Immunity, Vol. 11, 411-421, October, 1999, Copyright ©1999 by Cell Press


Live Cell Fluorescence Imaging of T Cell MEKK2:

Redistribution and Activation 4 Response to

Antigen Stimulation of the T Cell Receptor


Brian C. Schaefer,1,7 Margaret F. Ware,2

Philippa Marrack,1,3,4,5Gary R. Fanger,2

John W. Kappler,1,6Gary L. Johnson,2,6

and Collin R. F. Monks2

1Howard Hughes Medical Institute

and Department of Medicine

2Program in Molecular Signal Transduction

Division of Basic Sciences

National Jewish Medical and ResearchCenter

Denver, Colorado80206

3Departments of Biochemistry and Molecular Genetics

4Department of Immunology

5Department of Medicine

6Department of Pharmacology

University of ColoradoHealthSciencesCenter

Denver, Colorado80220




T cell activation requires engagement of the T cell receptor (TCR) at the interface of conjugates formed with antigen-presenting cell. TCR engagement is accompanied by a redistribution of specific signaling molecules to the cytoplasmicre9on of the TCR complex. In this study, immunocytochemistry and live cell fluorescence imaging demonstrate that T cell MEK kinase 2 (MEKK2) is translocated to the T cell/antigen presenting cell interface in response to antigen activation. MEKK2 translocation occurs more rapidly as the antigen concentration is increased. Biochemical activation of MEKK2 follows TCR stimulation, and expression of a dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK and p38 MAP kinase phosphorylation. Live cell fluorescence imaging thus enables characterization of signal transducers that are dynamically translocated following TCR engagement.




MEK kinases (MEKKs) are a family of serine-threonine kinases that have been shown to regulate mitogen-acti-vated protein kinase (MAPK) cascades, including the ERK, p38, and JNK pathways (Fanger et al., 1997 ;Widmann et al., 1999). In MAPK cascades, the MEKKs function as MAPK kinase kinases (MAPKKKs) that activate

a MAPK kinase (MAPKK), and the MAPKK then activates a MAPK. Many MAPKKKs, including the MEKKs, are capable of activating more than one MAPK pathway. Additionally, most cell types express multiple MAPKKKs, which, in transfection/overexpression experiments, often appear to be functionally interchangeable (Widmann et al., 1999). However, emerging evidence suggests that MAPK cascades are physically compartmentalized by scaffolding proteins that group specific MAPK signal transducers (Schaeffer et al., 1998;Whitmarsh et al., 1998). Such subcelluar organization may thus provide a means by which a receptor may be coupled to a specific MAPKKK, MAPKK, and MAPK.

        The transduction of signals from the T cell receptor (TCR) involves multiple cascades of many different signaling proteins that activate diverse targets, including MAPKs. Increasing evidence is accumulating that these signaling events are accompanied by a redistribution of some or all of the participating molecules into discrete, macromolecular signaling units. In T cells, antigen stimulation results in clustering of multiple cell surface molecules into discrete domains (Boniface et al., 1998 ;Monks et al., 1998;Wulfing and Davis, 1998; Wulfing et al, 1998;Penninger and Crabtree, 1999) and the concomitant translocation of signaling molecules to the cytoplasmic face of the clustered receptors (Monks et al., 1997).

Such protein redistribution represents a consequence of active signaling that can be visualized in fixed cells using immunocytochemistry (Monks et al., 1997, 1998). Redistribution in response to a specific signal provides evidence that the redistributed protein plays an active role in transducing that signal.

It is therefore possible to use immunocytochemistry as a screening method to determine if a given protein is likely to be involved in the response to a signal by assessing its redistribution. A recently developed tool for studies of this type is green fluorescent protein (GFP). which can be used as a covalently bound tag to allow the characterization of dynamic protein redistribution in single cells in real time.

Thus, analysis of the subcellular redistribution of MAPK signal transducers in response to the ligation of the TCR should provide an accurate means of determining whether a particular MAPK transducer is involved in the delivery of antigen-mediated signals.

We have used digital fluorescence imaging of fixed and live T cell Clones to determine whether MEKKs are involved in transducing antigen-dependent Signals in T cells. We have found that endogenous T cell MEKK2, but not MEKK1 or MEKK3, translocates to the cyto-plasmic face of the interface with antigen-loaded pre-senting cells. Experiments employing an MEKK2-GFP fusion protein show that translocation of MEKK2 occurs within seconds of exposure to presenting cells loaded with a highly stimulatory dose of antigen. However, as antigen becomes limiting, translocation of MEKK2-GFP does not occur until several minutes after initial contact with the presenting cell. MEKK2 is activated by TCR stimulation, and biochemical data indicate that MEKK2 activates T cell adhesion, as well as the ERK and p38 MAPK cascades. However, MEKK2 does not appear to directly regulate IL-4 production in response to antigen stimulation. Although a complete pathway for TCR-mediated MEKK2 activation remains to be defined, ex-perimental evidence suggests that PI3-Kinase may be an upstream modulator of MEKK2 activation. MEKK2 is thus a newly identified transducer of TCR signals that participates in the activation of specific antigen-regu-lated pathways.




