TAB-1 Modulates Intracellular Localization of p38 MAP Kinase and Downstream Signaling*

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1β-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.

Stress-activated protein kinase p38 is a member of a highly conserved subfamily of mitogen-activated protein (MAP) 2 kinases involved in a wide variety of stress responses in organisms ranging from yeast to mammals (1)(2)(3)(4). Activation of the p38 pathway is achieved by a cascade of phosphorylation events involving proximal upstream MAP kinase kinases, such as MKK3 and 6, and further upstream MAP kinase kinase kinases, such as TAK1 and ASK1 (3). Phosphorylation of both threonine and tyrosine at a conserved TGY motif leads to p38 kinase activation. This phosphorylation is therefore used widely as a biochemical marker for p38 activation status (5,6). Phosphorylated (activated) p38 phosphorylates a large number of transcription factors that include NFAT, CHOP, p53, ATF-2, and MEF-2 (3,4) to regulate gene expression. Through downstream kinases, such as MAP kinase-activated protein kinase (MAPKAP)-2/3 (MK2, MK3), MNK, and MSK1, p38 activity is also responsible for the phosphorylation of additional downstream targets that include cytosolic PLA2, heat shock proteins, and histone3/ HMG-14 (3,4).
p38 activation plays a critical role in the regulation of pro-inflammatory genes, including TNF␣ (7,8) and COX-2 (9 -12) in mammalian cells. The onset of heart failure is tightly associated with an elevated inflammatory response, both in human failing hearts arising from a wide variety of etiologies and in several animal models of heart failure (13,14). In addition, p38 activation is also observed in heart in a variety of pathological conditions, including mechanical stimulation, neural-hormonal stimulation, and cardiac ischemia injury (15)(16)(17). Therefore, it has been speculated that p38 activation and subsequent inflammatory induction contribute to pathological changes in the process of heart failure (18). Our recent studies also suggest a direct contribution of p38 activity to inflammatory induction, cardiac dysfunction, and pathological remodeling in heart (19,20).
In addition to MKK-dependent p38 kinase activation, an alternative pathway leading to p38 activation involving TAB-1 (TAK-1-binding protein) was recently identified (21). Although TAB-1 was originally found to interact with and activate an upstream MAP kinase kinase kinase, TAK-1, Ge et al. (21) showed that TAB-1 can also directly bind to p38 and promote SB203580-sensitive, but MKK-independent, p38 autophosphorylation. Recently, Tanno et al. (22) also implicated TAB-1 in p38 activation in ischemic mouse hearts with homozygous MKK3 null alleles, whereas Li et al. (23) showed an increase in TAB-1 recruitment by p38 in ischemia heart in response to activated AMP-activated protein kinase. Furthermore, TAB-1 expression induces p38␣ activation leading to IL-10 induction and ERK and IL-2 inhibition in anergic T-cells (24). Although TAB-1 is sufficient to activate p38 kinase in vitro (21) and is involved in p38 activation in cardiomyocytes and T-cells, other recent studies also suggest that TAB-1 might function as a negative feedback regulator between p38 and TAK-1 (25,26). Although increasing evidence suggests that TAB-1 participates in p38 signaling, the downstream effects of TAB-1-mediated p38 activity in stress signaling and gene regulation are unclear, particularly in comparison with MKK-induced p38 function.
In this report, we demonstrate that TAB-1 expression in cultured neo-natal cardiac myocytes is sufficient to induce p38 activity via autophosphorylation. However, TAB-1-mediated p38 activity does not lead to the classical downstream effects of p38 induced by MKK3 activation. In contrast, TAB-1 expression attenuates MKK3-induced downstream signaling, at least in part via competitive binding with p38, removal of p38 from the MKK3 signaling complex, and translocation of p38 to alternative intracellular compartments. Consequently, TAB-1 negatively modulates IL-1␤induced (MKK3/p38-mediated) inflammatory gene expression in cardiomyocytes. In contrast, knockdown of TAB-1 expression in wild-type MEF cells augments TNF␣-induced COX-2 expression. These data clearly suggest that TAB-1 not only induces p38 kinase activation but also has an important role in modulating downstream effects, at least in part by altering p38 intracellular localization and complex interaction. Thus, this study reveals a novel mechanism of stress-MAP kinase regulation and demonstrates that downstream effects of p38 activation can be modulated by different upstream activators to yield distinct intracellular localization and complex interaction.
