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The Journal of Immunology, 2006, 176: 1668-1674.
Copyright © 2006 by The American Association of Immunologists

Phosphatidylinositol 3-Kinase Functions as a Ras Effector in the Signaling Cascade That Regulates Dephosphorylation of the Actin-Remodeling Protein Cofilin after Costimulation of Untransformed Human T Lymphocytes1

Guido H. Wabnitz2, Gabriele Nebl2, Martin Klemke, Andreas J. Schröder3 and Yvonne Samstag4

Ruprecht Karls University, Institute of Immunology, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The activity of cofilin, an actin-remodeling protein, is required for T lymphocyte activation with regard to formation of the immunological synapse, cytokine production, and proliferation. In unstimulated T PBL (PB-T), cofilin is present in its Ser3-phosphorylated inactive form. Costimulation of TCR/CD3 and CD28 induces dephosphorylation and, thus, activation of cofilin. In this study we characterized the signaling cascades leading to cofilin activation in untransformed human PB-T. We show that a Ras-PI3K cascade regulates dephosphorylation of cofilin in PB-T. The GTPase Ras is a central mediator of this pathway; transient expression of an activated form of H-Ras in PB-T triggered the dephosphorylation of cofilin. Inhibition of either MAPK/ERK kinase or PI3K blocked both Ras-induced and costimulation-induced cofilin dephosphorylation in PB-T, showing that the combined activities of both signaling proteins are required to activate cofilin. That Ras functions as a central regulator of cofilin dephosphorylation after costimulation through CD3 x CD28 was finally proven by transient expression of a dominant negative form of H-Ras in primary human PB-T. It clearly inhibited costimulation-induced cofilin dephosphorylation, and likewise, activation of PI3K was diminished. Our data, in addition, demonstrate that regarding the downstream effectors of Ras, a clear difference exists between untransformed human PB-T and the T lymphoma line Jurkat. Thus, in PB-T the Ras signaling cascade is able to activate PI3K, whereas in Jurkat cells this is not the case. In addition to the insights into the regulation of cofilin, this finding discloses a to date unrecognized possibility of PI3K activation in T lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Full activation of T lymphocytes requires costimulation, i.e., engagement of the TCR/CD3 complex by antigenic peptide bound to a MHC molecule plus interactions of accessory receptors (e.g., CD2 or CD28) with their ligands (CD58 or CD80/CD86) on APCs. These processes involve the interplay of cell surface receptor proteins with intracellular signaling and adaptor proteins and lead to the production of cytokines, such as the growth factor IL-2, and subsequent proliferation of the T lymphocytes. Recent progress in this field demonstrates that the actin cytoskeleton plays an important functional role in the linkage of surface receptors to downstream events (1, 2, 3, 4).

The actin cytoskeleton represents a highly dynamic network that is influenced by a number of actin-regulating factors. Among these, the ubiquitously expressed actin-binding protein, cofilin, has a special state, because it regulates filament remodeling, thereby controlling both disassembly of existing and formation of new actin filaments (5). Phosphorylation of cofilin at Ser3 reduces the actin binding capacity of cofilin (6). In unstimulated T PBL (PB-T),5 cofilin exists in its inactive Ser3-phosphorylated form. T cell activation through costimulation of TCR/CD3 plus the coreceptor CD28 or CD2 stimulation induces the dephosphorylation of cofilin (7, 8). The activity of cofilin is crucially involved in T lymphocyte activation processes (9). Thus, cell-permeable peptides that block the binding of cofilin to actin in human PB-T impair the formation of the immunological synapse and inhibit the induction of cell proliferation and cytokine production. The serine kinases LIM motiv-containing protein kinases 1 and 2, testicular protein kinases 1 and 2, and Nck-interacting kinase-related kinase/Nck-interacting kinase-like embryo-specific kinase phosphorylate cofilin (10, 11, 12, 13, 14). Protein phosphatases that are able to dephosphorylate cofilin are the serine phosphatases PP1, PP2A (15), the slingshot phosphatases (16), and chronophin (17).