Translocation of T Cell MEKK2 in Response

to Antigen Stimulation

Because MAPK cascades are known to be involved in multiple aspects of T cell activation (Cantrell, 1996), ex-periments were performed to determine if MEKK family members are involved in early T cell signaling. Conju-gates between T cells and antigen-pulsed presenting cells were assayed at early timepoints for MEKK protein redistribution via digital immunofluorescence microscopy (Monks et al., 1997, 1998). The D10 T cell clone, which reacts with I-AK plus a peptide from conalbumin (Kaye et al., 1983), was incubated with l-AK-bearing CH12.LX B cells (APC) that had been either untreated or pulsed overnight with conalbumin. The T cell/APC conjugates that formed were fixed and stained for either MEKK1 or MEKK2 and digitally imaged. Figure 1 A shows that MEKK2 in the T cell translocated to the contact site between the T cell and APC, providing the APC had been pulsed with antigen, whereas MEKK1 remained distributed equally throughout the T cell cytoplasm. To verify these data, D10 T cells were infected with a retrovirus coding for MEKK2 fused in frame to green fluorescent protein (MEKK2-GFP), and a polyclonal cell line was established. Detection of GFP allowed assessment of the distribution of the MEKK2-GFP fusion protein in live cells. The D10/MEKK2-GFP line was incubated with conalbumin-loaded APC. T cell/APC conjugates were visualized by time lapse digital microscopy. Figure 1B shows that MEKK2-GFP rapidly translocated to the T cell APC interface and persisted for at least 30 min (Figure 1B). As shown in Figure 1C, MEKK2-GFP the APC were not pulsed with the specific antigen. Similar data were obtained when primary, conalbuminpulsed l-AK-bearing B cells were used (data not shown). The amount of MEKK2-GFP protein that redistributed to the T cell/APC interface in response to antigen stimulation was quantified (Figure 1 D). Approximately 9% of total cellular MEKK2 is translocated to the cytoplasmic region of the T cell/APC contact, which represents ap-proximately 1.25% of the total T cell volume. Our obser-vations indicate that the amount of MEKK2 translocated does not vary appreciably with antigen concentration (data not shown). Consistent with the observations in Figure 1C, no significant enrichment is observed in the absence of antigen. Taken together, these data demon strate that the MEKK2-GFP fusion protein behaves like endogenous MEKK2 in the D10 cells and that engage-ment of the TCR on T cells causes MEKK2 within the T cells to move rapidly and transiently to the interface  between the T cells and the APC, the site where the TCR is capped (Kupfer and Singer, 1989).


Imaging experiments were also performed on a polyclonal D10 T cell line expressing a kinase-inactive form of MEKK2, which, as shown in the experiments below, acts as a dominant-negative mutant (dnMEKK2-GFP). interestingly, dnMEKK2-GFP does not translocate to the T cell/APC contact site in response to antigen stimula- tion (Figure 1C), indicating that an active kinase domain is required for efficient MEKK2 translocation. To further investigate the translocation phenomenon, a third poly-clonal D10 T cell line was established that expresses an MEKK3 fusion protein (MEKK3-YFP). The kinase do-mains of MEKK2 and MEKK3 are highly homologous (94%), whereas their N-terminal domains are less related (65% homology) (Blank'et al., 1996). The observation that MEKK3-YFP does not redistribute in response to antigen stimulation (Figures 1C and 1D) suggests that unique sequences in the N terminus of MEKK2 are also required for translocation. Translocation in response to antigen stimulation thus appears to be a very specific property of MEKK2, since endogenous MEKK1 and ret-rovirally expressed MEKK3-YFP do not redistribute in D10T cells


The Kinetics of MEKK2 Translocation Are Depe-ndentupon Antigen Concentration

To establish how antigen concentration affects the ki-netics of MEKK2 translocation, live cell imaging experi-ments were performed on conjugates of D10/MEKK2-GFP T cells and APC, which had been pulsed overnight with various concentrations of antigen. As shown in Fig-ure 2A, translocation of MEKK2-GFP occured within 10 sec after exposure of T cells to APC that were loaded with 500 μg/ml conalbumin. At 20 μg/ml conalbumin, translocation was delayed, requiring approximately 40 sec following initial APC contact. When antigen concen-tration was lowered to 4 μg/ml, there was a delay of approximately 3 to 5 min before MEKK2-GFP transloca-tion was observed. To further support the live cell im-aging data, T cell/APC conjugates were synchronously initiated by brief centrifugation and were fixed 10 or 60 min later. Based on the data in Figure 2A, we expected that almost all conjugates at high antigen concentration (500 μg/ml) would also have translocated MEKK2-GFP But that this percentage would diminish as the antigen. Concentra-ion was lowered, and that no translocations would be observed in the absence of antigen. This is in fact what was observed (Figure 2B). Significantly, the most substantial drop in translocation percentage occured between 4.0 and 0.8 μg/ml conalbumin, which is the range of concentration in which antigen becomes limiting for functional responses (see Figure 4A).

At 60 min, the percentage of conjugates with MEKK2 translocations was generally less than at 10 min, suggesting that the enrichment of MEKK2 at the T cell/APC interface may be reversed before conjugate dissociation. This observation may suggest that MEKK2-GFP translocation is of shorter duration at lower antigen con-centration (Figures 2A and 2B). Although some of our live cell images seem to support the idea that MEKK2 translocation is transient, cell movement during imaging experiments of this duration complicates their interpre-tation. The data in Figures 2A and 2B do, however, strongly suggest that the rate and duration of MEKK2-GFP translocation is directly related to the number of Specific antigen/MHC complexes present on the APC.


TCP-Dependent Activation of MEKK2

To determine whether T cell MEKK2 is activated by TCR engagement, the kinase activity of the MEKK2-GFP fusion protein in the D10/MEKK2-GFP line was tested using a GST-SEK1 substrate. Figure 3A shows that the kinase activity of  MEKK2-GFP from D10 cells that had been incubated with fixed, conalbumin-pulsed APC was 4x higher than that of  MEKK2-GFP isolated from D10 cells incubated without APC or with fixed APC that had



Figure 1. Translocation of T Cell MEKK2 in Response to Antigen-Loaded APC

(A) D10 T cells were mixed with APC loaded with no antigen (no ag) or with 500 μg/ml conalbumin for 10 min at 37. Conjugates were bound to coverslips, and MEKK2 and MEKK1 were detected by immunofluorescence microscopy using either rabbit anti-MEKK2 or rabbit antiMEKK1 antibodies and a Cy3-conjugated anti-rabbit secondary reagent. The upper panels are Nomarski views of the same conjugates shown in the lower panels, which were imaged by immunofluorescence and digitally deconvolved. In each panel, the APC is the upper cell and the D10 T cell is the lower cell.

