Phosphatidylinositol 3-Kinase Functions As a Ras Effector in The

Phosphatidylinositol 3-Kinase Functions as a Ras Effector in the Signaling Cascade That Regulates Dephosphorylation of the Actin-Remodeling Protein Cofilin after This information is current as Costimulation of Untransformed Human T of October 1, 2021. Lymphocytes Guido H. Wabnitz, Gabriele Nebl, Martin Klemke, Andreas J. Schröder and Yvonne Samstag

J Immunol 2006; 176:1668-1674; ; Downloaded from doi: 10.4049/jimmunol.176.3.1668 http://www.jimmunol.org/content/176/3/1668

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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. Wabnitz,2 Gabriele Nebl,2 Martin Klemke, Andreas J. Schro¨der,3 and Yvonne Samstag4

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 Downloaded from 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 ؋ 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 like- http://www.jimmunol.org/ wise, 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. The Journal of Immunology, 2006, 176: 1668–1674.

ull activation of T lymphocytes requires costimulation, controlling both disassembly of existing and formation of new ac- 3

i.e., engagement of the TCR/CD3 complex by antigenic tin filaments (5). Phosphorylation of cofilin at Ser reduces the by guest on October 1, 2021 peptide bound to a MHC molecule plus interactions of actin binding capacity of cofilin (6). In unstimulated T PBL (PB- F 5 3 accessory receptors (e.g., CD2 or CD28) with their ligands (CD58 T), cofilin exists in its inactive Ser -phosphorylated form. T cell or CD80/CD86) on APCs. These processes involve the interplay of activation through costimulation of TCR/CD3 plus the coreceptor cell surface receptor proteins with intracellular signaling and adap- CD28 or CD2 stimulation induces the dephosphorylation of cofilin tor proteins and lead to the production of cytokines, such as the (7, 8). The activity of cofilin is crucially involved in T lymphocyte growth factor IL-2, and subsequent proliferation of the T lympho- activation processes (9). Thus, cell-permeable peptides that block cytes. Recent progress in this field demonstrates that the actin cy- the binding of cofilin to actin in human PB-T impair the formation toskeleton plays an important functional role in the linkage of sur- of the immunological synapse and inhibit the induction of cell face receptors to downstream events (1–4). proliferation and cytokine production. The serine kinases LIM mo- The actin cytoskeleton represents a highly dynamic network that tiv-containing protein kinases 1 and 2, testicular protein kinases 1 is influenced by a number of actin-regulating factors. Among and 2, and Nck-interacting kinase-related kinase/Nck-interacting these, the ubiquitously expressed actin-binding protein, cofilin, has kinase-like embryo-specific kinase phosphorylate cofilin (10–14). a special state, because it regulates filament remodeling, thereby Protein phosphatases that are able to dephosphorylate cofilin are the serine phosphatases PP1, PP2A (15), the slingshot phosphatases (16), and chronophin (17). Ruprecht Karls University, Institute of Immunology, Heidelberg, Germany In this study we have further characterized the signaling cascade Received for publication October 28, 2004. Accepted for publication November leading to cofilin dephosphorylation in untransformed human T 12, 2005. lymphocytes. Ras is known to be a central mediator of T lympho- The costs of publication of this article were defrayed in part by the payment of page cyte activation events (18). Interestingly, in anergic T lymphocytes charges. This article must therefore be hereby marked advertisement in accordance activation of Ras is blocked (19). Moreover, activated Ras is with 18 U.S.C. Section 1734 solely to indicate this fact. known to induce rearrangements of the actin cytoskeleton and al- 1 This work was supported by the Deutsche Forschungsgemeinschaft (Grant terations in the morphology of cells (20–22). Therefore, we ana- SFB405/A4). lyzed whether cofilin dephosphorylation in untransformed human 2 G.H.W. and G.N. contributed equally to this work. PB-T is related to Ras activation. We found that activated Ras 3 Current address: Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany. 4 Address correspondence and reprint requests to Dr. Yvonne Samstag, Ruprecht 5 Abbreviations used in this paper: PB-T, T PBL; EGFP, enhanced GFP; HA, hem- Karls University, Institute of Immunology, INF 305, 64293 Heidelberg, Germany. agglutinin; MEK, MAPK/ERK kinase; PdBU, phorbol 12,13-dibutyrate; PKB, pro- E-mail address: [email protected] tein kinase B; PKC, protein kinase C; wt, wild type.