Cell Culture-Neonatal ventricular cardiomyocytes from 1-2-dayold Sprague-Dawley rats were isolated using a Percoll gradient method as described previously (28). Cardiomyocytes were plated overnight in medium containing Dulbecco's modified Eagle's medium/medium 199 (4:1) supplemented with 10% horse serum, 5% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 10 mM glutamine. Subsequently, cardiomyocytes were infected with adenoviruses at MOI between 10 and 100 (optimized by protein expression level of the transgene) and incubated for 48 h in serum free medium supplemented with 1% ITS (BD Biosciences). p38-specific inhibitor SB203580 (Calbiochem) or IL-1␤ (BD biosciences) was added into the culture medium, either 2 or 12 h after adenovirus infection, as indicated in specific experiments. HEK293, Cos-1, and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. Wild-type MEF cells (C57Bl/6 background) were maintained in highglucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum. MEF cells were seeded 1 day before transfection, and 3-5 g of DNA was mixed with Lipofectamine 2000 (Invitrogen) in 200 l of Opti-MEM (Invitrogen) for 30 min before the addition to the cells. After 24 -48 h, the cells were incubated in a medium containing 100 g/ml of hygromycin and further cultured for 2 weeks and pooled.
Immunostaining and Fluorescence Microscopy-Cardiomyocytes and Cos-1 cells were cultured on 12-mm coverslips coated with 10 g/ml Laminin (Invitrogen). 48 h after adenoviral infection, the cells were washed with PBS, fixed for 5 min with 4% paraformaldehyde, permeablized for 5 min with 0.2% Triton X-100, and then blocked in PBS for 1 h with 3% bovine serum albumin and 5% donkey serum. The cells were then stained with primary antibodies and secondary antibodies diluted in 3% bovine serum albumin and 1% donkey serum in PBS for 2 h, respectively. The primary antibodies used were rabbit anti-rat ANF antiserum (Peninsula Laboratory) (1:1000), rabbit anti-HA polyclonal antibody (Santa Cruz) (1:1000), and mouse anti-FLAG M2 monoclonal antibody (Sigma) (1:5000). Secondary antibodies include Alexa568-conjugated donkey anti-rabbit IgG, Alexa568-conjugated donkey antimouse IgG, and Alexa640-conjugated donkey anti-rabbit IgG (Molecular Probes). F-actin was probed with fluorescein isothiocyanateconjugated phalloidin (Molecular Probes). Fluorescence images were obtained with a laser scanning confocal microscope (Olympus Fluoview) and analyzed with MetaMorph (Universal Imaging Corp.) and Auto Deblur (AutoQuant). The co-localization and the proximity of proteins were analyzed using custom made software described in Fig. 7.

TAB-1 Induces p38 Activity via Autophosphorylation in Cultured Rat
Myocytes-Rat neonatal ventricular cardiomyocytes (RNVC) have very low level expression of endogenous TAB-1, based on Western blotting. They have an abundant level of p38␣ protein, mostly in inactive form under basal condition (Fig. 1). These cells present a good model system to investigate the functional effect of TAB-1 on p38 kinase signaling. We expressed human wild-type, full-length TAB-1 in rat neonatal cardiomyocytes via a recombinant adenovirus vector. TAB-1 expression leads to significant activation of the p38 kinase, as determined either by antiphospho-p38 immunoblotting or by ATF-2 phosphorylation activity of p38 immunocomplexes (Fig. 1). p38 phosphorylation is partially blocked by p38 kinase-selective inhibitor SB203580 (Fig. 1A), in good agreement with previous observations suggesting that TAB-1-mediated p38 activation involves autophosphorylation (21). A splicing variant of TAB-1 (TAB-1␤) containing both the p38-binding domain and the protein phosphatase 2C (PP2C)-like domain but lacking the TAK-1-binding domain (27) also activates p38 kinase as reported (21). All of these results suggest that TAB-1 induces bona fide p38 kinase activation in a SB203580-sensitive manner via autophosphorylation.