(B) An MEKK2-GFP retrovirus was used to introduce an MEKK2-GFP fusion protein into D10 T cells. D10/MEKK2-GFP T cells were mixed with equal numbers of conalbumin (500 μg/ml)-loaded APC, and real-time fluorescent images were acquired. Exposures of 10 sec each were acquired at 30 sec intervals over a period of 36 min. Selected panels at the indicated timepoints are shown (APC and T cell are same orientation as in [A]). Three similar series can be viewed as Quicktime movies at figs.html. Some of these additional images show that multiple interactions result in multiple translocations, demonstrating that the observed translocations do not simply represent reorientation of the golgi and MTOC.

(C) D10 T cells expressing MEKK2-GFP. dnMEKK2-GFP, or MEKK3-YFP were mixed for 10 min at 37 with APC that had been loaded overnight with no antigen (no ag) or with 500 μg/ml conalbumin. Conjugates were bound to coverslips, fixed, and imaged as described in the Experimental Procedures. Upper panels are Nomarski images, and bottom panels are digitally deconvolved fluorescence images.

(D) Conjugates prepared as described in (C) were imaged in 0.2 μm steps through the entire cell volume (10-15 μ.m). The data shown represent the integral of the fluorescence intensity of the manually defined contact site over the total value of the cell. Error bars represent the standard deviation. The manually defined contact sites represent 6.9 ± 2.8% of the total cell volume. The relative enrichment of MEKK2-GFP at the manually defined contact site is 1.03 r 0.14 when stimulated with APC that had not been pulsed with antigen (no ag) and 2.38 ± 0.58 when stimulated with APC that had been loaded with 500 μ/ml conalbumin. The relative enrichment of MEKK3-YFP at the manually defined contact site is 1.08 ± 0.11. The digitally defined contact site (calculated only for MEKK2-GFP translocation in response to antigen stimulation; see Experimental Procedures) represents approximately 1.25% of the total cell volume and 9% of the total cellular MEKK2-GFP fluorescence. The relative enrichment of MEKK2-GFP at the digitally defined contact is 6.75 ±1.76.


not been pulsed with antigen. As expected, no phos-phorylation of GST-SEK1 was observed when cells that express the kinase-inactive mutant, dnMEKK2-GFP, were used (data not shown). Thus, engagement of D10 T cells by antigen-pulsed APC activates MEKK2-GFP. Furthermore, the observed activation was TCR medi-ated, because it was both antigen specific and accom-plished via the use of fixed APCs, which are incapable of


Figure 2. Effect of Antigen Concentration on Kinetics of MEKK2-GFP Translocation

(A) Live cell imaging is described in the Experimental Procedures. APC were loaded with the indicated concentrations of conalbumin.In each panel, the APC is the upper (dark) cell and the D10 T cell is the lower (bright) cell.

(B) T cell/APC conjugates were prepared, bound to coverslips, and fixed as described in the Experimental Procedures. Conjugates were identified visually using Nomarski optics, and fluorescence imaging was then used to determine whether there was enrichment of MEKK2-GFP at the T cell/APC contact site. A total of 100-160 conjugates were examined over two separate experiments for each conalbumin loading concentration, and the percentage of conjugates that exhibited MEKK2GFP transslcation is reported.








delivering costimulatory signals (Jenkins and Schwartz,

1987; Often and Germain, 1991; Harding et al., 1992).


A Dominant-Negative Form of MEKK2 Blocks TCR

Activation of the ERK and p38 MAPKs but Not the JNK MAPK Pathway


Previous studies of MAP kinase activation in T lympho-cytes have demonstrated that cross-linking of the TCR results in rapid activation of the ERK and p38 MAPK pathways, whereas activation of the JNK MAPK path-way requires cross-linking of both the TCR and CD28 or treatment with PMA and ionomycin (Whitehurst et al., 1992; Su et al., 1994; Cantrell. 1996; Li et al., 1996; Salmon et al., 1997). Moreover, MEKK2 has been shown to be a MAPK kinase kinase that can activate the MAPK kinases MEK-1 and MEK-2, which are the kinases directly upstream of the ERK MAPKs (Fanger et al., 1997). Thus, to establish whether MEKK2 has a role in T cell MAPK activation, we used the polyclonal D10/dnMEKK2-GFP line to look for evidence of inhibition of signals known to be downstream of TCR ligation.


D10/MEKK2-GFP, D10/dnMEKK2-GFP, and nonin-fected D10 cells were activated by treatment with the stimulatory anti-TCR monoclonal antibody, 3D3 (Kaye et al., 1983). The cells were then lysed and assayed by immunoblot using antibodies specific for activated, phosphorylated ERK-1 and ERK-2 and p38 MAPK. ERK-1 and ERK-2 were phosphorylated in D10 and D10/MEKK2-GFP T cells in response to treatment with the anti-TCR antibody (Figure 3B). ERK  phosphory-lation was substantially blocked by the MEK inhibitor, PD98059 (data not shown). In contrast, the stimulatory anti-TCR antibody  did not induce phosphorylation of ERK-1 and ERK-2 in cells expressing dnMEKK2-GFP (Figure 3B).


Figure 3. biochemical Analysis of Activities of MEKK2-GFP and dnMEKK2-GFP in D10 T Cells

(A) D10/MEKK2-GFP T cells were incubated at 37 for 10 min alone (control) or with paraformaldehyde-fixed APC that had previously been untreated (APC + no ag) or loaded overnight with 500 μg/ml conalbumin (APC '- conalb). Immunoprecipitated MEKK2-GFP was incubated with purified recombinant GSTSEK1 and y^-32 P-ATP, and products were sep-arated by SDS-PAGE. To measure MEKK2 kinase activity, phosphorimage analysis was performed to quantitate phosphorylated GSTSEK1. Relative phos-phorylation units are indicated beneath each lane.


