Guanine nucleotide exchange factor PIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells

nucleotide exchange factor PIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells. Background: Rac 1 GTPase mediates glycogen phosphorylase activation and controls IL-2-stimulated T cell proliferation. Results: PKC (cid:2) activates (cid:3) PIX by serine phosphorylation and now this Rho-GEF activates Rac 1. Conclusion: IL-2-stimulated T cells migration and proliferation require the involvement of the PKC (cid:2) / (cid:3) PIX/Rac 1/PYGM pathway. Significance: This new signaling cascade may be a viable therapeutic

Co-stimulation of T cell receptor and CD28 T cell receptors lead to IL-2 expression and secretion. An autocrine effect is necessary for expressing the IL-2 receptor (IL-2R) ␣ chain (1,2) and ultimately to culminate in T cell clonal expansion (3,4). The IL-2 effect on T cells is not only restricted to the induction of lymphocyte proliferation only but, in the inflammatory response it is also necessary for T lymphocytes differentiation into effector T lymphocytes as well as regulatory T lymphocytes (5).
Binding of IL-2 to its high affinity receptor (IL-2R) drives the activation of a signaling network giving rise to many cellular responses, among them the three major signaling cascades best characterized are the Janus kinase (Jak)/STAT and MAPK pathways, which modulate gene expression and PI3K-mediated cell survival (4). To accomplish these cellular responses, IL-2-dependent T cells possibly require not only activation of these pathways but also a complex cooperation with other signaling networks mediated by tyrosine kinases such as lck, BTK (6), PLC␥ (7,8), and serine/threonine kinases such as protein kinase C family members (9,10), and some GTPases of the Rho family. In fact, it has been reported that RhoA cooperates with ERK-dependent signaling pathways to transcribe c-fos in response to IL-2 (11). Moreover, Rac 1 has also been found to participate in IL-2-induced actin cytoskeleton rearrangement in a murine T cell line (12). More recently, our group reported that Rac 1 binds and activates the glycogen phosphorylase muscle isoform (PYGM) 3 and thus established a novel metabolic pathway that participates actively in IL-2stimulated cell proliferation in human T cells (13).
Signals emanating from a large variety of membrane receptors: growth factor receptors (14,15), G protein-coupled receptors (16,17), and tyrosine kinases-linked receptors such as TCR (5,18), BCR (19,20), and IL2-R (13), actively regulate Rho GTPase effects. Like other small GTPases, Rho GTPases function as molecular switches that cycle between the inactive GDP-bound and the active GTP-bound state. In the active state, GTPases interact with downstream effector molecules to promote a variety of biological responses, such as control of the appropriate actin cytoskeleton reorganization in response to extracellular signals, and their significant implications in additional biological processes, where gene expression regulation, cell polarity, and cell migration have also been reported (21)(22)(23).
The transition between the inactive to the active state is regulated by guanine nucleotide exchange factors (GEFs) (21)(22)(23).
A key factor in the functioning of small GTPases lies in their selection and regulation of these GEFs. It is well established that upon IL-2/IL-2R ligation, Ras GEF, Son of Seven (Sos), associates to Grb2 and it is recruited through the adapter protein Shc, to the tyrosine-phosphorylated IL-2R ␤ chain. In this configuration, Sos activates Ras and consequently the MAPK pathway (24,25). Therefore, Sos exchange activity is indirectly regulated by tyrosine phosphorylation. However, the exchange activity of some GEFs of the Dbl family that activates Rac 1 GTPase are directly regulated by phosphorylation. In fact, in the immune system, Vav (Rac 1-specific GEF) must be tyrosine phosphorylated at residue 174 to turn on its GTPase activity (26,27). Nevertheless, Tiam-1 and STEF, both members of the Tiam GEF family where the former is mainly expressed in the brain and in the immune system and the latter in the brain, are two additional GEFs with higher specificity for Rac 1 (28,29) that are activated by threonine (30) and serine/threonine phosphorylation (31), respectively. Like Tiam-1, ␣PIX (also known as ARH-GEF6 or Cool-2) (32-34), a Rho-GEF primarily expressed in neurons and hematopoietic cells (34), had its exchange activity predicted to be regulated by serine/threonine kinases phosphorylation by phosphoproteomic analysis (35)(36)(37)(38). In the last few years, GTPases of the Rac subfamily gained increasing relevance in T cell biology (39,40). In contrast to its well established Sos-mediated Ras activation mechanism in IL-2-stimulated T cells, the identity of the Rac GEF responsible for Rac activation in IL-2-stimulated signaling has not been determined.
