Characterization of NFκB Activation by Detection of Green Fluorescent Protein-tagged IκB Degradation in Living Cells*

Activation of the transcription factor NFκB requires rapid degradation of its inhibitor, IκBα. To facilitate the study of IκBα degradation, we fused IκBα protein to enhanced green fluorescent protein to construct IκBα-enhanced green fluorescent protein (IG). We demonstrated by both flow cytometry and Western blot analysis that the half-life of IG in the presence of human tumor necrosis factor (TNF) α is approximately 5 min, which is similar to the half-life of native IκBα. The degradation coincided with NFκB translocation from the cytoplasm to the nucleus and NFκB-mediated induction of transcription. Phorbol 12-myristate 13-acetate (PMA), but not forskolin, also induces degradation of IG fusion protein. The half-life of IG in the presence of PMA is approximately 15 min, longer than when induced with TNFα. Co-treatment with TNFα and PMA did not result in a synergistic effect on IG degradation, although they stimulate different kinases in two different signaling pathways. Degradation of IG was inhibited by mutations at serine residues 32 and 36, which are the target sites of the phosphorylation modification that initiates degradation of IκBα. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of IκBα also requires phosphorylation as the signal for degradation. Finally, we showed that the rate of TNFα-induced degradation of IG remains almost constant throughout the cell cycle, except at the mitotic phase, in which IG degrades more slowly.

Regulated degradation of specific proteins is part of the intracellular biochemical changes that contribute to the regulation of signal transduction pathways, cell proliferation, growth arrest, and apoptosis. For instance, tumor necrosis factor (TNF) 1 ␣-mediated NFB activation requires the rapid degradation of IB␣, the inhibitor of NFB (1)(2)(3)(4). NFB is a transcription factor that regulates the expression of a number of genes whose products contribute to inflammation and immune responses (5,6). Before activation, NFB is sequestered in the cytoplasm by forming a complex with IB␣ (7)(8)(9). The nuclear translocation signal of NFB is masked by the inhibitor (10). NFB can be activated by a number of stimuli, including TNF␣, interleukin 1␤, lipopolysaccharide, and phorbol esters (PMA). Initiated by TNF␣ binding, a number of proteins including TRADD, TRAF2, and RIP aggregate around the TNF␣ type 1 receptor (11)(12)(13), which triggers the phosphorylation of IB␣ and leads to the rapid dissociation of NFB from IB␣ (14 -17). A protein kinase complex, which includes NIK, IKK␣, and IKK␤, is involved in the phosphorylation of IB␣ (18 -22). The phosphorylated IB␣ is further modified by ubiquitination enzymes and degraded by the 26S proteasome (23). Serine residues 32 and 36 of IB␣ have been identified to be the specific target sites of phosphorylation (16, 24 -26). Mutations at these positions abolish both phosphorylation and degradation of IB␣ (16, 24 -26). Once released from the IB␣ complex, NFB immediately translocates from the cytoplasm to the nucleus, where it mediates the transcriptional activation of genes, such as interleukin 2 and IB␣. IB␣ degradation is rapid and is completed within 5-40 min after stimulation by TNF␣ (1,2,7). IB␣ also degrades in the absence of TNF␣, but this basal degradation is much slower than induced degradation. The half-life of the basal degradation is around 2 h (2,29). It is unclear whether the basal degradation of IB␣ also requires phosphorylation for initiation of the degradation process (30). Therefore, the relationship of basal degradation to induced degradation remains uncertain.