(B) D10 T cells, D10/MEKK2-GFP T cells, or D10/dnMEKK2-GFP T cells were mock treated or incubated for 5 or 10 min at 37 with the DIO-specific, TCR-activating monoclonal antibody 3D3. Protein extracts were pre pared and separated on an SDS/PAGE gel. Phosphorylated ERK-1 (phos ERK-1) and phosphorylated ERK-2 (phos ERK-2) were detected with an anti-phospho-ERK-1/-2 anti body (upper panel). This blot was then stripped and blotted with anti-ERK-1 and anti-ERK-2 antibodies, as shown in the lower panel


(C) D10 T cells, D10/MEKK2-GFP T cells, or D10/dnMEKK2-GFP T cells were mock treated or incubated for 5 or 10 min at 37 with the DIO-specific, TCP-activating monoclonal antibody 3D3. Protein extracts were prepared and separated on SDS-PAGE gels as described in (B). Phosphorylated p38 MAPK (phos p38) was detected with an anti-phospho-p38 MAPK antibody (upper panel). The same blot was stripped and blotted with an anti-p38 antiserum, as shown in the lower panel.


(D) D10 T cells or D10/dnMEKK2-GFP T cells were mock treated (control) or activated for 5 min at 37 by the addition of PMA and lonomycin (PMA + iono). cells were disrupted and cJun NH2-terminal kinase (JNK) activity was assayed using a GST-c-Jun kinase assay. Reaction products were separated by SDS-PAGE, and32 P-labeled GST-cJun was quantified using a phosphorimager. Relative phosphorylation units

are indicated beneath each lane.


        Stimulation of D10 or D10/MEKK2-GFP cells with the anti-TCP 3D3 antibody also rapidly induced p38 phosphorylation (Figure 3C). Expression of dnMEKK2-

GFP inhibited this phenomenon. This result was some-what surprising because transfection analyses in other cell types have not demonstrated p38 activation in response to transient overexpression of full-length MEKK2 or its kinase domain (Fanger et al., 1997; Widmann et al., 1999).

In contrast to the ERK and p38 MAPK pathways, PMA/ ionomycin-mediated activation of JNK was not significantly inhibited by expression of dnMEKK2-GFP (Figure 3D). Therefore, dnMEKK2-GFP selectively inhibits the ERK and p38 MAP kinase pathways that are activated by engagement of the TCR alone but does not inhibit the JNK pathway, which also requires costimulatory signals. Activation of MEKK2 is thus downstream of TCR ligation and is not dependent upon the cross-linking of additional cell surface molecules.


MEKK2 Influences T Cell Adhesion

To assay the effects of dnMEKK2-GFP on T cell function, D10 and its MEKK2-GFP infectants were tested for their ability to secrete IL-4 in response to antigen and APC. Figure 4A demonstrates that expression of dnMEKK2-GFP reduced the sensitivity of D10 cells to low concentrations of antigen. In time-lapse microscopy studies, we observed that expression of dnMEKK2-GFP by D10 both slowed the formation and reduced the number of conjugates that D10 formed with antigen-pulsed APC (data not shown). To address the possibility that D10/dnMEKK2-GFP T cells might have reduced levels of one or more cell surface proteins required for efficient interaction with APCs, FACS analyses were used to measure surface expression of molecules that contribute to conjugate formation. All but one of the proteins monitored (TCRβ, CD4, CD28, LFA-1, ICAM-1, and CD2) varied less than 2-fold in their levels on the three D10 cell lines (data not shown). The one notable exception was CD28, which, in comparison to D10 T cells, was substantially decreased on the D10/MEKK2-GFP line and modestly increased on the D10/dnMEKK2-GFP line Because CD28 probably contributes to regulation of T cell/B cell interactions, this CD28 phenotype did not correlate with the observed decrease in specific conjugate formation of the D10/dnMEKK2-GFP T cells.


        The fact that expression of dnMEKK2-GFP reduced the ability of T cells to respond to antigen and to form conjugates suggested that dnMEKK2-GFP affected a feature of T cell activation that contributes to both of these activities. Previous studies of T cell activation have shown that antibodies to T cell adhesion proteins, such as the integrin LFA-1, have effects similar to those shown here with dnMEKK2-GFP expression (Davignon et al., 1981; Golde et al., 1986). Engagement of TCRs on T cells leads to a rapid and transient increase in the avidity of T cell LFA-1 for its ligands on other cells (Dustin and Springer, 1989). In turn, this increase in avidity contributes to the ability of activated T cells to bind to target cells and produce cytokines. Anti-LFA-1 has a modest effect on the ability of T cells to secrete cytokines in response to antigen and a more dramatic effect on their ability to form conjugates with antigen-pulsed APCs









Figure 4. Effect of Expression of ME-KK2-GFP and dnMEKK2 GFP on AntigenDependent T Cell Responses


(A) The indicated D10 T cells were mixed with APC that had been loaded overnight with the indicated concentrations of conalbumin protein. Supernatants were harvested follow-ing 6 hr incubation at 37 IL-4 concentrations were measured by capture ELISA and calibrated to a recombinant IL-4 standard curve. Error bars represent SEM, and the majority are contained within the symbols.


(B) APC were labeled with the red dye PKH26 and then loaded with the indicated concentration of conalbu-min protein or with no antigen. The D10 and D10/dnMEKK2-GFP T cell lines were labeled with the  green dye CFSE. Each D10 T cell line was then incu - bated for at least 30 min at 37 with no antibody, or 100 μg/ml of blocking antibodies directed against LFA-1 orCD4. For each sample, T cells were mixed  with an equal number of APC and incubated for exactly 16 min at 37. Nonspe-cific aggregates were disrupted by vortexing, and samples were analy-zed by low cytometry. A representa-tive set of twodimensional plots of D10/MEKK2-GFP T cells (green) versus PKH-26-labeled APC (red) is shown. In this example, T cells were preincubated with no antibody or anti-LFA-1 antibody, and APC were loaded with conalbumin (20 μg/ml) or no antigen. The number in each plot is percent conjugates.


(C and D) Tabulation of data colllec-ted from flow cytometry experiments described in (B). In each graph, percent (%) conjugates versus concentration of conalbumin used for APC loading is shown. In (C), each untreated cell line (no antibody) is shown on a single graph. In (D), each cell line is shown on a separate graph, with all three treatment groups (no antibody, anti-LFA-1, and anti-CD4) shown. Error bars represent SEM, and the majority are contained within the symbols.