In this study we have further characterized the signaling cascade leading to cofilin dephosphorylation in untransformed human T lymphocytes. Ras is known to be a central mediator of T lymphocyte activation events (18). Interestingly, in anergic T lymphocytes activation of Ras is blocked (19). Moreover, activated Ras is known to induce rearrangements of the actin cytoskeleton and alterations in the morphology of cells (20, 21, 22). Therefore, we analyzed whether cofilin dephosphorylation in untransformed human PB-T is related to Ras activation. We found that activated Ras indeed induced the dephosphorylation of cofilin in PB-T. Downstream of Ras the combined activities of MAPK/ERK kinase (MEK) and PI3K are required to induce cofilin dephosphorylation. This finding was surprising, because it was reported that in T lymphocytes Ras is not able to activate PI3K (23, 24). Yet this conclusion resulted from experiments performed with the T lymphoma line Jurkat. Our data clearly show that, in contrast to the situation in Jurkat cells (23), in untransformed human T lymphocytes PI3K functions as a Ras effector.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

PB-T were prepared as described previously (25) and cultured in RPMI 1640 containing 10% FCS, 4 mM glutamine, 25 mM HEPES, penicillin, and streptomycin (Invitrogen Life Technologies). The enzyme inhibitors used were LY294002 (Sigma-Aldrich) at 50 µM, wortmannin (Sigma-Aldrich) at 100 nM, U0126 (Promega) at 20 µM, Ro32-0432 (Calbiochem) at 5 µM, and B581 (Calbiochem) at 250 µM.

Stimulation of PB-T

PB-T were stimulated via plate-bound Abs. The Abs used for coating of tissue culture plates were the CD3 Ab BMA030 (a gift from R. Kurrle, Aventis, Bridgewater, NJ; IgG2a; 1 µg/ml) and the CD28 Ab CD28.2 (BD Pharmingen; IgG1; 10 µg/ml) or the respective IgG2a and IgG1 control Abs (BD Pharmingen). PB-T were sedimented onto the Ab-coated plates (1500 rpm, 5 min) and subsequently incubated for the time periods indicated in the figure legends.

Transfections

Expression vectors were cloned in our laboratory: pEGFP-C1-H-RasV12, the coding sequence of H-RasV12 that is hemagglutinin (HA) tagged (26), was subcloned into pEGFP-C1 (BD Clontech) in frame with enhanced GFP (EGFP). The resulting RasV12-EGFP-C1 plasmid was taken for the reversion into H-Ras-wild type (H-Ras-wt; V12->G12) using the QuikChange Site-Directed Mutagenesis XL Kit (Stratagene) according to the manufacturer’s instructions. Subsequently, another point mutation at position 17 (S17->N17) was introduced, resulting in a dominant negative H-RasN17 mutant (27). Additional constructs were pEGFP-N1-cofilin (the coding sequence was subcloned into pEGFP-N1; BD Clontech) and pcDNA3-HA-protein kinase B (PKB)/Akt (the coding sequence of HA-PKB/Akt (28) was subcloned into pcDNA3 (Invitrogen Life Technologies)). Coexpression of GFP or GFP-H-RasV12 and the reporter proteins (cofilin-GFP and HA-PKB/Akt), respectively, was achieved through nucleofection of 20 x 106 PB-T with 9 µg of the reporter plasmid (pEGFP-N1-cofilin or pcDNA3-HA-PKB/Akt) plus 1 µg of pEGFP-C1-H-RasV12 or the control vector pEGFP-C1 following the protocol supplied by the manufacturer of the nucleofector (Amaxa). The transfection method led to ~50% GFP-expressing cells with a MFI between 200 and 300.

Determination of the phosphorylation state of cofilin

PB-T (3 x 106) were lysed in Laemmli sample buffer, sheared, and subjected to SDS-PAGE and Western blotting (polyvinylidene difluoride membranes). To determine the relative portion of phosphorylated cofilin, blots were stained with a phospho-Ser3-cofilin-specific antiserum (Cell Signaling Technology), stripped, and restained with a cofilin antiserum that detects all forms of cofilin (produced in our laboratory). Subsequently, the signals were quantified by densitometry. For each sample, the quotient of the signal intensities from phosphorylated cofilin vs total cofilin was calculated. It was set at 1 under control conditions, and all other values of the same experiment were calculated as multitudes (P-index). In addition, the mean and SEM were calculated. Alternatively, the phosphorylation state of cofilin was determined using a one-dimensional NEpHGE as previously described (8, 29). This method allows the detection of both phosphorylated and unphosphorylated forms of cofilin within one gel due to the difference in their isoelectric point without the need for a phosphospecific antiserum. The two methods, phospho-cofilin Western blot and one-dimensional NEpHGE, gave comparable results.