TAB-1 Does Not Induce p38 Downstream Signaling Events Associated with MKK3-p38 Activation-Expression of MKK3bE, an activated mutant of the p38 upstream activating kinase, in RNVC cells results in significant activation of p38 kinase activity as determined by phospho-p38-specific Western blot ( Fig. 2A) (28). As a consequence, TNF␣ and COX-2 expression are induced at both the protein and mRNA levels (Fig. 2, A and B). In addition, cardiac ANF gene expression is induced, and myofilament organization is enhanced as part of the p38-mediated stress response as reported previously (28) (Fig. 2, C and D). In contrast, TAB-1 expression in RNVC cells does not activate any of these well established p38 downstream responses or target gene induction in cardiomyocytes, despite the fact that comparable levels of p38 activation by MKK3bE and TAB-1 are achieved (Fig. 2).   MARCH 3, 2006 • VOLUME 281 • NUMBER 9 TAB-1 Antagonizes MKK3-mediated p38 Downstream Signals-We further investigated the impact of TAB-1 expression on the downstream signaling of p38 kinase in RNVC cells. As shown in Fig. 3A, MKK3bE expression induces phosphorylation of downstream kinase MAPKAPK-2 (MK2) and HSP27 as expected and SB203580 (10 M) significantly reduces MK2 and HSP27 phosphorylation without affecting the total p38 phosphorylation level (Fig. 3A). In contrast, TAB-1 expression does not result in any significant MK2 or HSP27 phosphorylation (Fig. 3B), suggesting that TAB-1-mediated p38 activation does not induce previously characterized downstream signaling. Furthermore, co-expression of TAB-1 with MKK3bE significantly reduces MK2 and HSP27 phosphorylation, without reducing the total level of phosphorylated p38 (Fig. 3B). TAB-1 also attenuates MKK3bE-induced COX-2 expression (Fig. 3C), further supporting the notion that TAB-1 antagonizes MKK3-dependent p38 downstream activity.

TAB-1 Modulates p38 Localization and Function
We also investigated whether TAB-1 binding to p38 was required for the observed antagonistic activity toward MKK3-induced downstream signaling. When co-expressed in RNVC cells, the full-length TAB-1 and C-terminal fragment (333-504) of TAB-1 can be readily detected in immunocomplexes with p38␣ following immunoprecipitation using anti-p38 antibodies (Fig. 4A). In contrast, no binding activity can be detected with the TAB-1 N-terminal fragment (residues 1-373), which contains a PP-2C-like domain, confirming the previous finding (21) that the binding motif for p38 is located in the C-terminal domain of TAB-1. Expression of the TAB-1 C-terminal (residues 333-504) fragment shows no ability to induce p38 phosphorylation but does retain potent inhibitory activity for MKK3bE-induced phosphorylation of MK2 and HSP27 (Fig. 4B). In contrast, expression of the TAB-1 N-terminal (1-373) fragment has neither activity for p38 phosphorylation nor any impact on downstream signaling in response to activated MKK3bE (Fig.  4B). These results suggest that, although both N-and C-terminal domains of TAB-1 are required to activate p38 kinase activity, only the C-terminal TAB-1 containing p38-binding domain is both necessary and sufficient to antagonize MKK3-mediated p38 downstream signaling.
TAB-1 Binds to p38 and Excludes p38 from the Nucleus-To determine why the TAB-1/p38 interaction down-regulates MKK3-induced downstream signaling, we investigated the effect of TAB-1 on the intracellular localization of p38 kinase. For these experiments, we used a p38-GFP fusion protein as a reporter. In RNVC cells, p38-GFP and HA-MKK3bE are located both in nuclei and in specific reticular patterned structures in the cytosol (Fig. 5A, panels a and b). In contrast, a TAB-1-RFP fusion protein is detectable exclusively in the cytoplasm but not in the nucleus (Fig. 5A, panel c). Co-expression of HA-MKK3bE and p38-GFP demonstrates extensive overlapping of their intracellular distribution in both cytosol and nuclear compartments (Fig. 5A, panels d-f). In contrast, co-expression of TAB-1-RFP with p38-GFP excludes p38-GFP from the nucleus and retains the kinase only in the cytosol (Fig. 5A, panels  g-i). This result suggests that TAB-1 is a cytosol localized protein and that TAB-1 expression can alter the intracellular location of p38.