indeed induced the dephosphorylation of cofilin in PB-T. Down- state of cofilin-GFP was determined using our standard protocol (see stream of Ras the combined activities of MAPK/ERK kinase above). Endogenous cofilin does not flaw this analysis, because its Mr ϳ (MEK) and PI3K are required to induce cofilin dephosphorylation. differs from that of cofilin-GFP (19 vs 50 kDa). This finding was surprising, because it was reported that in T lymphocytes Ras is not able to activate PI3K (23, 24). Yet this con- Determination of Ras activity clusion resulted from experiments performed with the T lym- The Ras activation assay was performed using a commercially available phoma line Jurkat. Our data clearly show that, in contrast to the system (Upstate Biotechnology). Briefly, cells (10 ϫ 106/sample) were situation in Jurkat cells (23), in untransformed human T lympho- lysed. Cleared lysates were incubated with bead-bound ras binding domain cytes PI3K functions as a Ras effector. 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 Materials and Methods revealed the level of Ras activation in the lysate. Cells and reagents Detection of the activated forms of ERK and PKB/Akt PB-T were prepared as described previously (25) and cultured in RPMI 1640 containing 10% FCS, 4 mM glutamine, 25 mM HEPES, penicillin, The activation levels of MEK and PI3K were determined using standard and streptomycin (Invitrogen Life Technologies). The enzyme inhibitors protocols; activation of MEK was analyzed through detection of the phos- used were LY294002 (Sigma-Aldrich) at 50 ␮M, wortmannin (Sigma- phorylated forms of the MEK substrates ERK1 and ERK2, whereas PI3K Aldrich) at 100 nM, U0126 (Promega) at 20 ␮M, Ro32-0432 (Calbiochem) activity was visualized through detection of the phosphorylated/activated at 5 ␮M, and B581 (Calbiochem) at 250 ␮M. form of the PI3K effector PKB/Akt. Cells were lysed in Laemmli sample buffer and sheared, and lysates were subsequently subjected to SDS-PAGE Stimulation of PB-T and Western blotting. Blots were stained sequentially with Abs against the Thr183/Tyr185-phosphorylated activated forms of ERK1/2 (Promega) and Downloaded from PB-T were stimulated via plate-bound Abs. The Abs used for coating of 473 tissue culture plates were the CD3 Ab BMA030 (a gift from R. Kurrle, the Ser -phosphorylated form of PBK/Akt (Promega). After stripping, Aventis, Bridgewater, NJ; IgG2a; 1 ␮g/ml) and the CD28 Ab CD28.2 (BD blots were reprobed with antisera detecting all forms of ERK1/2 (Promega) Pharmingen; IgG1; 10 ␮g/ml) or the respective IgG2a and IgG1 control and PKB/Akt (Santa Cruz Biotechnology). Abs (BD Pharmingen). PB-T were sedimented onto the Ab-coated plates (1500 rpm, 5 min) and subsequently incubated for the time periods indi- Flow cytometry cated in the figure legends. Cells were fixed and permeabilized before Ab staining. To this end, 5 ϫ http://www.jimmunol.org/ Transfections 105 PB-T were incubated for 5 min with 4% paraformaldehyde at 37°C. Expression vectors were cloned in our laboratory: pEGFP-C1-H-RasV12, The fixation was stopped by adding 2 vol of FACS buffer (0.5% BSA, 5% the coding sequence of H-RasV12 that is hemagglutinin (HA) tagged (26), FCS, and 0.07% NaN3). Cells were then spun down and permeabilized for was subcloned into pEGFP-C1 (BD Clontech) in frame with enhanced GFP 10 min at room temperature with 0.1% saponin in FACS buffer. After an ␮ (EGFP). The resulting RasV12-EGFP-C1 plasmid was taken for the rever- additional washing step, the cells were resuspended in 50 l of FACS ␮ sion into H-Ras-wild type (H-Ras-wt; V123G12) using the QuikChange buffer/0.1% saponin containing 1 l of the respective antiserum and incu- Site-Directed Mutagenesis XL Kit (Stratagene) according to the manufac- bated at room temperature for 15 min. Subsequently, cells were washed Ј turer’s instructions. Subsequently, another point mutation at position 17 and incubated with PE-labeled donkey anti-rabbit F(ab )2 (Dianova) for an (S173N17) was introduced, resulting in a dominant negative H-RasN17 additional 15 min. Finally, cells were washed and resuspended in 1% para- mutant (27). Additional constructs were pEGFP-N1-cofilin (the coding se- formaldehyde until quantification by flow cytometry using a FACSCalibur by guest on