Determination of the phosphorylation state of transiently expressed cofilin-GFP

Six to 8 h after nucleofection, cells were treated for 16 h with inhibitors. Then cell lysates (equivalent to 4–6 x 106 transfected cells) were prepared and subjected to SDS-PAGE and Western blotting. The phosphorylation state of cofilin-GFP was determined using our standard protocol (see above). Endogenous cofilin does not flaw this analysis, because its Mr differs from that of cofilin-GFP (19 vs ~50 kDa).

Determination of Ras activity

The Ras activation assay was performed using a commercially available system (Upstate Biotechnology). Briefly, cells (10 x 106/sample) were lysed. Cleared lysates were incubated with bead-bound ras binding domain of Raf-1 protein to precipitate Ras-GTP. The precipitates were subjected to SDS-PAGE and Western blotting. Staining of the blots with a Ras Ab revealed the level of Ras activation in the lysate.

Detection of the activated forms of ERK and PKB/Akt

The activation levels of MEK and PI3K were determined using standard protocols; activation of MEK was analyzed through detection of the phosphorylated forms of the MEK substrates ERK1 and ERK2, whereas PI3K activity was visualized through detection of the phosphorylated/activated form of the PI3K effector PKB/Akt. Cells were lysed in Laemmli sample buffer and sheared, and lysates were subsequently subjected to SDS-PAGE and Western blotting. Blots were stained sequentially with Abs against the Thr183/Tyr185-phosphorylated activated forms of ERK1/2 (Promega) and the Ser473-phosphorylated form of PBK/Akt (Promega). After stripping, blots were reprobed with antisera detecting all forms of ERK1/2 (Promega) and PKB/Akt (Santa Cruz Biotechnology).

Flow cytometry

Cells were fixed and permeabilized before Ab staining. To this end, 5 x 105 PB-T were incubated for 5 min with 4% paraformaldehyde at 37°C. The fixation was stopped by adding 2 vol of FACS buffer (0.5% BSA, 5% FCS, and 0.07% NaN3). Cells were then spun down and permeabilized for 10 min at room temperature with 0.1% saponin in FACS buffer. After an additional washing step, the cells were resuspended in 50 µl of FACS buffer/0.1% saponin containing 1 µl of the respective antiserum and incubated at room temperature for 15 min. Subsequently, cells were washed and incubated with PE-labeled donkey anti-rabbit F(ab')2 (Dianova) for an additional 15 min. Finally, cells were washed and resuspended in 1% paraformaldehyde until quantification by flow cytometry using a FACSCalibur (BD Biosciences).

Determination of the phosphorylation state of transiently expressed HA-PKB/Akt

Twenty-four hours after nucleofection, cells (equivalent to 5 x 106 transfected cells) were lysed in 500 µl lysis buffer (10 mM HEPES (pH 7.2), 140 mM KCl, 5 mM MgCl2, 2 mM EGTA, 0.2% Nonidet P-40, 0.1 mM PMSF, 1x proteinase inhibitor mix (Sigma-Aldrich; A-8340), and 1x phosphatase inhibitor mix (Sigma-Aldrich; A-2850)), sheared (five times pushing through a 27-gauge needle), and cleared (22,000 x g, 10 min, 4°C). To separate HA-PKB/Akt from endogenous PKB/Akt, HA-PKB/Akt was immunoprecipitated. For immunoprecipitation, 2.5 µg of HA Abs (Roche) were added to each lysate, and the lysate was incubated for 2 h at 4°C on a moving platform. After 1 h, protein G-Sepharose beads (Pharmacia Biotech; 50 µl of 50% slurry in lysis buffer) were added to the lysates. The beads were washed three times with lysis buffer, resuspended in 2x Laemmli sample buffer, and denatured (95°C, 5 min), and the eluates were subjected to SDS-PAGE (10%). The gels were blotted onto polyvinylidene difluoride membranes. Blots were stained with Abs against the Ser473-phosphorylated form of PKB/Akt (Promega). After stripping, blots were reprobed with the HA Abs to detect total HA-PKB/Akt. Staining with a GFP-antiserum (Invitrogen Life Technologies) indicates the presence of GFP-H-rasV12 that is coprecipitated, because this construct contains an HA tag. Subsequently, the signals were quantified by densitometry. For each sample the quotient of the signal intensities from phosphorylated HA-PKB/Akt vs total HA-PKB/Akt was calculated. It was set at 1 under control conditions, and all other values of the same experiment were calculated as multitudes (P-index). In addition, the mean and SEM were calculated.