To further demonstrate that TAB-1 can directly modulate p38 intracellular distribution, we generated nucleus-targeted mutants of TAB-1 (TAB-1-NLS-FLAG) and the TAB-1(333-504) C-terminal fragment (TAB-1(333-504)-NLS-FLAG) by adding a nuclear localization signal at the C-terminal of the coding regions (Fig. 5B). The nucleus-targeted TAB-1 and the TAB-1 C-terminal fragment retain p38 binding activity in cells, as determined from co-immunoprecipitation assays (Fig. 5C) and are effectively targeted to nuclei in cardiomyocytes (Fig. 5D, panels b and c). Coexpression of p38␣-GFP with either nucleus-targeted TAB-1 or the nucleus-targeted TAB-1 C-terminal fragment results in nearly complete co-localization of p38 and TAB-1 in the nucleus (Fig. 5D, panels d-i). Despite their ability to target p38 into nucleus, both proteins fail to induce p38 downstream signaling, as measured by MK2 phosphorylation HSP27 phosphorylation or COX-2 expression (Fig. 5E). These data demonstrate clearly that (i) the wild-type TAB-1 protein is exclusively localized in the cytosol, (ii) TAB-1 can modulate the subcellular localization of p38 via direct interaction, and (iii) TAB-1 is not sufficient to activate p38 downstream, even if it is targeted to the nucleus.
TAB-1 Modulates p38 Intracellular Distribution and the Interaction of p38 with MKK3-The above results suggest that TAB-1 not only modulates p38 intracellular localization but also changes the MKKmediated downstream effects of p38. To investigate the underlying mechanism, we analyzed the effects of TAB-1 expression on the protein-protein interaction between p38 and its upstream activating kinase MKK3. Co-immunoprecipitation analysis demonstrates that TAB-1 expression significantly reduces p38 binding to MKK3 in COS-1 cells (Fig. 6). Supporting this finding, intracellular localization of MKK3bE and p38␣ were analyzed by immunofluorescent confocal microscopy (Figs. 7, panels a-f). The protein proximity index analyses of these images (Fig. 7, panels m and n) indicate a highly significant degree of overlap between MKK3bE and p38␣-GFP proteins (Fig. 7, panel q). However, in the presence of TAB-1, p38 is retained in the cytosol where its co-localization with MKK3 is significantly disrupted (Fig. 4B, panels  g-l). The MKK3/p38 protein proximity index changes from 0.96 Ϯ 0.02 to 0.59 Ϯ 0.04 (mean Ϯ S.E., n ϭ 6, p Ͻ 0.001) when TAB-1 is coexpressed (Fig. 7, panel q). As shown more clearly in the high magnitude images (Fig. 7, panels d-f and j-k), p38␣-GFP and MKK3bE both have a reticular distribution pattern in the cytosol. However, in the presence of TAB-1, the degree of overlap is substantially reduced (illustrated by the appearance of red and green areas). Therefore, both immunoprecipitation and co-localization analysis provide clear evidence that TAB-1 directly competes with MKK3 for p38 protein binding and restricts p38 intracellular distribution in the cytosol to a specific compartment away from the MKK3 kinase. All of these observations, along with previous functional studies, suggest a previously unknown mechanism in p38 kinase regulation in which different upstream activators can dictate the downstream signaling of p38 kinase by modulating its intracellular localization and complex interactions.
TAB-1 Modulates Cytokine-induced Inflammatory Gene Expression-p38 is known to mediate cytokine-induced inflammatory gene expression, such as COX-2, in cultured cardiac myocytes (12). To investigate the functional significance of TAB-1/p38 interaction in inflammatory gene regulation, we determined the effect of TAB-1 p38 binding activity on IL-1␤ induced COX-2 expression. As shown in Fig. 8, IL-1␤ potently induces COX-2 expression in cultured myocytes infected with a control AdvGFP vector. In contrast, COX-2 induction is significantly attenuated by the TAB-1 C-terminal fragment (333-504) (Fig. 8), which contains only the p38-binding motif but lacks p38 autophosphorylation activity (Fig. 4). The ability of TAB-1⅐p38 complex to inhibit COX-2 expression is correlated with an inhibition of MK2 phosphorylation. However, the level of total p38 activity, as measured by p38 phosphorylation, is not affected.
