spleens were isolated from naïve BALB/c mice and lysed and prepared for co-immunoprecipitation as described above. Use of the animals has been approved by the Intramural Animal Use and Care Committee (IACUC) of the National Cheng Kung University.

Results
Calcium Ionophore/PMA (IoP) Rapidly Induced IB␣ and ERK Phosphorylation, Which Led to CD3 Expression in MOLT-4 T Cell-To investigate the role of WWOX in T cell growth and differentiation, MOLT-4 cell was mainly used in this study, with Jurkat T cell for comparison. MOLT-4 is considered less differentiated than Jurkat. We have shown that Jurkat expresses CD3, but MOLT-4 does not (12). A combination of PMA (0.55 nM) and A23187 (0.30 M) (or IoP) is known to induce differentiation in MOLT-3, mononuclear cells, and HPB-ALL cells (10). When MOLT-4 and Jurkat cells were exposed to IoP for 15 h, expression of CD3 was significantly increased in whole cell lysates in MOLT-4, but not in Jurkat, 15-24 h post stimulation (Fig. 1, A and B). CD3 is a differentiation marker of T cell whose subunits accumulate in the intracellular compartments of immature thymocytes (2, 30). How-ever, CD3 is on the cell surface of mature cells. IoP significantly increased membrane localization of CD3 in both non-permeabilized MOLT-4 and Jurkat cells after stimulation for 15-24 h as determined by flow cytometry (Fig. 1, C and D), suggesting that cells have reached maturation. 15 h post stimulation, IoP promoted MOLT-4 to enter the S and G 2 /M phases of the cell cycle, as determined by flow cytometry (Fig. 1E). Also, this is supported by up-regulation and nuclear accumulation of G 2 /M-specific cyclin B in the IoP-treated MOLT-4 (Fig. 1F). The IoP-stimulated cells continued to grow 72 h after exposure (data not shown), which is in agreement with other reports (10,31).
PMA increased ERK phosphorylation rapidly in 1-3 h and induced differentiation of human myeloblastic leukemia ML-1 cells and Jurkat and MOLT-4 T cells (12,32). The upstream MEK phosphorylates ERK (p-ERK), whereas p-ERK down-regulates several anti-proliferative genes and promotes the G 0 /G 1 phase to the S phase transition (33,34). IoP increased ERK phosphorylation at Thr-202 and Tyr-204 in MOLT-4 in 5 min or less, and the phosphorylation lasted for 5 h and was then reduced to a basal level of phosphorylation or non-phosphorylated in 15-24 h (Fig. 2, A and B). To block MEK/ERK signaling, MOLT-4 cells were pretreated with U0126, a MEK inhibitor, and then exposed to IoP for 24 h. Again, IoP induced CD3 and CD8 expression and ERK phosphorylation (Fig. 2, C and D). U0126 significantly suppressed ERK and WWOX phosphorylation and abolished CD3 and CD8 expression (Fig. 2, C and D).
In comparison, human monocytic U937 and THP-1 cells were exposed to IoP. ERK was rapidly phosphorylated in Ͻ5 min in both cells (Fig. 2, E and F). IoP induced CD8␣ expression in the less differentiated U937 cells in 5 min, whereas the more differentiated THP-1 cells showed a constitutive expression of CD8␣ (35). In addition, IoP induced the second phase of ERK phosphorylation in both U937 and THP-1, but not in MOLT-4, post stimulation for 15 h (Fig. 2G).
IoP Up-regulated Ser-14 Phosphorylation in WWOX and Then Induced IB␣ Degradation-In time-course experiments, IoP induced degradation of endogenous IB␣ in MOLT-4, which started to occur approximately in 3 h post stimulation (Fig. 3A). IB␣ disappearance lasted approximately for 12 h. However, IB␣ degradation did not occur effectively in the differentiated Jurkat (Fig. 3B). When MOLT-4 cells were pretreated with MG-132 for 30 min followed by treating with IoP for 5 h, IB␣ degradation was blocked (Fig. 3C). Similar experiments were conducted using Jurkat cells, and no degradation of IB␣ was observed (Fig. 3D).
Using cytosolic and nuclear fractions for Western blotting, we showed that IoP induced translocation of p-ERK to the nuclei in MOLT-4 in 5-7 h post stimulation (Fig. 3G). In contrast, no nuclear accumulation of NF-B/p65 was shown (Fig.  3G). WWOX regained phosphorylation at Tyr-33 in 7 h, as it relocated to the nucleus, whereas nuclear relocation of p-ERK occurred somewhat earlier, which was 5 h after IoP stimulation (Fig. 3G).