Statistical analysis

Where indicated, data are presented as the mean ± SEM, and the probability of differences was assessed using Students paired t test. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Costimulation-dependent cofilin dephosphorylation in PB-T is sensitive to inhibitors of Ras, PI3K, and MEK

To obtain information about the costimulation-triggered signaling cascade leading to cofilin dephosphorylation, we analyzed untransformed human T cells in the presence of synthetic inhibitors for signaling molecules. To this end, PB-T were incubated with the Ras inhibitor B581 (250 µM), the MEK inhibitor U0126 (20 µM), or the PI3K inhibitors LY294002 (50 µM) and wortmannin (100 nM), respectively. Subsequently, cells were costimulated via plate-bound Abs directed against the TCR/CD3 complex and CD28 (CD3 x CD28) or settled on an isotype control Ab mix. After 30 min, whole-cell lysates were prepared, and the phosphorylation state of cofilin was determined via Western blot analysis using a phosphospecific antiserum. Fig. 1 shows that the cofilin dephosphorylation induced through CD3 x CD28 costimulation was clearly diminished by LY294002, wortmannin, B581, and U0126. Thus, these experiments strongly suggested that Ras, MEK, and PI3K are involved in the signaling cascade leading to cofilin dephosphorylation upon costimulation of untransformed human PB-T.


Figure 1
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  		FIGURE 1. Ras, MEK, and PI3K are involved in the regulation of cofilin dephosphorylation after costimulation of human PB-T. Primary human T cells were preincubated for 40 h with the Ras inhibitor B581 (B; 250 µM; lane 5) or for 30 min with the PI3K inhibitor LY294002 (LY; 50 µM; lane 3) or wortmannin (W; 100 nM; lane 4) or the MEK inhibitor U0126 (U0; 20 µM; lane 6). As solvent, control cells were treated with DMSO (lanes 1 and 2). Subsequently, cells were left unstimulated (IgG; lane 1) or were costimulated for 30 min via plate-bound CD3 plus CD28 Abs (CD3 x CD28; lanes 2–6). The phosphorylation state of cofilin in postnuclear lysates was determined through one-dimensional Western blot analysis using an antiserum specific for the phosphorylated form of cofilin (upper panel; p-cofilin). After stripping, the blot was reprobed with an antiserum that recognizes cofilin independent of its phosphorylation state (lower panel; total cofilin).

 
The activity of Ras in untransformed PB-T was analyzed through precipitation of the activated GTP-bound form of Ras from cell lysates. Fig. 2 demonstrates that costimulation through CD3 x CD28 induces an activation of Ras similar to that observed after treatment with the phorbol ester PMA. In unstimulated PB-T, no Ras activity could be detected.


Figure 2
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  		FIGURE 2. Costimulation through CD3 x CD28 or PMA stimulation induces activation of Ras in human PB-T. PB-T were stimulated for 10 or 30 min via cross-linked CD3 and CD28 Abs (CD3 x CD28) or 10–8 M PMA. In parallel, cells were settled on isotype control Abs (IgG). Activated Ras (Ras-GTP) was immunoprecipitated and detected as described in Materials and Methods.

 
Transiently expressed activated Ras protein induces the dephosphorylation of cofilin in PB-T

For analysis of the role of Ras in the regulation of cofilin dephosphorylation in untransformed PB-T, transient transfection of an expression vector for a constitutively active variant of H-Ras (H-RasV12) was performed. Subsequently, the effects of activated H-RasV12 on the phosphorylation state of cofilin were analyzed. To be able to selectively analyze alterations in the phosphorylation state of cofilin in transfected cells, an expression vector for a tagged form of cofilin was cotransfected, and the phosphorylation indices of the tagged cofilin (cofilin-GFP) were determined. As shown in Fig. 3, the expression of H-RasV12 in PB-T indeed induced dephosphorylation of the coexpressed cofilin-GFP (p < 0.001).