October 1, 2021 quence was subcloned into pEGFP-N1; BD Clontech) and pcDNA3-HA- (BD Biosciences). protein kinase B (PKB)/Akt (the coding sequence of HA-PKB/Akt (28) was subcloned into pcDNA3 (Invitrogen Life Technologies)). Coexpres- sion of GFP or GFP-H-RasV12 and the reporter proteins (cofilin-GFP and Determination of the phosphorylation state of transiently HA-PKB/Akt), respectively, was achieved through nucleofection of 20 ϫ expressed HA-PKB/Akt 106 PB-T with 9 ␮g of the reporter plasmid (pEGFP-N1-cofilin or Twenty-four hours after nucleofection, cells (equivalent to 5 ϫ 106 trans- pcDNA3-HA-PKB/Akt) plus 1 ␮g of pEGFP-C1-H-RasV12 or the control fected cells) were lysed in 500 ␮l lysis buffer (10 mM HEPES (pH 7.2), vector pEGFP-C1 following the protocol supplied by the manufacturer of 140 mM KCl, 5 mM MgCl , 2 mM EGTA, 0.2% Nonidet P-40, 0.1 mM the nucleofector (Amaxa). The transfection method led to ϳ50% GFP- 2 PMSF, 1ϫ proteinase inhibitor mix (Sigma-Aldrich; A-8340), and 1ϫ expressing cells with a MFI between 200 and 300. phosphatase inhibitor mix (Sigma-Aldrich; A-2850)), sheared (five times Determination of the phosphorylation state of cofilin pushing through a 27-gauge needle), and cleared (22,000 ϫ g, 10 min, 4°C). To separate HA-PKB/Akt from endogenous PKB/Akt, HA-PKB/Akt PB-T (3 ϫ 106) were lysed in Laemmli sample buffer, sheared, and sub- was immunoprecipitated. For immunoprecipitation, 2.5 ␮gofHAAbs jected to SDS-PAGE and Western blotting (polyvinylidene difluoride (Roche) were added to each lysate, and the lysate was incubated for2hat membranes). To determine the relative portion of phosphorylated cofilin, 4°C on a moving platform. After 1 h, protein G-Sepharose beads (Phar- blots were stained with a phospho-Ser3-cofilin-specific antiserum (Cell macia Biotech; 50 ␮l of 50% slurry in lysis buffer) were added to the Signaling Technology), stripped, and restained with a cofilin antiserum that lysates. The beads were washed three times with lysis buffer, resuspended detects all forms of cofilin (produced in our laboratory). Subsequently, the in 2ϫ Laemmli sample buffer, and denatured (95°C, 5 min), and the eluates signals were quantified by densitometry. For each sample, the quotient of were subjected to SDS-PAGE (10%). The gels were blotted onto polyvi- the signal intensities from phosphorylated cofilin vs total cofilin was cal- nylidene difluoride membranes. Blots were stained with Abs against the culated. It was set at 1 under control conditions, and all other values of the Ser473-phosphorylated form of PKB/Akt (Promega). After stripping, blots same experiment were calculated as multitudes (P-index). In addition, the were reprobed with the HA Abs to detect total HA-PKB/Akt. Staining with mean and SEM were calculated. Alternatively, the phosphorylation state of a GFP-antiserum (Invitrogen Life Technologies) indicates the presence of cofilin was determined using a one-dimensional NEpHGE as previously GFP-H-rasV12 that is coprecipitated, because this construct contains an described (8, 29). This method allows the detection of both phosphorylated HA tag. Subsequently, the signals were quantified by densitometry. For and unphosphorylated forms of cofilin within one gel due to the difference each sample the quotient of the signal intensities from phosphorylated HA- in their isoelectric point without the need for a phosphospecific antiserum. PKB/Akt vs total HA-PKB/Akt was calculated. It was set at 1 under control The two methods, phospho-cofilin Western blot and one-dimensional conditions, and all other values of the same experiment were calculated as NEpHGE, gave comparable results. multitudes (P-index). In addition, the mean and SEM were calculated. Determination of the phosphorylation state of transiently expressed cofilin-GFP Statistical analysis Six to 8 h after nucleofection, cells were treated for 16 h with inhibitors. Where indicated, data are presented as the mean Ϯ SEM, and the proba- Then cell lysates (equivalent to 4–6 ϫ 106 transfected cells) were prepared bility of differences was assessed using Students paired t test. A value of and subjected to SDS-PAGE and Western blotting. The phosphorylation p Ͻ 0.05 was considered statistically significant. 1670 Ras ACTIVATES PI3K IN T LYMPHOCYTES