Here we show that subsequent to IL-2 stimulation ␣PIX-Rho-GEF mediates PYGM activation in Kit 225 T cells; an IL-2dependent human T cell line. Serines 225 and 488 of ␣PIX are critical to active Rac 1 and mediate PYGM activation in IL-2stimulated cells. By combining pharmacological and genetic approaches, we identified PKC as the serine/threonine kinase that controls the phosphorylation of these serines and consequently the Rac 1/PYGM axis.
These results reveal that Rac 1/PYGM pathway activation stimulated by IL-2 is achieved through ␣PIX. Furthermore, our results identify PKC as the intermediary between the activated IL-2⅐IL-2R complex and ␣PIX. This novel intracellular signaling pathway actively participates in the regulation of the IL-2stimulated T cell migration and proliferation.

EXPERIMENTAL PROCEDURES
Reagents-PKA inhibitor H-89 dihydrochloride, PKC inhibitors Gö6976 and Rottlerin, and PI 3-kinase inhibitor LY29004 hydrochloride were obtained from Sigma. Mouse monoclonal anti-HA antibody was obtained from Covance, mouse monoclonal anti-phosphoserine clone PSR-45 and rabbit monoclonal anti-glutathione S-transferase (GST) antibodies were from Sigma, mouse monoclonal anti-Rac 1 clone 23A8 antibody was obtained from Millipore, rabbit monoclonal anti-PKC␣ was obtained from Cell Signaling, and enhanced chemiluminescence (ECL) reagent was obtained from GE Healthcare. Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma. MISSION esiRNA human PRKCQ (gene synonym PKC, reference EHU093601), MISSION esiRNA human ARHGEF6 (gene synonym ␣PIX, reference EHU133681), and MISSION esiRNA targeting EGFP (reference EHUEGFP) were from Sigma. IL-2 cytokine was provided by the "AIDS Research and Reference Reagent Program," Division of AIDS (NIAD, National Institutes of Health).
Cell Culture and DNA/esiRNA Transfections-Kit 225 T cells were cultured as described by Hori et al. (41) in the presence of 16 units/ml of recombinant human IL-2. For transient transfections, cells were cultured in complete RPMI 1640 medium in the absence of IL-2 for 24 h. Thereafter, cells were washed, resuspended in 200 l of serum-free medium, and placed in an electroporation cuvette (0.4 mm, Sigma) containing 10 -20 g of different plasmids, or 15 ng of esiRNAs. Electroporation was carried out in a Gene Pulser Xcell Electroporator (Bio-Rad) at 260 V and 950 microfarads (13). The cuvette content was collected into 10 ml of complete RPMI 1640 medium and cultured in the absence of IL-2 for an additional 24 h.
Agonists and Inhibitors-Kit 225 T cells were maintained in the absence of IL-2 for 48 h and subsequently stimulated with 500 units/ml of IL-2 at 37°C (13). In some experiments, Kit 225 T cells were pretreated with 10 M H-89 (PKA inhibitor) or 20 M LY29004 (PI3K inhibitor) or 100 nM Gö6976 and different concentrations of Rottlerin (PKC inhibitors) for 1 h prior to IL-2 or PMA stimulation (13,42).
Activity Assay for Glycogen Phosphorylase-The glycogen phosphorylase activity assay was performed as previously described (43,44) with some modifications. Briefly, cells were washed twice with cold PBS and resuspended in 500 l of TES buffer (20 mM Tris, pH 7.4, 1 mM EDTA, 225 mM sucrose, 2.5 mM DTT, 0.1 mM PMSF, 1 g/ml of leupeptin, 1 g/ml of aprotinin). Samples were sonicated and centrifuged at 12,300 ϫ g for 10 min at 4°C. Total protein (100 g) was used to measure PYGM activity in assay buffer (50 mM KH 2 PO 4 , pH 7.5, 10 mM MgCl 2 , 5 mM EDTA, pH 8, 0.5 mM NADP ϩ , 1.5 units/ml of glucose-6-phosphate dehydrogenase, 1 unit/ml of phosphoglucomutase, 0.1 mg/ml of glycogen (all from Sigma). Assay buffer containing 300 l of TES without NADP ϩ , glycogen, phosphoglucomutase, and glucose-6-phosphate dehydrogenase was added to 100 g of total protein as a blank control. To carry out the metabolic activity assay the mixture was incubated at 37°C for 20 min. By placing samples on ice the reaction was stopped. Sample absorbances were detected at 340 nm in a spectrophotometer (Ultrospec 3100 pro, Amersham Biosciences). The amount of NADPH formed was determined using a standard curve of known NADPH concentrations (Sigma).