WWOX Bound IB␣ and ERK in the Mitochondria, and IoP Dissociated the WWOX⅐IB␣⅐ERK Complex-WWOX is localized in many organelles in resting cells, including mitochondria (22), cell membrane (26), and lysosome (12,37). Tyr-33-phosphorylated or activated WWOX is mainly localized in the mitochondria and nuclei (21,22,23,38,39). Translocation of WWOX from the cell membrane, cytoplasm, or lysosome to the mitochondria and nuclei in vitro and in vivo has been doc-  , n ϭ 3). The bar graph shows the normalized p-ERK levels relative to ␣-tubulin (mean Ϯ S.D.; n ϭ 3). C ϭ non-treated control. C and D, MOLT-4 cells were pretreated with U0126 (10 M) for 30 min and then exposed to IoP for 24 h. U0126 blocked the expression of CD3 and CD8 in MOLT-4 T cells (C). As shown in the bar graph (D), IoP significantly increased the expression of CD3 and CD8 and ERK phosphorylation in 24 h and that U0126 suppressed the increases (mean Ϯ S.D.; n ϭ 3). E and F, human monocytic U937 and THP-1 cells were exposed to IoP and shown to have a rapidly increased ERK phosphorylation in 5 min or less. CD8␣ expression was induced in the less differentiated U937 cells in 5 min. THP-1 has a constitutive expression of CD8␣. G, IoP induced the second phase of ERK phosphorylation in U937 and THP-1, but not in MOLT-4, in 15 h post stimulation.
We then isolated the cytoplasmic and mitochondrial fractions from MOLT-4. WWOX, IB␣, and ERK were present in the cytoplasm and mitochondria (Fig. 4C). IoP rapidly decreased the protein levels of WWOX and its Tyr-33 phosphorylation in the mitochondria (Fig. 4C), suggesting release of these proteins from the mitochondria. In additional experiments, WWOX and IB␣ were found in the isolated mitochon-dria, and the protein levels were reduced during exposure to IoP for 1 h followed by increase after 2-3 h (Fig. 4D).
By confocal microscopy, IoP significantly reduced the mitochondrial localization of IB␣ and WWOX in MOLT-4 (Fig.  4E), again suggesting their translocation to the cytoplasm from the mitochondria. In comparison, TNF-␣ did not effectively reduce the levels of WWOX in the mitochondria (Fig. 4E). However, TNF-␣ significantly decreased the level of mitochondrial IB␣ (Fig. 4E).
WWOX Protected IB␣ from Degradation-By co-immunoprecipitation, IB␣ was shown to physically interact with WWOX and ERK in resting MOLT-4 (Fig. 5A). When cells were treated with half-strength IoP, ERK exhibited a delayed phosphorylation in 2 h, and this led to dissociation of the IB␣⅐ERK⅐WWOX complex (Fig. 5A). Malignant breast  ; Fig. 5B). PMA at 10 M induced ERK phosphorylation in Ͻ1 min, and the phosphorylation led to rapid reduction of the IB␣⅐ERK⅐WWOX complex (Fig. 5C).
MOLT-4 cells were transiently overexpressed with EYFP-WWOX and ECFP-tagged IB␣, IB␣295N-(1-295), or FIGURE 4. IoP decreased colocalization of WWOX and IB␣ in the mitochondria. A and B, MOLT-4 cells were treated with IoP for the indicated times followed by processing fixation, permeabilization, and antibody staining for confocal microscopy. The nuclei were stained with DAPI. WWOX and IB␣ colocalized in cytosol, and IoP significantly reduced the colocalization in 3 h (ϳ200 cells counted, mean Ϯ S.D., n ϭ 3; Student's t test). Also, IoP decreased the colocalization of WWOX and ERK2 with time. In comparison, IoP increased the colocalization of WWOX and ERK1 initially followed by reduction. C, MOLT-4 cells were treated with IoP for the indicated times followed by preparing cytosolic and mitochondrial fractions. WWOX, ERK, and IB␣ were present in the mitochondria. Mitochondrial IB␣ was decreased upon IoP stimulation. Cont ϭ non-treated control. D, similarly, IoP initially reduced the levels of WWOX and IB␣ in the mitochondria followed by up-regulation with time. E, MOLT-4 cells were treated with IoP or TNF-␣ (50 ng/ml). Cells were processed for immunofluorescent staining and confocal microscopy. IoP induced the release of IB␣ and WWOX from the mitochondria in 3 h; however, TNF-␣ was less effective (mean Ϯ S.D., n ϭ 3, Student's t test).