Both the gene and cDNA of the green fluorescent protein (GFP) have been cloned from the jellyfish Aequorea victoria (31). GFP has been widely used to study gene expression and protein localization (32-35) because its fluorescence emission does not require substrates or cofactors (36), and fluorescence detection can be made in real time. The key sequence of Ser-Tyr-Gly (amino acids 65-67) within GFP undergoes spontaneous oxidation to form a cyclized chromophore that emits fluorescence (37). Mutation of Ser to Thr in the chromophore (S65T) leads to a higher fluorescence intensity of GFP. Enhanced GFP (EGFP) is one such mutant. It contains the mutations S65T and F64L and is encoded by a gene with humanoptimized codons (38 -40). Crystallographic structures of wildtype GFP and the mutant S65T reveal that the tertiary structure of GFP resembles a barrel (41,42), and this compact structure makes GFP a very stable protein. Fusing EGFP to the degradation domain of mouse ornithine decarboxylase, we were able to generate a destabilized EGFP with a half-life of 2 h (43). This study indicates that EGFP is able to degrade in vivo when fused with a degradation domain and suggests that EGFP can be used as a general reporter to measure protein degradation in vivo when fused to a full-length rapid turnover protein.
In the present study, we used EGFP to monitor IB␣ degradation by making a fusion protein of IB␣ and EGFP. The fusion protein was found to be degraded as well as IB␣ upon TNF␣ treatment. Therefore, IB␣ degradation can be analyzed simply by monitoring the change of the fluorescence intensity of the fusion protein without cell disruption. Using EGFP, we were able to analyze the degradation more efficiently and rapidly than possible with other methods. We showed that both TNF␣ and PMA, but not forskolin, induce degradation of the fusion protein. Degradation induced by PMA is slower than that induced by TNF␣, and no synergistic effect was detected when cells were treated with both TNF␣ and PMA simultaneously. During the study, we also found that the basal degradation of IB␣, like the induced degradation, also requires phosphorylation at serines 32 and 36 for initiation of the degradation process. Finally, we found that IG degradation at the mitotic phase is slower than IG degradation at the other phases.

EXPERIMENTAL PROCEDURES
The cDNAs encoding IB␣ and EGFP were amplified with Pfu DNA polymerase. IB␣ was amplified with primers that incorporated a SacII recognition sequence at the 5Ј end and a BamHI sequence at the 3Ј end. The stop codon of IB␣ was deleted during polymerase chain reaction amplification to make an open reading frame with EGFP. EGFP was amplified with primers that incorporated a BamHI recognition sequence at the 5Ј end and an EcoRI sequence at the 3Ј end. The amplified polymerase chain reaction products were ligated at the BamHI site, and the resulting fusion construct (IB␣-EGFP) was cloned into the SacII and EcoRI sites of the pTRE expression vector (CLONTECH, Palo Alto, CA) for use in the tetracycline-regulated expression system (44). The IB␣ mutant S32A/S36A was made with overlap extension mutagenesis (45).
pTRE-IG and pTRE-IGS32A/S36A expression vectors were transfected into HeLa Tet-Off TM cells (CLONTECH) for degradation studies. HeLa Tet-Off TM cells stably express a fusion protein of the tet repressor and the herpes simplex virus VP16 (tTA) and thus can be used for tetracycline-regulated expression of a gene cloned into the pTRE vector (44). 24 h after transfection, the transfected cells were subject to functional analyses. To make stable cell lines, cell were co-transfected with pTK-Hyg and cultured in medium containing 200 g/ml hygromycin. Drug-resistant colonies were then screened for green fluorescence under a Zeiss Axioskop Model 50 fluorescence microscope.
To examine the fluorescence intensity of EGFP or IG, the cells were cultured on coverslips. After transfection, the cells were incubated at 37°C for 24 h and then fixed with 4% paraformaldehyde for 30 min. The coverslips were mounted on a glass slide for examining fluorescence under a fluorescence microscope. To determine protein turnover, the cells were treated with 0.1 g/ml recombinant human TNF␣ (CLON-TECH) for varying times before paraformaldehyde fixation. The transfected cells with/without TNF␣ treatment were collected by EDTA treatment, and the cell pellets were resuspended in 0.5 ml of PBS. Cell suspensions were analyzed for fluorescence intensity using a FACS-Calibur TM flow cytometer (Becton Dickinson, Inc., San Jose, CA). EGFP was excited at 488 nm, and emission was detected using a 510/20 bandpass filter.