(Davignon et al., 1981; Golde et al., 1986 Dustin and Springer, 1989). interestingly, there is at leaast one report that the ERK MAPK pathway contributes to TCR regulation of integrin avidity (Mobley et al., 1996).

Several additional findings suggest a relationship between the effects of dnMEKK2-GFP and activation of LFA-1. For example, both antigen-stimulated MEKK2-GFP translocation and increases in LFA-1 adhesion to its ligand, ICAM-1, occur with rapid kinetics and have a duration of at least 30 min (Figures 1A, 1B, and 2A; data not shown Dustin and Springer, 1989). Addition-ally, both biochemical activation of MEKK2-GFP and upregulation of LFA-1 avidity occur rapidly in response to treatment with either PMA or anti-T cell receptor antibodies. Finally, after a primary TCP-mediated stimulation, both MEKK2-GFP translocation and LFA-1 avidity upregulation can recur if an activation signal is delivered a second time (data not shown Dustin and Springer, 1989). Thus, MEKK2 is likely to be involved in delivering the TCR-mediated signals that directly or indirectly result in increased avidity of T cell LFA-1 for its ligand on APCs.

To test this hypothesis, we measured the ability of wild-type D10 cells and their MEKK2-GFP or their dnMEKK2-GFP infectants to form conjugates with anti-gen-pulsed APC in the absence or presence of anti-LFA-1 antibodies. Wild-type D10 cells were labeled with the green fluorescent dye CFSE (Molecular Probes), and the APC were labeled with the red fluorescent dye PKH26 (Sigma). T cells and B cells were mixed in equal numbers and incubated for 16 min. Conjugates were counted by measuring the numbers of cell aggregates that fluoresced both green and red. As shown in Figure 4B, many such conjugates were formed when D10/MEKK2-GFP cells were incubated with conalbuminpulsed APC. Formation of these conjugates was almost entirely dependent on the presence of antigen, since very few conjugates appeared in mixtures of D10/MEKK2-GFP cells and APC that had not been pulsed with antigen. Conjugate formation was also dependent to a large extent on LFA-1 activity, since anti-LFA-1 antibody reduced the numbers of conjugates substantially, from 21.7% to 3.5% of the cells (Figure 4B).

        When these methods were applied in an antigen titration experiment, it was clear that dnMEKK2-GFP affected TCR regulation of adhesion. Wild-type D10 cells and D10/MEKK2-GFP T cells formed substantial numbers of con-jugates at all antigen doses tested, whereas cells ex-pressing dnMEKK2-GFP formed significantly fewer con-

jugates when incubated with antigen-pulsed APC, par-ticularly as the antigen concentration was decreased (Figure 4C). Significantly, at all but the lowest dose of antigen, expression of MEKK2-GFP increased the num-ber of conjugates formed, demonstrating that overex-pression of enzymatically active MEKK2 enhances con-jugate formation.

To investigate the mechanism of dnMEKK2-GFP inhi-bition of adhesion, conjugate assays were performed on the D10 cell lines following preincubation with anti-LFA-1 or anti-CD4 blocking antibodies. In general, D10,D10/MEKK2-GFP, and D10/dnMEKK2-GFP formed sub-stantially fewer conjugates when preincubated with ei-ther antibody (Figure 4D). These observations demon-strate that although conjugate formation is strongly inhibited by dnMEKK2-GFP, there is not a complete

 block in either LFA-1-medisted adhesion or in CD4-mediated signaling events that participate in antigen-regulated adhesion.





P13-Kinase is a Probable Upstream Mediator


of TCR Activation of MEKK2



Previous studies have shown that phosphatidylinositol 3-Kinase (Pl3-K) is involved in antigen-regulated T cell adhesion. Evidence from several groups indicates that P13-K can increase LFA-1 avidity by activating cytoskel-etal modifying proteins that facilitate cell spreading and membrane ruffling (Pardi et al., 1992; Shimizu and Hunt,1996 Stewart and Hogg, 1996 SH et al., 1997 Han et 3., 1998). To determine whether P13-K may be upstream Of the MEKK2-driven regulation of T cell avidity , we studied the effects of the P13-K inhibitor wortmannin on the binding of D10 and its derivatives to antigen-pulsed APC (Figure 5A). Consistent with the findings in Figure 4C, D10/dnMEKK2-GFP cells made fewer conjugates than either D10 or D10/MEKK2-GFP T cells. Wortmannin inhibited conjugate formation by all three cell types,and the  amount of inhibition was comparable to that observed with the anti-LFA-1 antibody, consistent with the model that P13-K is upstream of LFA-1. Addition-ally, the binding of anti-TCR-stimulated D10 T cells to ICAM-1 expressing fibroblasts was inhibited almost com-pletely by wortmannin, demonstrating that TCR activa-tion of LFA-1 -mediated adhesion requires P13-K activity (data not shown). A combination of wortmannin and anti-LFA-1 did not inhibit conjugate formation by D10 cells much more potently than either agent alone, demonstrat-  ing that the majority of the effect of P13-K on conjugate


formation is via LFA-1 (data not shown). Finally, the observation that inhibition of conjugate formation by dnMEKK2-GFP dramatically reduces the ability of wort-mannin to block adhesion (Figure 5A) suggests that MEKK2 and P13-K may be in a common pathway that connects the TCR to a cellular function that contributes to LFA-1 avidity.



In other studies, we have found that wortmannin inhib its MEKK2 activation in response to EGF or high-affinity IgE receptor ligation in T47D breast carcinoma and mast cells, respectively, demonstrating that MEKK2 activa-tion is dependent upon P13-K (G. J. et al., unpublisheddata). Figure 5B shows that TCR activation of ERK and Figure 5. Effect of the P3-Kinase Inhibitor Wortmannin on T Cell/APC Conjugates and MAPK Activation.