To further investigate the functional role of TAB-1 in cytokine-mediated signaling, we studied mouse embryonic fibroblast in which significant endogenous TAB-1 is present (Fig. 9A). Two MEF cell lines are established using different small interfering RNAs against endogenous TAB-1 mRNA. As shown in Fig. 9A, TAB-1 protein expression is significantly lowered in both small interfering RNA-treated MEF cell lines. TNF␣-induced COX-2 expression is augmented in TAB-1 knockdown MEF cells compared with the control cells (Fig. 9B). Similar results are observed using anisomycin as an alternative p38 activator (data not shown). Therefore, both gain-of-function and loss-of-function studies support a potentially important role for TAB-1 as a signaling modulator in inflammatory response.

DISCUSSION
Stress-activated MAP kinase p38 is a highly conserved signaling molecule responsible for a variety of stress responses in different mammalian cells. Like other MAP kinase family members, the p38 kinase catalytic activity is activated by upstream MAP kinase kinases, including MKK3, MKK6, and, with lesser specificity, by MKK4 (3,6). p38 activation is implicated in many critical cellular functions, including gene regulation, apoptosis, energy metabolism, cellular differentiation, and proliferation, presumably via an array of downstream target molecules (4). However, it is unclear how these diverse downstream signaling pathways are regulated in response to a variety of different stimuli that activate p38 kinase activity. Our data suggest that TAB-1 is not only a MKK3-independent activator of p38 MAP kinase but is also a potent modulator of p38 intracellular localization and p38 signal complex formation in the cytosol. By competing with MKK3 for p38 binding, TAB-1 antagonizes MKK3-mediated activation of downstream kinases and, consequently, inflammatory gene induction. Our observations demonstrate that the differential intracellular localization of activated p38 MAP kinase can be dictated by two different upstream molecules, MKK3 versus TAB-1, and result in dramatically different downstream consequences. Thus, the modulating effect of TAB-1 may offer a potentially important molecular mechanism contributing to the fine tuning and the functional diversity of p38 pathways in mammalian cells.
Our study redefines the role of TAB-1 in p38 regulation. TAB-1␤, a splicing variant of TAB-1 lacking the TAK-1-binding motif (27), has the same capacity as full-length TAB-1 to activate p38 kinase activity in cardiomyocytes. Truncated N-and C-terminal portions of TAB-1 that lack either the p38-binding domain or PP2C-like domain do not activate p38 kinase activity. Therefore, the p38 MAP kinase-binding motif and the PP2C-like domain of TAB-1 are both required for p38 activation in cardiomyocytes as reported (21). In contrast, the C-terminal portion of TAB-1(333-504) containing the p38-binding motif is sufficient to retain p38 in the cytosol, dictates the intracellular distribution of p38, and inhibits MKK3-mediated signaling. In the cytosol, TAB-1 and MKK3 direct p38 into different intracellular compartments with distinct localizations. Therefore, TAB-1 can function both as an activator

TAB-1 Modulates p38 Localization and Function
and a tethering factor for p38 kinase. Activation of p38 by TAB-1 requires both p38 binding and PP2c-like domain of TAB-1, whereas tethering requires only the C-terminal domain containing the p38binding motif. The potent inhibitory function of TAB-1 (333-504) for MKK3-mediated signaling indicates clearly that TAB-1/p38 interaction is both necessary and sufficient to modulate p38 downstream signaling. It seems likely that TAB-1-mediated changes in p38 kinase cellular localization contribute to the previously reported negative feedback function for TAB-1 in p38 signaling (25). A significant number of scaffold proteins have been identified for MAP kinase signaling cascades, all of which possess the hallmark of interacting with multiple components of the kinase signaling complex (29). Some of them, including KSR, MP1, JIP-1, and JIP2, can target MAP kinase complexes to specific subcellular locations, such as plasma membrane, late endosome, or kinesin cargo (transport vehicles) (30 -33). However, unlike TAB-1, none of them has an intrinsic activation function toward the targeted protein kinases.
In addition, local signaling complex interaction appears to be critical to achieve spatio-temporal regulation of other protein kinases, including cAMP-dependent protein kinase, protein kinase C, and tyrosine kinases, as revealed by genetic fluorescent probes (34 -38). Indeed, nuclear localized TAB-1 fails to activate p38 downstream signaling, suggesting that TAB-1 and MKK form different signaling complexes for p38. TAB-1 appears to be a unique signaling molecule that functions both as an upstream MKK-independent activator of p38 activity and as a scaffold protein that modulates the intracellular localization and signal complex interaction of the activated p38 kinase.