The transfected cells with/without CHX treatment were collected in PBS, and cell lysates were prepared by sonication. Proteins were resolved by SDS gel electrophoresis and transferred onto a membrane. IG fusion proteins were detected using a monoclonal antibody against GFP (CLONTECH). Bands were visualized with the Western Exposure chemiluminescent detection kit (CLONTECH).
To examine NFB translocation, cells stably expressing IG were treated with TNF␣. The treated cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature and rinsed with PBS three times. The fixed cells were then permeabilized with a blocking solution for 1 h. The cells were then incubated with a 1:250 dilution of anti-Rel p65 polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) in the blocking solution for 2 h. After washing with PBS three times, the cells were incubated with a 1:250 dilution of rhodamine-conjugated anti-rabbit IgG (Roche Molecular Biochemicals, Indianapolis, IN) for 45 min in PBS containing 4% bovine serum albumin. After rinsing three times with PBS, the stained cells were mounted in Citifluor (Ted Pella, Inc., Redding, CA). To determine the induction of SEAP mediated by NFB, IG cells were transfected with pNFB-SEAP. TNF-mediated induction was determined by comparing SEAP levels within cells with and without TNF␣ treatment, using the Great EscA-Pe TM chemiluminescent detection kit (CLONTECH).
We treated cells with different compounds for 16 h to arrest them at various stages in the cell cycle. The G 1 -phase-arrested cells were obtained by treatment of the cells with 100 M mimosine, the G 1 -S-phasearrested cells were obtained by treatment of the cells with 2 mM thymidine, and the mitotic phase-arrested cells were obtained by treatment of the cells with 50 ng/ml nocodazole. The cell cycle-arrested cells were then challenged with TNF␣ to determine the degradation rate of IG.

RESULTS
To investigate whether the biochemical degradation of IB␣ can be monitored by fluorescence decay of the EGFP fusion protein, we appended EGFP to the C terminus of IB␣. The fusion was designated IG. The fusion IG was transiently expressed in HeLa Tet-Off TM or 293 Tet-Off TM cells by transfecting the expression vector pTRE-IG. The transfected cells were treated with TNF␣ for 1 h and collected for analysis by flow cytometry. As shown in Fig. 1, TNF␣ treatment resulted in decreased fluorescence of transfected cells, indicating sensitivity of IG fluorescence to TNF␣ treatment. To examine whether TNF␣-mediated fluorescence decay of IG requires the phosphorylation of IB␣, we mutated serines 32 and 36 to Ala to make IG-S32A/S36A. The results from flow cytometry analysis demonstrated no decrease in the green fluorescence of IG-S32A/ S36A after TNF␣ treatment in both HeLa and 293 cells (Fig. 1). Therefore, we concluded that TNF␣-mediated fluorescence decay of IG, like IB degradation, needs the specific phosphorylation modification at serines 32 and 36.
To establish stable expression of the IG fusion protein in HeLa cells, we co-transfected pTRE-IG with pTK-Hyg, selected drug-resistant colonies in the presence of hygromycin, and screened for fluorescence under a fluorescence microscope. One of green fluorescent clones, designated as HeLa-Off/IG, is shown in Fig. 2. HeLa-Off/IG cells were treated with TNF␣ for 0, 5, 10, 15, 20, and 25 min and subjected to fluorescence microscopy. Green fluorescence was not detectable within 20 min after treatment with TNF␣ ( Fig. 2A). Next we used flow cytometry to quantitatively analyze the half-life of IG fluorescence. Both total fluorescence and the percentage of fluorescent cells declined to a basal level after 15 min of treatment. Fluorescence declined to 50% of uninduced levels 5 min after induction. This half-life is similar to that of endogenous IB␣ protein in HeLa cells (1,2,7). The results suggest that the EGFP tag changes neither TNF␣-mediated regulation of IB␣ turnover nor the protein's half-life.