MEK1 Inhibitor U0126 Decreased the Binding of IB␣ with WWOX-U0126 inhibited IoP-induced CD3 and CD8 expression in MOLT-4 cells (Fig. 2C). To investigate whether U0126 blocks the interactions between WWOX, IB␣, and ERK, coimmunoprecipitation was carried out. Again, IB␣ physically bound WWOX and ERK in resting MOLT-4 cells, and IoP induced the dissociation of IB␣ with WWOX and ERK in 2-3 h. U0126 disrupted the binding of IB␣ with WWOX but not with ERK in MOLT-4 during pretreatment for 30 min (Fig. 7E). IoP and U0126 reduced the IB␣/ERK interaction with time (Fig. 7E).
The presence of the IB␣⅐ERK⅐WWOX complex in the spleen is shown (Fig. 7, F-H). By using whole spleen lysates from naïve BALB/c mice for co-immunoprecipitation, binding of IB␣ with WWOX and ERK along with Tyr(P)-33-WWOX interacting with ERK, was found in the spleen lysates of naïve BALB/c mice.
Time-lapse FRET Microscopy of IB␣⅐ERK⅐WWOX Signaling-IoP stimulated rapid phosphorylation of IB␣ and ERK in 5 min or less ( Fig. 2A). By measuring three-way protein/ protein interactions (29, 43), ECFP-IB␣ was excited to allow the energy transfer from a high to a low level, i.e. to EGFP-ERK and then to DsRed-monomer WWOX. Positive signals were observed in IoP-stimulated COS7 cells expressing ECFP-IB␣, EGFP-ERK, and DsRed-monomer WWOX (Fig. 8 and supplemental Video S1; 36 positive cells of 40 counted in end-point experiments). The emission energy from ECFP could not directly go to the recipient DsRed monomer without an EGFP bridge. The reason for using monomer expression for WWOX is that this protein may undergo self-binding during overexpression (data not shown). . All yeast cells grow at room temperature. The binding activates the Ras pathway in cdc25 mutant yeast that allows their growth at 37°C. Binding of WWOX with p53 is regarded as positive controls. In negative controls, Sos protein did not bind the Myr tag expressed on the cell membrane. E, by FRET microscopy, EYFP-WWOX bound the N terminus of IB␣ (ECFP-IB␣295N) but not the PEST domain (ECFP-IB␣243C) in MOLT-4 cells. Percent pixel changes for the strongest binding areas (red color) were quantified and tabulated (mean Ϯ S.D., n ϭ 8 for IB␣/WWOX, n ϭ 9 for IB␣295N/WWOX, n ϭ 6 for IB␣243C/ WWOX; Student's t test; *, p Ͻ 0.05). In the negative control, ECFP did not bind EYFP. F, also, by FRET microscopy, EYFP-tagged the first WW domain (EYFP-WWOXww) physically bound ECFP-IB␣ compared with the ECFP/EYFP controls (****, p Ͻ 0.001). A dominant-negative (dn) construct dn-WWOXww at the WW domain had a significantly reduced binding with IB␣ (mean Ϯ S.D., n ϭ 6 for EYFP/ECFP, n ϭ 4 for WWOXww/IB␣, n ϭ 5 for dn-WWOXww/IB␣). G, HEK293 cells transiently overexpressed EGFP-WWOX or dominant-negative WWOX. By immunoprecipitation, IB␣ was shown to bind ERK, p-ERK, and WWOX but not dn-WWOX. Pre-IP ϭ 1 ⁄10 of each of the whole cell lysates (ϳ30 g) was loaded onto gels. The extent of IB␣ binding with ERK and WWOX was quantified.