TAB-1 gene inactivation leads to an embryonic lethal phenotype with impaired TGF-␤ signaling and cardiovascular defects (39), suggesting a FIGURE 6. TAB-1 inhibits p38␣ interaction with MKK3. TAB-1 reduces MKK3 binding to p38. COS-1 cells were infected with adenoviruses expressing HA-MKK3bE, TAB-1-RFP, or a kinase dead mutant of p38␣ (FLAG-dnp38␣) as indicated. Immunoprecipitation (IP) was performed using M2 anti-FLAG antibody followed by immunoblot for HA, TAB-1, or p38 as indicated. WB, Western blot.  MARCH 3, 2006 • VOLUME 281 • NUMBER 9 vital role of TAB-1 in cardiovascular development and function. However, the specific role of TAB-1 in p38-mediated function versus TGF-␤/TAK-1 signaling in heart or other cell systems is unknown. Tanno et al. (22) demonstrated that TAB-1 binding to p38 was induced in ischemic hearts but not in TNF␣-treated hearts, suggesting that different mechanisms are involved in p38 activation under different stress conditions. Li et al. (23) also recently reported some interesting observations on AMP-activated protein kinase-mediated p38 activation that involves TAB-1. The data from this study indicate that these different mechanisms of p38 activation would lead to distinct downstream signaling events. Although a negative modulating role of TAB-1 for MKK3-mediated signaling has been clearly demonstrated in our study, the selective downstream effect of TAB-1-mediated p38 activity in the cytosol remains unclear and requires further studies. Given the diverse roles of p38 in various cellular functions, it is conceivable that TAB-1mediated p38 activity is responsible for a specific subset of these activ-FIGURE 7. TAB-1 disrupts p38␣⅐MKK3 complex in cells. TAB-1 disrupts p38 and MKK3 complex in cytosol. COS-1 cells were infected with GFP-p38␣ and HA-MKK3bE without (panels a-f) and with TAB-1-RFP (panels g-l). p38␣ and MKK3bE were visualized by GFP and anti-HA labeling, respectively. THE right panels are the corresponding overlays of the left and center panels. Panels d-f and j-l are magnified regions of the squares in panels a-c and g-i, respectively. The images were restored with three-dimensional deconvolution, and the degree of association between p38␣ and MKK3bE was quantified in the absence (panels m and n) and presence (panels o and p) of TAB-1. For the quantification of protein proximity index of p38 versus MKK3 and vice versa, cell planes excluding the nucleus were divided in 2 ϫ 2-m squares by a grid, and the squares of p38 and MKK3 images with values larger than 0.5 of the total image intensity were selected. The correlation coefficient and its statistical significance (P) of each square were calculated after thresholding to 0.25 of its maximum intensity. The protein proximity index was calculated from (number of squares with positive correlation coefficient and p Ͻ 0.05)/(total number squares). Panels m-p show distribution histograms of square correlation coefficient (panels m and o) and P SIGN VALUE (Ref. 40) correlation coefficient plots (panels n and p) in the absence (panels m and n) and presence of TAB-1 (panels o and p) In these bar graphs (panels m and o), red areas indicate significant positive correlation coefficient. Note the reduction of the red area by TAB-1, which is quantified in panel q. Panel q shows quantitative values of protein proximity index (mean Ϯ S.E., n ϭ 6) for p38 versus MKK3 (green) and MKK3 versus p38 (red) without TAB-1 (no pattern) or with TAB-1 (diagonal line pattern). From 10 to 20 planes were analyzed for each cell and total of six cells were analyzed from each treatment group.

TAB-1 Modulates p38 Localization and Function
ities. In fact, early studies from our laboratory and from others have demonstrated a potent negative inotrophic effect of p38 activity on cardiomyocyte contractility (19) and cytokine release (20) in addition to gene regulation. Clearly, although our study implicates a negative modulatory role for TAB-1 in the p38 kinase pathway, further studies are needed to identify the specific downstream effect of TAB-1-induced p38 kinase activity and its physiological implication in normal and pathological stress responses.