To confirm that the decay in fluorescence correlates with a decrease in IG protein levels, we measured the amount of the fusion protein by Western blot analysis with a monoclonal antibody against GFP. At 0 min, we saw a band at 65 kDa, corresponding to the predicted size of the IG protein (Fig. 2C). The intensity of this band declined with increasing length of TNF␣ treatment. The band's intensity was reduced to about 50% at approximately 5-10 min of treatment, confirming the IG protein half-life of 5-10 min. Therefore, the intracellular fluorescence intensity of the IG fusion protein correlates directly to the IG protein level.
To examine whether IG degradation leads to NFB translocation, we detected NFB protein in IG cells with polyclonal antibodies against p65 as the primary antibody and rhodamine-conjugated secondary antibody. Before TNF␣ treatment, both NFB and IB␣ are localized to the cytoplasm. Degradation of IG was induced by adding TNF␣, which leads to immediate translocation of NFB from the cytoplasm to the nucleus (Fig. 3A). No difference in translocation of NFB was found in IG cells and control HeLa cells. NFB-mediated transcription was measured by the induction of the NFB response elements, B 4 , located on a reporter vector transiently transfected into the cells. The TNF-induced induction of the reporter protein SEAP in the IG cells is the same as that in control HeLa cells (Fig. 3B). These results indicated that degradation of the IG fusion protein is associated with NFB translocation and transcription induction. Therefore, the EGFP tag on IB␣ does not change normal NFB function.
It has been shown that protein kinase A in vitro (46) and protein kinase C both in vitro (46) and in vivo (1, 2) are able to activate NFB. We first examined whether protein kinase A can induce degradation of IG by treatment of HeLa-Off/IG cells with forskolin. Analysis by flow cytometry showed that fluorescence of the treated cells remained the same during the 3-h period of treatment (Fig. 4A). This result indicates the insensitivity of IB degradation to protein kinase A, which does not agree with the in vitro study in which protein kinase A activates NFB. We also tested whether protein kinase C can activate NFB by treatment of HeLa-Off/IG cells with PMA. As shown in Fig. 4B, the fluorescence of IG rapidly decayed after PMA treatment, and the half-life of IG fluorescence is about 15 min. The sensitivity of IG degradation to protein kinase C is similar to that seen in the previous studies. The degradation rate of IG fluorescence induced by PMA is slower that induced by TNF␣ (Figs. 2B and 4B). To examine the difference more carefully, we did a side-by-side comparison of the degradation of IG fluorescence induced by TNF␣ and PMA. Treatment of HeLa-Off/IG cells with TNF␣ resulted in rapid degradation of IG, but treatment with PMA produced a slightly delayed degradation response (Fig. 5). This delay is approximately 5 min. TNF␣-induced degradation of IB proceeds via the activation of the IKK complex, whereas PMA-induced degradation of IB requires activation of the 90-kDa ribosomal S6 kinase (pp90 rsk ) (27,28). A dominant negative mutant of pp90 rsk inhibits PMAinduced but not TNF␣-induced degradation of IB, further suggesting that these two pathways are different. To examine whether PMA has any additive effect on TNF␣-induced degradation, we co-treated HeLa-Off/IG cells with TNF␣ and PMA. The resultant degradation rate is identical to that induced by TNF␣ (Fig. 5). Therefore, there is no synergistic effect of these two inducers on the degradation of IG.