Results Costimulation-dependent cofilin dephosphorylation in PB-T is sensitive to inhibitors of Ras, PI3K, and MEK To obtain information about the costimulation-triggered signaling FIGURE 2. Costimulation through CD3 ϫ CD28 or PMA stimulation cascade leading to cofilin dephosphorylation, we analyzed untrans- induces activation of Ras in human PB-T. PB-T were stimulated for 10 or formed human T cells in the presence of synthetic inhibitors for 30 min via cross-linked CD3 and CD28 Abs (CD3 ϫ CD28) or 10Ϫ8 M signaling molecules. To this end, PB-T were incubated with the PMA. In parallel, cells were settled on isotype control Abs (IgG). Activated Ras inhibitor B581 (250 ␮M), the MEK inhibitor U0126 (20 ␮M), Ras (Ras-GTP) was immunoprecipitated and detected as described in Ma- or the PI3K inhibitors LY294002 (50 ␮M) and wortmannin (100 terials and Methods. nM), respectively. Subsequently, cells were costimulated via plate- bound Abs directed against the TCR/CD3 complex and CD28 (CD3 ϫ CD28) or settled on an isotype control Ab mix. After 30 min, whole-cell lysates were prepared, and the phosphorylation MEK and PI3K are required downstream of Ras to induce state of cofilin was determined via Western blot analysis using a cofilin dephosphorylation phosphospecific antiserum. Fig. 1 shows that the cofilin dephos- To identify Ras effectors participating in the cofilin dephosphory- phorylation induced through CD3 ϫ CD28 costimulation was clearly lation pathway PB-T transfected with the Ras construct were in- diminished by LY294002, wortmannin, B581, and U0126. Thus, cubated in the presence or the absence of the MEK inhibitor these experiments strongly suggested that Ras, MEK, and PI3K are U0126 or the PI3K inhibitor LY294002. Each inhibitor alone was involved in the signaling cascade leading to cofilin dephosphorylation Downloaded from able to inhibit H-RasV12-induced cofilin dephosphorylation (Fig. upon costimulation of untransformed human PB-T. 4), revealing that the activities of both MEK and PI3K are required The activity of Ras in untransformed PB-T was analyzed downstream of Ras to induce cofilin dephosphorylation. This find- through precipitation of the activated GTP-bound form of Ras ing was surprising, because it contradicted the current opinion that, from cell lysates. Fig. 2 demonstrates that costimulation through unlike in other cell types, in T lymphocytes Ras was not able to CD3 ϫ CD28 induces an activation of Ras similar to that observed activate PI3K (23, 24, 30, 31). This opinion resulted from exper- after treatment with the phorbol ester PMA. In unstimulated PB-T, http://www.jimmunol.org/ iments performed with the human T lymphoma line Jurkat (23) in no Ras activity could be detected. which phorbol ester treatment led to the activation of Ras and Transiently expressed activated Ras protein induces the MEK, but not PI3K. dephosphorylation of cofilin in PB-T To analyze whether the discrepancy regarding PI3K activation through Ras in T lymphocytes stems from the use of different types For analysis of the role of Ras in the regulation of cofilin dephos- of T lymphocytes, we first reproduced the decisive experiments of phorylation in untransformed PB-T, transient transfection of an the previous study (23). Jurkat cells were treated with phorbol expression vector for a constitutively active variant of H-Ras (H- 12,13-dibutyrate (PdBU; Fig. 5A) or PMA (data not shown, results RasV12) was performed. Subsequently, the effects of activated H- are similar to those obtained with PdBU), and subsequently the by guest on October 1, 2021 RasV12 on the phosphorylation state of cofilin were analyzed. To activities of Ras, MEK, and PI3K were determined using standard be able to selectively analyze alterations in the phosphorylation protocols. Activation of Ras was analyzed through precipitation of state of cofilin in transfected cells, an expression vector for a the activated GTP-bound form of Ras, MEK activity through de- tagged form of cofilin was cotransfected, and the phosphorylation tection of the phosphorylated forms of the MEK substrates ERK1 indices of the tagged cofilin (cofilin-GFP) were determined. As and -2, whereas PI3K activity was visualized through detection of shown in Fig. 3, the expression of H-RasV12 in PB-T indeed in- the phosphorylated/activated form of the PI3K effector PKB/Akt. Ͻ duced dephosphorylation of the coexpressed cofilin-GFP ( p As shown in Fig. 5A, PI3K activity, which was already detect- 0.001). able 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 FIGURE 1. Ras, MEK, and PI3K are involved in the regulation of co- treatment induces not only Ras, MEK, and cofilin activation, but filin dephosphorylation after costimulation of human PB-T. Primary hu- also PI3K activity (Fig. 5B). In addition, the PKC dependency of man T cells were preincubated for 40 h with the Ras inhibitor B581 (B; 250 the observed phorbol ester effects was confirmed by pretreatment ␮M; lane 5) or for 30 min with the PI3K inhibitor LY294002 (LY; 50 ␮M; of the cells with the PKC inhibitor Ro32-0432. lane 3) or wortmannin (W; 100 nM; lane 4) or the MEK inhibitor U0126 To prove that PI3K functions as a Ras effector in untransformed (U0; 20 ␮M; lane 6). As solvent, control cells were treated with DMSO human T lymphocytes, H-RasV12 was again expressed in PB-T. (lanes 1 and 2). Subsequently, cells were left unstimulated (IgG; lane 1)or To monitor PI3K activity, we coexpressed a tagged form of PKB/ were costimulated for 30 min via plate-bound CD3 plus CD28 Abs Akt (HA-PKB/Akt). Subsequently, phosphorylation at the PKB/ (CD3 ϫ CD28; lanes 2–6). The phosphorylation state of cofilin in post- Akt sites known to reflect PI3K activity (amino acids Ser473 and nuclear lysates was determined through one-dimensional Western blot 308 analysis using an antiserum specific for the phosphorylated form of cofilin Thr of the wt PKB/Akt protein) was determined in the HA- (upper panel; p-cofilin). After stripping, the blot was reprobed with an tagged PKB/Akt. Coexpression of H-RasV12 and HA-PKB/Akt antiserum that recognizes cofilin independent of its phosphorylation state clearly induced phosphorylation of HA-PKB/Akt at the position (lower panel; total cofilin). corresponding to the phosphorylation site Ser473 (Fig. 6) and the The Journal of Immunology 1671 Downloaded from