(A) The indicated T cell lines were meek treated (control) or preincubated for 30 min with 100 nM wortmannin or with 100 μg/ml of the anti-LFA-1 antibody 121/7.7. APC were loaded with 500 μg/ml conalbumin or no antigen, and conjugate assays were as described in Figures 3B and 3C. Values are plotted as percent (%) specific conjugates. Error bars represent SEM. Similar results were obtained with another P13-K inhibitor, LY294002 (data not shown).


(B) D10 T cells were mock treated or incubated for 30 min with 100 nM wortmannin, as indicated. T cells were then incubated with media for 10 min (control) or activated with supernatant from TCP-activating antibody (3D3) for the indicated time periods. Western


blots for phosphorylated and total ERK and p38 MAPKs were per-formed and are labeled as in Figures 2B and 2C.



p38 is also inhibited by wortmannin in D10 cells, indicating that MEKK2 regulation of ERK and p38 is downstream of Pl3-K. Thus, several lines of evidence indicate that MEKK2 is a PI3-Kinase-dependent transducer of TCR signals that activate T cell adhesion and the ERK and p38 MAPK cascades.







We have used live-cell imaging to provide real-time analysis of the intracellular redistribution of a signaling protein in response to antigen stimulation of the T cell receptor. This study also provides evidence that MEKK2 is activated and is required for activation of the ERK and p38 MAPK pathways in response to TCR ligation. Although other MAPKKKs, particularly Raf-1, have previously been suggested to activate the ERK pathway in response to TCR stimulation (Siegel et al., 1990; Owaki et al.. 1993; Siegel et al., 1993; Franklin et al., 1994; Gupta et al., 1994; Izquierdo et al., 1994), a study employing primary murine T cells has also shown that ligation of additional receptors, including CD2, CD4, and CD28, is also capable of activating Raf-1 (Siegel et al., 1993). However, ligation of CD28 does not result in p38 phosphorylation (Salmon et al., 1997). Our findings would suggest that Raf-1 is more likely to play an accessory or costimulatory role in T cell activation, whereas MEKK2 is a MAPKKK that directs antigen-regulated responses.



We have demonstrated that antigen ligation of the TCR activates MEKK2 and that dnMEKK2-GFP inhibits  aspects of TCR signaling. importantly, the effect of dn-


MEKK2-GFP in D10 T cells is selective for specific aspects of TCR signaling. Activation of the ERK and p38 pathways are blocked by dnMEKK2-GFP ex-

pression, but activation of JNK is not perturbed (Figures 3B-3D). Conjugate stability is inhibited, but D10/dnMEKK2-GFP cells make an amount of IL-4 essentially indistinguish able from the D10 and D10/MEKK2-GFP cell lines when activated by APC loaded with high concentrations of antigen (see Figure 4A). Because the production of IL-4 in response to TCR stimulation is a complex biological response that involves the integration of numerous signaling pathways, it seems extremely unlikely that dnMEKK2-GFP has nonspecifically perturbed signal transduction pathways. The ERK MAPK pathway has been shown to have little influence on IL-4 production (Dumont et 8., 1998), consistent with our results. Addi-tionally, we would emphasize that the kinetics of the signal transduction events inhibited by dnMEKK2-GFP (Figures 3B and 3C) are consistent with the kinetics of translocation of endogenous MEKK2 and transloca-tion and activation of MEKK2-GFP (Figures 1A, 1B, 2A,

and 3A).


Although it is reasonable to postulate that the eff-ects of MEKK2 on T cell adhesion might be mediated by the ERK or p38 MAPK pathways, our analyses with the MEK inhibitor PD98059 and the p38 inhibitor SB-203580 have shown no significant effect of these compounds on conjugate formation (data not shown). Thus MEKK2, like other MAPKKKs, including MEKK1 and PAK, appears capable of activating non-MAPK signaling pathways (Widmann et al., 1999).



As shown in Figure 5A, MEKK2 is a required signal transducer for a PI3-K-regulated adhesion pathway that contributes to increased LFA-1 avidity. Because the MEKK2 protein does not include a pleckstrin-homology domain, we postulate that TCP-mediated MEKK2 acti-vation is downstream of one or more proteins that are directly acted upon by products of PI3-K. Given that LFA-1 avidity can be affected by diverse processes including cell spreading, LFA-1 clustering, and LFA-1 conformational changes, the mechanism(s) by which MEKK2 contributes to increased LFA-1 avidity may be quite indirect. The discovery that MEKK2 is recruited to the T cell/APC interface and regulates adhesion will allow a genetic and biochemical dissection of this TCR-regulated response, a response that has been largely refractory to analysis up to this time.

The finding that MEKK2 translocation can occur within seconds (Figures 1 B and 2A) indicates that this relocalization is one of the earliest known TCP-depen-dent activation events (Chan et @., 1991). interestingly, MEKK2-GFP translocation is delayed at low antigen concentration (Figures 2A and 2B), while the net per-centage of total cellular MEKK2-GFP translocated to the contact site appears to be independent of antigen concentration (Figures 1D and 2A; data not shown).These observations may suggest that MEKK2 translocation reaches maximal levels only after a signaling threshold has been reached or an active signaling complex has formed. MEKK2-GFP tran-


slocation may thus be a visual proxy of TCR triggering, which occurs within seconds after the TCR has been functionally engaged. A potentially important applica-tion of our current findings would be to use recombinant MHC complexes of specific valency and geometry to define agonists that are capable of effecting TCP-mediated MEKK2-GFP translocation. Such studies may provide important informa tion regarding the nature of the activated TCR signaling complex (Germain, 1997) and facilitate resolution of the debate concerning the minimal requirements for initiation of the TCR signaling cascade (Boniface et al., 1998 Delon et al., 1998 Vignali and Vignali, 1999).


Live cell fluorescence imaging proved to be a pow-erful Diagnostic tool for defining subcellular relocaliza-tion and potential functions of signaling proteins during a complex biological response. This method should facilitate the characterization of other crucial signaling proteins that redistribute in response to receptor liga-tion or other biochemical stimuli. Beyond confirming results obtained by immunocytochemistry, live cell fluorescence imaging allows investigation of the dynamics of protein redistribution and reorganization in single cells in real time. Because this technology makes possble the visualization and dissection of rapidly occurring, complex redistribution events, live cell fluorescence imaging is a technique that makes possible the study of the temporal component of subcelluar protein reorganization.