IB␣ is subject to both TNF␣-induced and basal degradation. The basal degradation of IB␣ has been characterized in the presence of CHX. In the absence of de novo protein synthesis, IB␣ exhibits a baseline half-life of 2 h in HeLa cells (2) and 2.5 h in the 70Z/3 murine pre-B cell line (29). To determine whether basal degradation of IG has a half-life similar to that of IB␣, we treated HeLa-Off/IG cells with CHX for 0, 1, 2, 3, and 4 h and collected the cells for analysis by flow cytometry. The time-course fluorescence decay was plotted and is shown in Fig. 6A. The data revealed that the half-life of the fusion in the constitutive degradation is about 2 h, which is similar to that of IB␣. Pretreatment of IG cells with CHX for 1 h decreased the total fluorescence (initial fluorescence intensity of 80) but did not change the degradation patterns of TNF␣ and PMA (Figs. 5 and 6B), suggesting that TNF␣-or PMA-induced degradation of IG is not affected by the basal degradation.
It was unclear whether the basal degradation of IB␣ requires the same phosphorylation events as the TNF␣-induced degradation (30). To examine this, we transiently expressed both IG and IG-S32A/S36A in HeLa-Off cells. The fluorescence intensity of IG, but not IG-S32A/S36A, decreased in the presence of CHX, indicating that the basal degradation of IB␣ also requires the phosphorylation sites (Fig. 6C). Therefore, both basal degradation and induced degradation of IB␣ require the same phosphorylation event to initiate degradation.
To examine TNF␣-mediated degradation of IB␣ at the dif-ferent stages of cell cycle, we pretreated HeLa/IG cells with the following compounds: (a) mimosine that arrests cells at the G 1 phase, (b) excess thymidine at the G 1 -S phase, and (c) nocodazole at the mitotic phase. Induced HeLa/IG cells at the G 1 phase arrested by mimosine did not change the sensitivity of IB␣ to TNF␣, although the fluorescence intensity of the cells at the G 1 phase (initial fluorescence intensity of 111) is lower than that of dividing cells (initial fluorescence intensity of 197) (Fig. 7). Treatment of HeLa/IG cells with excess thymidine did not change the fluorescence intensity, as well as TNF␣-induced degradation of IB␣ (Fig. 7). As at the G 1 phase, HeLa/IG cells at the mitotic phase induced by treatment with nocodazole showed a lower initial fluorescence intensity of 106, but TNF␣induced degradation of IB␣ was slower than that of the dividing cells or the other arrested cells. The half-life of IB␣ is extended to be a few minutes longer. These results suggested that the degradation rate of IB␣ varies at different phases of the cell cycle.

DISCUSSION
The intracellular biochemical changes that mediate NF-B activation are well documented. The key element in the NFB activation pathway that determines NF-B activation is IB␣ phosphorylation and degradation. Recent studies indicate that the kinases that mediate IB␣ phosphorylation are a large complex (18 -22). Analysis of NF-B activation is often monitored by its transcriptional induction of a reporter in vivo, but the time required for the induction is as long as 4 -6 h. Because IB degrades very quickly after stimulation, it would be an ideal target to be monitored for analysis of NFB signaling pathways. We have demonstrated in our previous study (43) that EGFP can be used as a reporter for protein degradation. In   FIG. 3.

TNF␣-mediated NFB localization and NFB-mediated induction of SEAP in HeLa-Off/IG cells.
HeLa-Off/IG cells were treated with TNF for 50 min. NFB translocation was examined with antibodies against p65 and rhodamine-labeled IgG, and IB␣ degradation was examined by green fluorescence (A). HeLa-Off/IG cells were transfected by the reporter construct pNFB-SEAP. 24 h after transfection, the cells were treated with 0.1 g/ml human TNF␣ and collected for SEAP detection (B). SEAP activities of HeLa-Off and HeLa-Off/IG cells were plotted.