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 http://www.jimmunol.org/ 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 ␮M; lanes 5 and 6) or the PI3K inhibitor LY294002 (LY; 50 ␮M; 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. by guest on October 1, 2021

FIGURE 3. Expression of activated Ras (H-RasV12) in untransformed dominant active (H-RasV12) mutant. The different transfectants ϫ human PB-T induces cofilin dephosphorylation. Freshly isolated PB-T were costimulated for 30 min (CD3 CD28) or left unstimulated were transfected with an expression vector for activated Ras (H-RasV12; (IgG). The phosphorylation states of cofilin and PKB/Akt in GFP- lane 2 and bar 2) or the respective empty expression vector (vector; lane positive cells were determined by staining with phosphospecific 1 and bar 1) plus an expression vector for GFP-tagged cofilin. Twenty-four Abs, followed by flow cytometry. Again, the expression of dom- hours after transfection, whole-cell lysates were prepared, and the phos- inant active H-RasV12 alone, without stimulation, was sufficient to phorylation state of cofilin-GFP was determined. A, Immunoblot detecting induce dephosphorylation of cofilin (Fig. 7A; IgG). Concomi- phosphorylated cofilin-GFP (P-cofilin-GFP) with a phospho-cofilin-spe- tantly, phosphorylation of Akt could be observed (Fig. 7B; IgG), cific antiserum. After stripping, cofilin-GFP (cofilin-GFP) was stained with again confirming that Ras induces the activation of PI3K. an antiserum detecting total cofilin. In addition, the expression of H-rasV12 Upon costimulation via CD3 ϫ CD28 H-Ras-wt-expressing was confirmed through staining of the blots with a Ras antiserum. B, Im- munoblot signals for P-cofilin-GFP and cofilin-GFP were quantified by cells were able to dephosphorylate cofilin. As expected, costimu- densitometry, and the P-indices of cofilin-GFP were calculated as de- lation led to phosphorylation of Akt in these cells. Note that the scribed in Materials and Methods. The P-index of cofilin-GFP in vector- phosphorylation of PKB/Akt was completely prevented if PB-T transfected cells was set at 1. The figure shows the mean and SEM P- were costimulated in the presence of the PI3K inhibitor Ly294002 -p Ͻ 0.001. or wortmannin (data not shown), confirming that this phosphory ,ءءء .indices of cofilin-GFP from 11 independent experiments lation event is PI3K dependent. In marked contrast, cells expressing the dominant negative H-Ras form (H-RasN17) exhibited sig- site corresponding to Thr308 (data not shown). Because these phos- nificantly reduced cofilin dephosphorylation in response to CD3 ϫ phorylation events depend on upstream PI3K activity (32), this CD28 costimulation compared with H-Ras-wt-expressing cells result definitely confirms that Ras is able to induce PI3K activity ( p Ͻ 0.05). In line with these findings, phosphorylation of Akt in human PB-T. (Fig. 7B) was also inhibited within these cells ( p Ͻ 0.05). Con- clusively, our experiments demonstrate that in untransformed hu- Costimulation through TCR/CD3 and CD28 induces cofilin man T lymphocytes, Ras induces activation of PI3K and dephos- dephosphorylation through a Ras-PI3K signaling cascade phorylation of the actin-remodeling protein cofilin. Final proof of the key function of Ras in the costimulatory signaling pathway leading to activation of PI3K and cofilin activation in Discussion human PB-T is provided by experiments using the expression of a Activation of T lymphocytes requires a costimulation-dependent GFP-tagged, dominant negative mutant of Ras (H-RasN17). In remodeling of the actin cytoskeleton. We have shown previously parallel, GFP-tagged H-Ras was expressed either as H-Ras-wt or that cofilin, a central actin-remodeling protein, is activated through 1672 Ras ACTIVATES PI3K IN T LYMPHOCYTES Downloaded from