Figure 3
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  		FIGURE 3. Expression of activated Ras (H-RasV12) in untransformed human PB-T induces cofilin dephosphorylation. Freshly isolated PB-T were transfected with an expression vector for activated Ras (H-RasV12; lane 2 and bar 2) or the respective empty expression vector (vector; lane 1 and bar 1) plus an expression vector for GFP-tagged cofilin. Twenty-four hours after transfection, whole-cell lysates were prepared, and the phosphorylation state of cofilin-GFP was determined. A, Immunoblot detecting phosphorylated cofilin-GFP (P-cofilin-GFP) with a phospho-cofilin-specific antiserum. After stripping, cofilin-GFP (cofilin-GFP) was stained with an antiserum detecting total cofilin. In addition, the expression of H-rasV12 was confirmed through staining of the blots with a Ras antiserum. B, Immunoblot signals for P-cofilin-GFP and cofilin-GFP were quantified by densitometry, and the P-indices of cofilin-GFP were calculated as described in Materials and Methods. The P-index of cofilin-GFP in vector-transfected cells was set at 1. The figure shows the mean and SEM P-indices of cofilin-GFP from 11 independent experiments. ***, p < 0.001.

 
MEK and PI3K are required downstream of Ras to induce cofilin dephosphorylation

To identify Ras effectors participating in the cofilin dephosphorylation pathway PB-T transfected with the Ras construct were incubated in the presence or the absence of the MEK inhibitor U0126 or the PI3K inhibitor LY294002. Each inhibitor alone was able to inhibit H-RasV12-induced cofilin dephosphorylation (Fig. 4), revealing that the activities of both MEK and PI3K are required downstream of Ras to induce cofilin dephosphorylation. This finding was surprising, because it contradicted the current opinion that, unlike in other cell types, in T lymphocytes Ras was not able to activate PI3K (23, 24, 30, 31). This opinion resulted from experiments performed with the human T lymphoma line Jurkat (23) in which phorbol ester treatment led to the activation of Ras and MEK, but not PI3K.


Figure 4
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  		FIGURE 4. H-RasV12-induced cofilin dephosphorylation depends on the combined activities of MEK and PI3K. Freshly isolated PB-T were transfected with an expression vector for activated Ras (RasV12) or the respective empty expression vector (vector) plus an expression vector for GFP-tagged cofilin. Six to 8 h after transfection, cells were either left untreated (lanes 1 and 2) or were treated for 16 h with the MEK inhibitor U0126 (U0; 20 µM; lanes 5 and 6) or the PI3K inhibitor LY294002 (LY; 50 µM; lanes 3 and 4). A, Whole-cell lysates were prepared, and the P-indices of cofilin-GFP were determined. B, The P-index of cofilin-GFP in vector-transfected cells was set at 1. The figure shows the means and SEM P-indices of cofilin-GFP from five experiments from P-cofilin and cofilin immunoblots.

 
To analyze whether the discrepancy regarding PI3K activation through Ras in T lymphocytes stems from the use of different types of T lymphocytes, we first reproduced the decisive experiments of the previous study (23). Jurkat cells were treated with phorbol 12,13-dibutyrate (PdBU; Fig. 5A) or PMA (data not shown, results are similar to those obtained with PdBU), and subsequently the activities of Ras, MEK, and PI3K were determined using standard protocols. Activation of Ras was analyzed through precipitation of the activated GTP-bound form of Ras, MEK activity through detection of the phosphorylated forms of the MEK substrates ERK1 and -2, whereas PI3K activity was visualized through detection of the phosphorylated/activated form of the PI3K effector PKB/Akt.


Figure 5
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  		FIGURE 5. Phorbol esters induce the activation of Ras, PI3K, MEK, and cofilin in PB-T. A, Jurkat cells were treated for 5, 10, or 15 min with 50 ng/ml PdBU (lanes 2–4) or were left untreated (lane 1). B, Freshly isolated PB-T were pretreated for 15 min with the PKC inhibitor Ro32-0432 (5 µM; lanes 3 and 4) or were left untreated (lanes 1 and 2). Subsequently, cells were stimulated for 15 min with 10 ng/ml PMA (lanes 2 and 4) or were left unstimulated (lanes 1 and 3). A and B, The activation of Ras was determined through precipitation of Ras-GTP from cell lysates (IP). Immunoprecipitates were analyzed on SDS-PAGE, and the amounts of precipitated Ras-GTP were determined by staining the blot with a Ras antiserum (Ras-GTP). In parallel, whole cell lysates were prepared and subjected to SDS-PAGE and Western blotting (lysate). The blots were stained with Abs against the activated Ser473-phosphorylated form of the PI3K substrate PBK/Akt (P-PKB/Akt) and the activated dually phosphorylated (Thr183/Tyr185) forms of the MEK-substrates ERK1 and -2 (P-Erk1, 2), respectively. For the control, total amounts of PKB/Akt (PKB/Akt) and ERK1 and -2 (Erk1, 2) were determined. B, The blot was also stained with an antiserum directed against Ser3-phosphorylated cofilin and, after stripping, was reprobed with a cofilin-antiserum. The cofilin signal intensities were quantified by densitometry. The bars show P-indices of cofilin in the presented experiment. The P-index of cofilin in untreated cells was set at 1. The data are representative of three independent experiments.