Experimental Procedures






The polyclonal rabbit anti-MEKKI and anti-MEKK2 anti-bodies used in fluorescence microscopy experiments have been previously described (Fanger et al., 1997), and the Cy3-conjugated anti-rabbit secondary antibody was purchased from Jackson Laboratories. For some T cell/APC conjugate analyses, blocking antibodies directed against LFA-1 (121/7.7) orCD4 (GK1.5) (Trowbridge and Omary, 1981; Dialynas et al., 1983) were used. These were prepared from hybridoma supernatants by purification over protein G agarose columns. In ELISA experments, the monoclonal antibody BVD4-1D11 (PharMingen) was used for IL-4 capture and the biotinylated monoclonal antibody BVD6-24G2 (PharMingen) was used for IL-4 detection.


For Western blotting experiments, T cells were activated by incubation with the anti-DIO TCR monoc-lonal antibody 3D3, which does not require addition of cross-linking secondary antibody (Kaye et al., 1983). Phosphorylated ERK-1 and phosphorylated ERK-2 were detected with a rabbit polyclonal anti-phospho-MAPK antibody (New England Biolabs), and phosphorylated p38 MAPK was detected with a rabbit polyclonal anti-phospho-p38 MAPK antibody (New England Biolabs). Total ERK-1 and ERK-2 were detected using polyclonal goat anti-ERK-1 and rabbit anti-ERK-2 antibodies (Santa Cruz Biotechnology). Total p38 was detected using a rabbit polyclonal anti-p38 antiserum.



Cells and Retroviral Infection


The T cell clone D10 (Kaye et al., 1983) and all describ-ed D10derived lines were maintained in Cick's (EHAA) media (Irvine Scientific) supplemented with 10% fetal bovine serum and 8 U/ml IL-2. Cells were transferred to media lacking IL-2 approximately 20 hr before use in experiments. The CH12.LX B cell line (APC) was main tained in minimal essential media (MEM) (Gibco/BRL)supplemented as described (Kappler et al., 1981).

Murine cDNA clones encoding either MEKK2 or a F502L kinase inactive mutant (dnMEKK2) were modified by the truncation of the 5' untranslated region and introduction of an Ncol restriction site at the initial methionine (CCATGG). The MEKK2 stop codon was also removed and replaced with a synthetic linker, adding the residues Gly Arg. These modified MEKK2 cDNAs were then fused to the N terminus of an open reading frame encoding the red-shifted, humanized green fluorescent protein variant EGFP (Clontech), altered by deletion of the first GFP codon and introduction of the "cycle 3" mutations described by Crameri et al. (1996). MEKK2-GFP fusion genes were cloned upstream of an internal ribosome entry site (IRES)-neomycin resistance (NeoR) cassette, and the MEKK2-GFP/IRES-NeoR inserts were cloned into the Ncol and BamHI sites of the MFG retroviral vector (Riviere et al., 1995). The MEKK3-YFP retrovirus was created by identical methodology. YFP is a previously described spectral variant of GFP (Ormo et al., 1996). The MEKK3-YFP fusion was cloned upstream of an IREShygromycin (HygR) resistance cassette in the MFG vector. Retroviral plasmid constructions were transfected into the packaging line. Phoenix-Eco (Kinsella and Nolan, 1996). The D10 T cell line was infected bv coculture with the packaging line. followed by G418 or hygromycin selection. The resulting G418R or HygR cultures were sorted for high GFP or YFP fluorescence by the National Jewish Flow Cytometry Core Facility.

In certain T cell/APC conjugate assays and West-ern blotting experiments, T cells were preincubated for 30 min with 100 nM wortmannin. This dose of wortman-nin over the 30 min preincubation plus 16 min conjugate formation period (or 10 min 3D3 antibody treatment period) was judged to be nontoxic because flow cytometry analysis of the conjugate assay samples did not demonstrate a significantly higher percentage of dead cells in the wortmannintreated samples versus untreated or antibody-treated samples.


Immunocytochemistry and Cell Imaging Analysis

Conjugates were formed by mixing'a 1:1 or 1:2 ratio of T cells to APC. For fixed cell experiments, mixing was followed immediately by centrifugation in a Stratagene PicoFuge for 20 sec, and cell mixtures were placed in a CO; incubator at 37 for the indicated time periods. For immunocytochemistry experiments, T cell/APC conjugates were bound to washed, poly-D-lysine-coated coverslips (3 mg/ml), fixed for 10 min with 3% paraformaldehyde/3% sucrose, and stripped of membranes by 5 min treatment with 0.2% Triton X-100 in 1 x PBS. Conjugates were stained with indicated antibodies and were observed using a Leica DMXRA epifluorescence microscope. Visual data were acquired using a Cooke Corporation SensiCam CCD camera and were digitally deconvolved using a nearest neighbors algorithm with SlideBook software (Intelligent Imaging Corporation). In live-cellimaging experiments, T cells and APC (each at approximately 2 × 105/ml) were mixed in medium buffered with 25 mM HEPES (pH 7.4), and real-time fluorescent images were gath-ered with a 40 × water immersion objective and a Bi-optechs stage heater set to 37 Short exposure times and a xenon light source were used to minimize bleach-ing effects.


Conjugate contact analysis was performed using the masking and statistics capabilities of SlideBook. Initially, stacks of images were acquired in 0.2 urn steps throughout the contact volume. Stacks were decon-volved using a nearest neighbors algorithm, and the contact site was defined either manually or digitally. Digital definition was available only for the antigen-stimulated D10/MEKK2-GFP cell line, since MEKK2-GFP localizes to the contact interface and generates a contact site identifiable by fluorescence. Therefore, since translocation was not a feature of all cell lines and conditions under comparison, manual definition was used. Manual definition results in a much larger contact volume, resulting in an artificially inflated localization percentage for MEKK2. We therefore reported both the manual and digital values for percent localization. We have also reported the relative enrichment (see the legend to Figure 1 D), which is the fluorescence per unit volume at the contact site divided by the fluorescence per unit volume of the entire cell.