FIGURE 6. H-RasV12 induces HA-PKB/Akt phosphorylation. A, Freshly isolated PB-T were transfected with an expression vector for ac-

tivated Ras (RasV12) or the respective empty expression vector (vector) http://www.jimmunol.org/ 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 by guest on October 1, 2021 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 .p Ͻ 0.05 ,ء .FIGURE 5. Phorbol esters induce the activation of Ras, PI3K, MEK, from three independent experiments 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- have identified a signaling pathway that regulates the activation of 0432 (5 ␮M; lanes 3 and 4) or were left untreated (lanes 1 and 2). Sub- cofilin in untransformed human PB-T. The central mediator of this sequently, cells were stimulated for 15 min with 10 ng/ml PMA (lanes 2 pathway is the GTPase Ras. The induction of cofilin dephosphor- and 4) or were left unstimulated (lanes 1 and 3). A and B, The activation ylation requires the combined activities of two Ras effectors, of Ras was determined through precipitation of Ras-GTP from cell lysates namely, MEK and PI3K. PKC and/or RasGRP (33, 34) may link (IP). Immunoprecipitates were analyzed on SDS-PAGE, and the amounts costimulation to Ras activation. From our data we cannot distin- of precipitated Ras-GTP were determined by staining the blot with a Ras antiserum (Ras-GTP). In parallel, whole cell lysates were prepared and guish whether one or both of them are involved, because phorbol subjected to SDS-PAGE and Western blotting (lysate). The blots were esters not only bind to and activate PKC, but also associate with stained with Abs against the activated Ser473-phosphorylated form of the and activate RasGRP (35–37). Moreover, inhibitors of PKC may PI3K substrate PBK/Akt (P-PKB/Akt) and the activated dually phosphor- down-regulate the activity of RasGRP by preventing the activa- ylated (Thr183/Tyr185) forms of the MEK-substrates ERK1 and -2 (P-Erk1, tion-related phosphorylation of RasGRP (38). 2), respectively. For the control, total amounts of PKB/Akt (PKB/Akt) and Initially we analyzed the signaling cascades regulating the phos- ERK1 and -2 (Erk1, 2) were determined. B, The blot was also stained with phorylation state of cofilin in murine fibroblasts because essential 3 an antiserum directed against Ser -phosphorylated cofilin and, after strip- experimental protocols (e.g., feasible transfection systems) were ping, was reprobed with a cofilin-antiserum. The cofilin signal intensities not available for untransformed human PB-T. In fibroblasts, Ras were quantified by densitometry. The bars show P-indices of cofilin in the and the combined activities of its effectors, MEK and PI3K, were presented experiment. The P-index of cofilin in untreated cells was set at 1. The data are representative of three independent experiments. 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 ac- T lymphocyte costimulation (7, 8). We also demonstrated that the tivate PI3K in T lymphocytes (23, 24). Through a newly developed activity of cofilin is essential for the execution of T lymphocyte approach using nucleofection of untransformed resting human activation processes, e.g., formation of the immunological syn- PB-T, we were able to demonstrate that Ras is indeed a central apse, cytokine production, and proliferation (9). In this study we regulator in the cofilin dephosphorylation pathway. Considering The Journal of Immunology 1673