When dominant-negative EGFP-ERK was used, no positive signals were observed (Fig. 8 and   . WWOX bound the non-PEST region of IB␣. A, HEK293 cells were transiently overexpressed with ECFP and ECFP-tagged constructs, respectively. After culturing for 48 -72 h, WWOX was precipitated by a specific antibody as shown below. The GFP antibody, which cross-reacts with CFP, was used for probing the ECFP-tagged proteins. B, C, and D, by co-immunoprecipitation (IP), WWOX was shown to bind the full-length IB␣, IB␣295N, IB␣-(1-67), IB␣-(68 -243), IB␣-(1-243), and IB␣-(244 -314). WWOX did not bind the PEST-containing IB␣243C. Binding of WWOX with ERK was also shown. Negative control ϭ non-immune IgG used for immunoprecipitation. IB, immunoblot. E, MOLT-4 cells were pretreated with U0126 (10 M) for 30 min before exposure to IoP for indicated times. U0126 disrupted the binding of WWOX with ERK and IB␣. Similarly, IoP dissociated WWOX from the complex ERK and IB␣. U0126 and IoP in combination further reduced the binding of IB␣ with ERK. C ϭ non-treated control or resting cells. Pre-IP ϭ 1 ⁄10 of each of the whole cell lysates (ϳ30 g) was loaded onto gels. Percent changes in binding were calculated as indicated. F-H, by using whole spleen lysates from naïve BALB/c mice for co-immunoprecipitation, IB␣ interacted with WWOX and ERK, and Tyr(P)-33-WWOX interacted with ERK are shown. Control ϭ non-immune serum used for immunoprecipitation. IB␣, ERK, and WWOX are needed to transduce the IoP signal.
In summary, an endogenous IB␣⅐ERK⅐Tyr(P)-33-WWOX complex is present in the immature MOLT-4 cells (Fig. 9). IoP induced rapid phosphorylation of endogenous ERK and IB␣ in 5 min or less. Meanwhile, WWOX underwent dephosphorylation at Tyr-33 and Tyr-287 and phosphorylation at Ser-14 in 1-2 h, which led to dissociation of WWOX from the p-IB␣⅐p-ERK complex. Degradation of IB␣ and de-phosphorylation of ERK occurred in the next 3-5 h and lasted for the next 12 h. Meanwhile, a portion of WWOX and ERK relocated to the nucleus. When the level of IB␣ returned to normal, up-regulation of CD3 and CD8 along with re-formation of the IB␣⅐ERK⅐Tyr(P)-33-WWOX occurred in 15-24 h (Fig. 9).

Discussion
Here, we have discovered for the first time that an endogenous complex of Tyr(P)-33-WWOX, ERK, and IB␣ plays a critical role in driving T cell acute lymphoblastic leukemia maturation. A portion of this complex is present in the mitochondria. We have previously reported the presence of WWOX and p53 in the mitochondria (22,39,44). In addition, WWOX is involved in mitochondrial respiration and metabolism (45,46). By co-immunoprecipitation, yeast two-hybrid analysis, timelapse FRET microscopy, and expression of cloned plasmid vectors, we deciphered how the component proteins in the complex interact with each other and how the action contributes to MOLT-4 maturation. IoP rapidly stimulates phosphorylation of ERK and IB␣ in Ͻ5 min, and then endogenous WWOX undergoes significant dephosphorylation at Tyr-33 and Tyr-287 in 1-2 h in MOLT-4 along with significantly increased Ser-14 phosphorylation. WWOX then dissociates from ERK and IB␣ in 1-2 h. Later, ERK becomes dephosphorylated to a basal level or non-phosphorylated, and p-IB␣ is degraded in 3-15 h. Ultimately, CD3 ϩ and CD8 ϩ MOLT-4 cells are present in 15-24 h. There are 25% of cells are at the G 2 /M phase of the cell cycle. ERK has been implicated in the growth factor-independent cell cycle progression toward the G 2 /M phase (33). CD4 ϩ cells are barely detectable, although IoP may rapidly induce CD4 expression in 1-3 h followed by reduction (data not shown).
Supporting evidence is provided that there is a time-related release of WWOX and IB␣ from the mitochondria in 1-2 h post IoP stimulation, whereas ERK appears to remain in the mitochondria. IB␣ may undergo degradation upon release from mitochondria. We have recently reported the shuttling of TRAPPC6A (trafficking protein particle complex 6A) in between nucleolus and mitochondrion (42,43). TRAPPC6A is a carrier for WWOX to undergo nuclear translocation. Whether IoP-induced translocation of WWOX and ERK to the nucleus requires TRAPPC6A remains to be established.
Similarly, p-ERK interacts with p-IB␣, which lasts for 2 h. The interaction blocks the degradation of p-IB␣. Clearly, the initial events for IB␣, ERK, and WWOX are needed for MOLT-4 maturation.
Together, both WWOX and ERK protect IB␣ from degradation. Phosphorylation of IB␣ and ERK along with altered WWOX phosphorylation destabilizes the IB␣⅐ERK⅐WWOX complex. p-ERK apparently has a conformal change that allows its dissociation from the IB␣⅐ERK⅐WWOX complex.