The indicated D10 T cell lines (l06/ml) were mixed with an equal volume of APC (l06/ml) that had been loaded overnight with the indicated concentrations of conal-bumin protein. Supernatants were harvested and froz-en following 6 hr incubation at 37. IL-4 concentra-tions were measured by capture ELSA, using recom-binant murine IL-4 (R&D Systems) to generate a standard curve. The monoclonal antibody BVD4-1D11 was bound to the wells of a 96-wGl ELSA Pate, IL-4 was captured from 200 μl of each supernatant by incubation at ambient temperature for 1 hr, and IL-4 was detected using the biotinylated monoclonal antibody BVD6-24G2. Following incubation with extravidin alkaline phosphatase and hydrolysis of the substrate p-nitropheny phosphate, ELSA samples were read at 405 nm on a microplate reader (BioTek).


Conjugate Assays

APC were labeled with the dye PKH-26 (Sigma) acc-rding to the instructions of the manufacturer and then loaded for 16 hr with 500, 100, 20, or 4 μg/ml conalbumin protein or with no antigen. The D10 and D10/dnMEKK2-GFP T cell lines were labeled with 0.1 and 0.025 μM, respectively, CFSE (Molecular Probes)  for 15 min at 37in 1×BSS, approximately 16 hr prior to initiation of the experiment. Each D10 T cell line was then incubated for at least 30 min at 37 with no antibodv or 100 μg/ml of blocking antibodies directed against LFA-1 (121 /7.7) or CD4 (GK1.5) (Trowbridge and Omary, 1981: Dialynas et al., 1983). For each sample, 20 μl of T cells (107/ml) was mixed in a 1.5 ml microcentrifuge tube with an equal volume of APC (107ml) and incubated for exactly 16 min at 37. Nonspecific aggregates were disrupted by vortexing, and samples were analyzed using a FACScan flow cytometer (Beckton-Dickinson). For each sample, 4 x 104 live-gated events (i.e., live cells or live cell con-jugates) were collected and analyzed. Two-dimensional plots of T cells (FL1 channel, GFP and/or CFSE fluor-escence) versus APC (FL2 channel, PKH-26 fluore-scence) were generated. Percent conjugates, defined as the number of live-gated, double-positive events in the upper right quadrant divided by the total number of live-gated events, was then determined for each sample. In Figure 5A, values are expressed as percent specific conjugates, which is defined as percent conjugates in the APC/no antigen samples subtracted from the percent conjugates calculated for each APC/conalbumin sample.


MEKK2 and JNK Assays

To assay kinase activity of MEKK2-GFP, D10/MEKK2

-GFP T cells (2 x l07) were incubated at 37 for 10 min alone or with the indicated paraformaldehyde-fixed APC (2 x 107). Cells were disrupted in lysis buffer (20 mM Tris [pH 7.6], 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, and 3 mM EGTA), nuclei were pelleted by centrifugation at 12,000 ×g for 10 min at 4, and MEKK2-GFP was immunoprecipitated using a polyclonal rabbit anti-GFP antibody (Clontech) and protein A sepharose. immunoprecipitates were washed twice with lysis buffer and once with kinase buffer (20 mM HEPES [pH 7.5], 20 mM p-glycerophosphate, 10 mM p-nitrophenyl phosphate, 10 Mm MgCl2, 10 mM MnCl2, 1 mM DTT, and 50 μM Na3VO4). A MEKK2 kinase assay was performed by incubating the immunoprecipitate with bacterially produced recom-binant GST-SEK1 and γ-32 P-ATP at 30 for 20 min in kinase buffer. Phosphorylation of GST-SEK1 was determined by boiling the products of the kinase reaction in 1 ×Laemmli buffer, followed by SDS-PAGE electrophoresis and quantitation of 32P-labeled GST-SEK1 using a phosphorimager.



For the cJun NH;-terminal kinase (JNK) assay, D10 T cells or D10/dnMEKK2-GFP T cells (107) were mock treated or activated for 5 min at 37 by the addition of 10 ng/ml PMA (Sigma) and 1 μM lonomycin (Calbiochem). Cells were disrupted in lysis buffer, JNK was precipitated from lysates using GST-cJun-sepharose beads, and a GST-c-Jun kinase assay was performed as previously described (Fanger et al., 1997). Reaction products were separated on a 10% SDS-PAGE gel, and 32P-labeled GST-cJun was quantified using a phosphorimager.




Western Blotting


D10 T cells or D10/dnMEKK2-GFP T cells (107) were pretreated as indicated in the figure legends and were then incubated for the indicated times at 37 without stimulation or with the DIO-specific TCP-activating monoclonal antibody 3D3 (10 μg/ml of Protein G-purified antibody). Protein extracts were prepared by disrupting cells in lysis buffer followed by pelleting of the nuclei by centrifugation at 12,000 ×g for 10 min at 4. Proteins were denatured by boilling in Laemmli buffer, and 2.5 × 106 cell equivalents per sample were separated on a 10% SDS/PAGE gel. Proteins were transferred to nitrocellulose and detected using the indicated anti-phosphoMAP kinase antibody and a standard chemiluminescence protocol. Where indicat-ed, Blots were stripped and reblotted.






The authors thank M. Sadelain and R. Mulligan for pro-viding the MFG retrovirus and sequence information, G. Nolan for the Phoenix Eco retroviral packaging line, C. Janeway for the 3D3 antibody, A. Kupfer for the IL-2-dependent subclone of the D10 T cell line, W. Townend and S. Sobus of the National Jewish Flow Cytometry Core Facility for cell sorting, K. Teague for advice concerning integrin experiments, and J. Cambier, J. Freed, T. Mitchell, J. Bender, and K. Teague for review of the manuscript. This work was supported by grants AM 8785, Al-17134, Al-22295, DK-37875, and GM-30324.




Received February 12, 1999; revised August (0, 1999








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