 
As shown in Fig. 5A, PI3K activity, which was already detectable at a low level in untreated Jurkat cells (Fig. 5A), was not enhanced, but was even reduced, after phorbol ester treatment, whereas Ras and MEK were clearly activated. Thus, in line with the previously published data (23), in Jurkat T lymphoma cells the protein kinase C (PKC)/Ras signaling cascade does not induce PI3K activity. Next, we performed a similar set of experiments with untransformed human PB-T (Fig. 5B). PB-T from different donors were stimulated with phorbol ester, and the activation states of Ras, MEK, PI3K, and cofilin were determined. These experiments revealed that in untransformed human PB-T, phorbol ester treatment induces not only Ras, MEK, and cofilin activation, but also PI3K activity (Fig. 5B). In addition, the PKC dependency of the observed phorbol ester effects was confirmed by pretreatment of the cells with the PKC inhibitor Ro32-0432.

To prove that PI3K functions as a Ras effector in untransformed human T lymphocytes, H-RasV12 was again expressed in PB-T. To monitor PI3K activity, we coexpressed a tagged form of PKB/Akt (HA-PKB/Akt). Subsequently, phosphorylation at the PKB/Akt sites known to reflect PI3K activity (amino acids Ser473 and Thr308 of the wt PKB/Akt protein) was determined in the HA-tagged PKB/Akt. Coexpression of H-RasV12 and HA-PKB/Akt clearly induced phosphorylation of HA-PKB/Akt at the position corresponding to the phosphorylation site Ser473 (Fig. 6) and the site corresponding to Thr308 (data not shown). Because these phosphorylation events depend on upstream PI3K activity (32), this result definitely confirms that Ras is able to induce PI3K activity in human PB-T.


Figure 6
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  		FIGURE 6. H-RasV12 induces HA-PKB/Akt phosphorylation. A, Freshly isolated PB-T were transfected with an expression vector for activated Ras (RasV12) or the respective empty expression vector (vector) plus an expression vector for HA-tagged PKB/Akt. Twenty-four hours after transfection, cell lysates were prepared. Subsequently, anti-HA immunoprecipitation was performed, and the precipitates were analyzed by SDS-PAGE and Western blotting. The blots were stained with an antiserum against the activated Ser473-phosphorylated form of the PI3K-substrate PBK/Akt (P-HA-PKB/Akt). To control for equal loading, the blots were stripped and subsequently stained with HA Abs detecting all forms of HA-PKB/Akt (HA-PKB/Akt). The expression of H-RasV12 was demonstrated by restaining the blots with a GFP antiserum (H-RasV12). The H-RasV12-construct is HA-tagged and thus also precipitated. B, Western blot signals for P-HA-PKB/Akt and HA-PKB/Akt were quantified, and the P-indices of HA-PKB/Akt were calculated as described in Materials and Methods. The P-index of HA-PKB/Akt in vector-transfected cells was set at 1. The figure shows the mean and SEM P-indices of P-HA-PKB/Akt from three independent experiments. *, p < 0.05.

 
Costimulation through TCR/CD3 and CD28 induces cofilin dephosphorylation through a Ras-PI3K signaling cascade

Final proof of the key function of Ras in the costimulatory signaling pathway leading to activation of PI3K and cofilin activation in human PB-T is provided by experiments using the expression of a GFP-tagged, dominant negative mutant of Ras (H-RasN17). In parallel, GFP-tagged H-Ras was expressed either as H-Ras-wt or dominant active (H-RasV12) mutant. The different transfectants were costimulated for 30 min (CD3 x CD28) or left unstimulated (IgG). The phosphorylation states of cofilin and PKB/Akt in GFP-positive cells were determined by staining with phosphospecific Abs, followed by flow cytometry. Again, the expression of dominant active H-RasV12 alone, without stimulation, was sufficient to induce dephosphorylation of cofilin (Fig. 7A; IgG). Concomitantly, phosphorylation of Akt could be observed (Fig. 7B; IgG), again confirming that Ras induces the activation of PI3K.