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 con- sequences 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 (41–43). Yet, to date, an agreement about the impor- Downloaded from tance of PI3K binding to CD28 for lymphocyte functions has not FIGURE 7. Costimulation-induced PI3K activation and cofilin dephos- been achieved, because numerous studies yielded discrepant re- phorylation are dependent on H-Ras activation. Human PB-T were trans- sults (40). Furthermore, of the factors that have been implicated to fected with GFP-tagged H-Ras-wt (f), H-RasN17 (u), or H-RasV12 (^). ϫ ϫ be key for the recruitment/activation of PI3K during T lymphocyte Subsequently, cells were costimulated via CD3 CD28 (CD3 CD28) or ␨ settled on isotype control Abs (IgG) for 30 min. After harvesting, cells activation (the -chain (44), CD3 chains (45, 46), linker for acti- http://www.jimmunol.org/ were stained for phospho-cofilin (A) or phospho-Akt (B) and analyzed by vation of T cells, T cell receptor-interacting molecule, and Shc flow cytometry. For quantification, events were gated on GFP-positive (47–49)), only TRIM contains the requisite YXXM motif in its cells (note that all constructs were expressed at the same level; data not sequence; for the other proteins, alternative activation mechanisms shown). Depicted is the mean phosphorylation of the respective proteins have to be assumed. Taken together, although it is without ques- (calculated from mean fluorescence intensities) of three independent ex- tion that PI3K activity is induced in T lymphocytes upon Ag rec- periments (ϮSEM); the values of the unstimulated wt control were set at ognition on APC (50, 51), the molecular mechanisms by which 1. Statistical calculation was performed for influence of dominant negative PI3K is activated remain unclear. Our data open up a new aspect H-RasN17 compared with H-Ras-wt on costimulation-induced cofilin de- of this field. Ras-mediated activation of PI3K in T lymphocytes phosphorylation (A;*,p Ͻ 0.05) and Akt phosphorylation (B;*,p Ͻ 0.05). provides an as yet unrecognized mechanistic basis for how com- by guest on October 1, 2021 ponents downstream of the TCR complex that do not contain YXXM motifs, but are known to be key for PI3K recruitment/ the fact that cofilin activation enhances actin dynamics, this ob- activation, may regulate PI3K. servation may provide a mechanistic explanation for the ability of activated Ras to alter the morphology of T lymphocytes when Acknowledgments expressed constitutively (22). We thank B. Burgering and M. White for providing constructs, and Surprisingly, PI3K was also activated by Ras in human PB-T. In S. Meuer for critical reading of the manuscript. this study the two Ras effectors, MEK and PI3K, needed to work in combination, because inhibition of either of them abolished Ras- Disclosures or costimulation-induced cofilin dephosphorylation in PB-T. 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