the present study, we demonstrated that EGFP can be used for monitoring IB␣ degradation and demonstrated that the IG fusion protein is functionally identical to IB␣. Like IB␣, the IG fusion protein responds to TNF␣ very quickly and specifically. The half-life of IG is 5 min in the presence of TNF␣ and 2 h in the presence of CHX, which agrees with previously described rates of degradation for IB (1, 2, 7, 29). Furthermore, appending EGFP to IB did not change the specific requirement of phosphorylation for initiation of TNF␣-induced degradation. TNF␣-induced degradation of the IG fusion protein is also prevented by the mutations at serine residues 32 and 36 of IB␣. Lastly, both NFB translocation and NFBmediated transcription in IG-expressing cells are the same as those in control HeLa cells, suggesting that the addition of the EGFP tag and the expression of IG in the cells do not change the downstream events in NFB signaling. Therefore, IB␣ degradation can be analyzed simply by monitoring the change NFB can be activated by a number of stimuli, and the activation requires phosphorylation and degradation of IB␣. By monitoring the degradation of IG by flow cytometric analysis, we showed that the degradation cannot be induced by forskolin, indicating that protein kinase A is not involved in the activation of NFB. Our conclusion is different from that of an in vitro study (46) in which translocation of NFB can be induced by protein kinase A. This might result from the two different assay systems used. Protein kinase A induction of NFB translocation may have been an artificial effect caused by assaying in a cellular extract and not in intact cells. In our study, we show that degradation of IG can be activated by protein kinase C via PMA treatment of HeLa-Off/IG cells. We demonstrate that PMA-induced degradation is slower than induction with TNF␣. Although previous studies demonstrated that the activation pathway of PMA is different from that of TNF␣, it was unclear whether these two pathways had any synergistic effect on IG degradation. We show in this study that co-treatment of PMA and TNF␣ results in an identical degradation pattern to that of TNF␣, suggesting that an additive effect of PMA on TNF␣-induced degradation of IG does not exist. One possible explanation for this observation is that the degradation of IG is dominantly mediated by TNF␣.
There are two types of IB␣ degradation: TNF␣-induced degradation, and basal degradation. The basal degradation of IB␣ is also believed to contribute to the regulation of NFB activation. In the absence of TNF␣, IB␣ has a relatively fast turnover rate, with a half-life of 2 h. This turnover is necessary in preventing IB␣ from accumulation. Degradation of IB␣ is stimulated by TNF␣ treatment, and the half-life of IB␣ is greatly shortened to 5-30 min. We have demonstrated for the first time that phosphorylation on serine residues 32 and 36 is needed for both basal and TNF␣-induced degradation. Based on our results, we propose that the signaling mechanisms that initiate basal and TNF␣-mediated degradation of IB␣ are the same, and TNF␣ treatment only leads to an increased rate of basal degradation.
TNF␣-induced degradation of IB␣ has been examined at the different phases of cell cycle. The fluorescence intensity varies at the different phases of cell cycle. It is lower at the G 1 and M phases, possibly due to a slower protein synthesis or a higher basal turnover rate. Our results also indicated that the degradation rate of IB␣ under the treatment with TNF␣ does not change dramatically, except in the cells that are arrested at the mitotic phase by nocodazole. We found that the degradation rate of IB␣ is slower at the mitotic phase than at the G 1 or S phase. For degradation to occur, IB␣ must be phosphorylated at serines 32 and 36. Only this phosphorylated protein can be degraded by the 26S protease. During M phase, the phosphorylation cascade may be altered or impeded, resulting in the impaired degradation of IB␣. Secondly, the slower degradation could be due to inactivation of the degradation machinery. The second scenario is unlikely, because some proteins, such as cyclin B1, use the same machinery and degrade well in the same phase. The third possibility can be due to the existence of an inhibitory protein that is released from nucleus in the mitotic phase and prevents IB␣ degradation. Additional studies need to be done to distinguish the possibilities.
Many regulatory proteins are short-lived. Rapid turnover provides a possible means to regulate their protein levels. Degradation of these proteins can be detected by the methods described here, which avoid complicated biochemical analysis, such as pulse-chase immunoprecipitation, and substitute a simple fluorescence assay. Because emission of EGFP does not require any cofactors or substrates, detection can be made in real time, and the data can be collected at multiple time points. Therefore, the method provides simple and quick detection of protein degradation in living cells. It can facilitate the analysis of a large number of samples, such as in high throughput screening.