Figure 7
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  		FIGURE 7. Costimulation-induced PI3K activation and cofilin dephosphorylation are dependent on H-Ras activation. Human PB-T were transfected with GFP-tagged H-Ras-wt ({blacksquare}), H-RasN17 ({cjs2108}), or H-RasV12 ({cjs2113}). Subsequently, cells were costimulated via CD3 x CD28 (CD3xCD28) or settled on isotype control Abs (IgG) for 30 min. After harvesting, cells were stained for phospho-cofilin (A) or phospho-Akt (B) and analyzed by flow cytometry. For quantification, events were gated on GFP-positive cells (note that all constructs were expressed at the same level; data not shown). Depicted is the mean phosphorylation of the respective proteins (calculated from mean fluorescence intensities) of three independent experiments (±SEM); the values of the unstimulated wt control were set at 1. Statistical calculation was performed for influence of dominant negative H-RasN17 compared with H-Ras-wt on costimulation-induced cofilin dephosphorylation (A; *, p < 0.05) and Akt phosphorylation (B; *, p < 0.05).

 
Upon costimulation via CD3 x CD28 H-Ras-wt-expressing cells were able to dephosphorylate cofilin. As expected, costimulation led to phosphorylation of Akt in these cells. Note that the phosphorylation of PKB/Akt was completely prevented if PB-T were costimulated in the presence of the PI3K inhibitor Ly294002 or wortmannin (data not shown), confirming that this phosphorylation event is PI3K dependent. In marked contrast, cells expressing the dominant negative H-Ras form (H-RasN17) exhibited significantly reduced cofilin dephosphorylation in response to CD3 x CD28 costimulation compared with H-Ras-wt-expressing cells (p < 0.05). In line with these findings, phosphorylation of Akt (Fig. 7B) was also inhibited within these cells (p < 0.05). Conclusively, our experiments demonstrate that in untransformed human T lymphocytes, Ras induces activation of PI3K and dephosphorylation of the actin-remodeling protein cofilin.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Activation of T lymphocytes requires a costimulation-dependent remodeling of the actin cytoskeleton. We have shown previously that cofilin, a central actin-remodeling protein, is activated through T lymphocyte costimulation (7, 8). We also demonstrated that the activity of cofilin is essential for the execution of T lymphocyte activation processes, e.g., formation of the immunological synapse, cytokine production, and proliferation (9). In this study we have identified a signaling pathway that regulates the activation of cofilin in untransformed human PB-T. The central mediator of this pathway is the GTPase Ras. The induction of cofilin dephosphorylation requires the combined activities of two Ras effectors, namely, MEK and PI3K. PKC and/or RasGRP (33, 34) may link costimulation to Ras activation. From our data we cannot distinguish whether one or both of them are involved, because phorbol esters not only bind to and activate PKC, but also associate with and activate RasGRP (35, 36, 37). Moreover, inhibitors of PKC may down-regulate the activity of RasGRP by preventing the activation-related phosphorylation of RasGRP (38).

Initially we analyzed the signaling cascades regulating the phosphorylation state of cofilin in murine fibroblasts because essential experimental protocols (e.g., feasible transfection systems) were not available for untransformed human PB-T. In fibroblasts, Ras and the combined activities of its effectors, MEK and PI3K, were required to induce dephosphorylation of cofilin (39). However, the existence of an identical regulatory cascade in T lymphocytes seemed unlikely, because it was reported that Ras would not activate PI3K in T lymphocytes (23, 24). Through a newly developed approach using nucleofection of untransformed resting human PB-T, we were able to demonstrate that Ras is indeed a central regulator in the cofilin dephosphorylation pathway. Considering the fact that cofilin activation enhances actin dynamics, this observation may provide a mechanistic explanation for the ability of activated Ras to alter the morphology of T lymphocytes when expressed constitutively (22).