WWOX and ERK relocate to the nucleus 5 h post IoP exposure, suggesting that both proteins are committed to MOLT-4 maturation by working in the nucleus. It has been well documented that activated ERK controls the activity of many transcription factors. The N-terminal first WW domain of WWOX induces transcriptional activation of NF-B, and WWOX interacts with many transcription factors to control neuronal survival via its WW domain in vivo (28).
How Ser(P)-14-WWOX works in vivo is largely unknown. In our preliminary study, we made Ser-14-phosphorylated and non-phosphorylated WWOX peptides of 15 amino acid residues and found that the phosphorylated peptide is a potent inducer of T cell expansion in BALB/c mice growing with melanoma. 3 The T cells include CD3ϩ, CD4ϩ, and CD8ϩ populations. Without phosphorylation, there is no induction of T cells, again suggesting a role of pSer-14-WWOX in committing T cell grow and expansion in vivo.
The WW, ANK and PEST domains are involved in signal transduction (13, 26, 27, 33, 37, 48 -52). We have extensively investigated how WWOX binds IB␣ and determined that the first N-terminal WW domain binds the non-PEST region in IB␣. First, binding of WWOX with IB␣ is shown in yeast two-hybrid analysis. Second, by FRET analysis, the N terminus of IB␣ interacts much stronger with WWOX than the C-terminal PEST domain. A dominant-negative WWOX construct possessing alterations in the WW domain loses its binding with IB␣, indicating that the N-terminal first WW domain of WWOX interacts with the N-terminal ankyrin-repeat motif of IB␣. By co-immunoprecipitation, the binding interactions are confirmed by using designed expression constructs of IB␣ for interacting with WWOX. Finally, time-lapse FRET analysis confirms that functionally active IB␣, ERK, and WWOX together are critical for IoP signaling and MOLT-4 maturation.
Tyrosine phosphorylation is known in many WW domaincontaining proteins (52). Unknown kinases and phosphatases are involved in the IoP-mediated phosphorylation and dephosphorylation in WWOX. We have utilized specific inhibitors to block the function of ERK and IB␣, and the inhibition leads to blockade of MOLT-4 maturation. As a partner of WWOX (21)(22)(23), p53 participates in T cell maturation (53). However, the precise molecular action needs further investigation.
Inhibition of MEK kinase by U0126 results in blocking of ERK phosphorylation and MOLT-4 maturation. Whether MEK is in the IB␣⅐ERK⅐WWOX complex is unknown. Indeed, WWOX physically binds MEK in many types of cancer cells (12). Dissociation of the WWOX⅐MEK complex by high concentrations of PMA allows WWOX relocation to the mitochondria for causing cancer cell death. IB␣ is a key inhibitor of NF-B (7, 54) and appears to play an inhibitor role in preventing the phosphorylation of ERK in response to IoP. Thus, blocking of IB␣ degradation by proteasome inhibitor MG132 halts MOLT-4 maturation.
In addition to MOLT-4, we investigated whether IoP induces maturation in monocytic U937 and THP-1 cells. The less differentiated U937 is susceptible to IoP for induction of CD8␣. Unlike MOLT-4 and Jurkat T cells, monocytic cells undergo an early and a late phase of ERK phosphorylation. Functional rel- FIGURE 9. IoP signaling. A schematic graph is illustrated for IoP-induced signal transduction leading to MOLT-4 maturation. An endogenous IB␣⅐ERK⅐Tyr(P)-33-WWOX complex is present in the immature MOLT-4. IoP rapidly induces ERK and IB␣ phosphorylation in 5 min or less, and then WWOX undergoes dephosphorylation at Tyr-33 and Tyr-287 and phosphorylation at Ser-14. WWOX is released from the p-ERK⅐p-IB␣ complex in 1 h. p-IB␣ is then degraded, and ERK phosphorylation returns to a basal level or non-phosphorylation in 5-15 h. Meanwhile, both ERK and WWOX relocate to the nucleus (to induce gene transcription for cell maturation). Finally, expression of CD3 and CD8 occurs along with reappearance of the IB␣⅐ERK⅐WWOX complex in the cells in 15-24 h. CD4 is barely detectable. evance for ERK phosphorylation remains to be established. Taken together, immature leukemia cells are susceptible to maturation by IoP. This raises promising therapeutic considerations and designing approaches to induce leukemia cell maturation in patients.