Surprisingly, PI3K was also activated by Ras in human PB-T. In this study the two Ras effectors, MEK and PI3K, needed to work in combination, because inhibition of either of them abolished Ras- or costimulation-induced cofilin dephosphorylation in PB-T. The earlier assumption that Ras was not able to activate PI3K in T lymphocytes (23, 24, 30, 31) resulted from experiments using the human T lymphoma line Jurkat as an experimental model for T lymphocytes. In these cells, phorbol ester treatment did not induce PI3K activity while activating Ras and MEK (23). These data led to the conclusion that Ras signaling in T lymphocytes differs from that in other cell types such as fibroblastic and epithelial cells, where Ras is able to activate PI3K (24). Our data clearly show that such T cell-specific peculiarities are not observed when Ras signaling is investigated in untransformed human PB-T. First, as opposed to Jurkat lymphoma cells, in PB-T phorbol esters clearly activate not only Ras, but also the PI3K substrate Akt. Second, activated Ras induces phosphorylation of the PI3K substrate PKB/Akt. Third, PI3K inhibitors block the dephosphorylation of cofilin induced by transiently expressed activated Ras protein (Fig. 4 and data not shown demonstrating similar effects of the PI3K inhibitor wortmannin) or induced by costimulation through CD3 x CD28. Finally, dominant negative Ras prevents the costimulation-induced phosphorylation of PKB/Akt and dephosphorylation of cofilin in untransformed human PB-T. Thus, with respect to Ras effectors, the T lymphoma line Jurkat is exceptional; however, this does not apply to T lymphocytes in general. Whether other T lymphoma cells also show this peculiarity and whether this finding has consequences for the respective malignant phenotype remain to be investigated.

The identification of PI3K as a Ras effector in T lymphocytes may in addition answer important questions regarding mechanisms of PI3K activation in T lymphocytes. T lymphocytes express members of the four known PI3K classes (1A, 1B, 2, and 3). As a basic principle (40), PI3K activation can be mediated either via Ras (applies to PI3K classes 1A, 1B, and 2) or via regulatory subunits that recruit the catalytic PI3K subunit through interactions of Src homology 2 domains with tyrosine-phosphorylated YXXM motifs into activated receptor complexes (applies to class 1A). In light of the earlier assumption that Ras was not able to activate PI3K in T lymphocytes (23), only PI3K activation via recruitment of regulatory subunits could be explained, leaving unsettled how class 1B and class 2 PI3K are activated. The coreceptor CD28 contains a YXXM motif in its cytoplasmic tail, hinting at recruitment of PI3K via CD28 (41, 42, 43). Yet, to date, an agreement about the importance of PI3K binding to CD28 for lymphocyte functions has not been achieved, because numerous studies yielded discrepant results (40). Furthermore, of the factors that have been implicated to be key for the recruitment/activation of PI3K during T lymphocyte activation (the {zeta}-chain (44), CD3 chains (45, 46), linker for activation of T cells, T cell receptor-interacting molecule, and Shc (47, 48, 49)), only TRIM contains the requisite YXXM motif in its sequence; for the other proteins, alternative activation mechanisms have to be assumed. Taken together, although it is without question that PI3K activity is induced in T lymphocytes upon Ag recognition on APC (50, 51), the molecular mechanisms by which PI3K is activated remain unclear. Our data open up a new aspect of this field. Ras-mediated activation of PI3K in T lymphocytes provides an as yet unrecognized mechanistic basis for how components downstream of the TCR complex that do not contain YXXM motifs, but are known to be key for PI3K recruitment/activation, may regulate PI3K.


   Acknowledgments
 
We thank B. Burgering and M. White for providing constructs, and S. Meuer for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft (Grant SFB405/A4). Back

2 G.H.W. and G.N. contributed equally to this work. Back

3 Current address: Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany. Back

4 Address correspondence and reprint requests to Dr. Yvonne Samstag, Ruprecht Karls University, Institute of Immunology, INF 305, 64293 Heidelberg, Germany. E-mail address: yvonne.samstag{at}urz.uni-heidelberg.de Back

5 Abbreviations used in this paper: PB-T, T PBL; EGFP, enhanced GFP; HA, hemagglutinin; MEK, MAPK/ERK kinase; PdBU, phorbol 12,13-dibutyrate; PKB, protein kinase B; PKC, protein kinase C; wt, wild type. Back

Received for publication October 28, 2004. Accepted for publication November 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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