Rac 1 Activation Assay-Rac 1 pulldown assay was performed using a GST fusion protein containing the Rac 1 binding domain of PAK1 (GST-RBD-PAK1). Transfected and untransfected cells kept in the absence of IL-2 for 48 h were stimulated with IL-2 for 10 min and lysed as described in Ref. 45. Cell lysates were centrifuged at 12,300 ϫ g for 10 min at 4°C and incubated for 1 h at 4°C with 50 g of GST-RBD-PAK1 fusion protein coupled to glutathione-Sepharose beads. Precipitated proteins were eluted from beads using 2ϫ loading buffer (12 mM Tris, pH 6.8, 5% glycerol, 0.4% SDS, 140 mM 2-mercaptoethanol, 0.02% bromphenol blue), separated by SDS-PAGE, and analyzed by immunoblot with specific monoclonal antibodies. Immunoreactive bands were visualized using ECL.
Immunoprecipitation Assay-Transfected Kit 225 T cells with cDNA encoding for pMT2-HA-␣PIX or empty vector (pMT2-HA) or esiRNAs to knock down PKC as indicated were treated or not with 500 units/ml of IL-2 for 10 min. Cells were washed three times in ice-cold PBS and lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.25% Na-deoxycolate, 1 mM EDTA, 1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, 1 g/ml of aprotinin, 1 g/ml of leupeptin). Ectopic HA-␣PIX and endogenous ␣PIX were immunoprecipitated for 2 h at 4°C using anti-HA or anti-␣PIX antibodies. Immune complexes were recovered using Gamma Bind Plus-Sepharose beads (GE Healthcare, Pittsburgh, PA), washed, and eluted from beads and resolved electrophoretically by SDS-PAGE and analyzed by Western blot with anti-phosphoserine, anti-HA, or anti-␣PIX antibodies. Immunoreactive bands were visualized using ECL.
In Vitro Kinase Assay-Kit 225 T cells transfected with PKC (wild type), PKC K409R (dominant-negative), and/or the constitutively active forms of PKC␣ (PKC␣ A25E ), PKC⑀ (PKC⑀ A159E ), and PKC (PKC A148E ) were incubated in the presence or absence of 500 units/ml of IL-2 for 10 min at 37°C and washed twice with cold PBS. Thereafter, cells were lysed with lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 5 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM sodium pyrophosphate, 100 mM ␤-glycerophosphate, 10 g/ml of aprotinin, 1 mM PMSF, 10% glycerol, and 1% (v/v) Triton X-100) and lysates were clarified by centrifugation for 10 min at 12,300 ϫ g at 4°C. PKCs were immunoprecipitated with specific antibodies and immunocomplexes were recovered using Gamma Bind Plus-Sepharose beads (GE Healthcare). The immunocomplexes were washed twice with cold lysis buffer, twice with cold washing buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 20 g/ml of aprotinin, and 0.5% IGEPAL-360) and twice with reaction buffer (20 mM Tris, pH 7.4, 20 mM NaCl, 1 mM DTT, 10 mM MgCl 2 , and 1 mM MnCl 2 ). 500 ng of purified recombinant GST-␣PIX 204 -532 , which encompasses the two potential serine phosphorylation sites (Ser 225 and Ser 488 ) of ␣PIX (33), and 20 M ATP was then added to the reaction mixture. The in vitro kinase reaction was carried out for 30 min at 30°C after which it was stopped by adding 30 l of 2ϫ loading buffer. Proteins were separated by SDS-PAGE, followed by Western blot. Immunoreactive bands were visualized with anti-phosphoserine antibody and ECL.
Cell Migration Assay-esiRNA-transfected cell suspensions (2.5 ϫ 10 5 cells in a 100-l volume) were placed into the upper chamber, whereas 600 l of medium with or without IL-2 (500 units/ml) was introduced into the lower chamber. Both chambers were incubated overnight at 37°C in 5% CO 2 and 95% air. Cells in the upper and bottom chamber were recovered separately into equal volumes for cell counting. The percentage of migrating cells was determined as follows: [number of cells migrating (lower chamber)/total number of cells (cells in the lower chamber ϩ remaining cells in the upper chamber)]. Assay was performed using pore filters (8 m, Corning Costar Transwell and cell culture inserts were from Sigma) and cell counts were done in triplicate.
Cell Proliferation Measurement-esiRNA-transfected cells were seeded in 24-well plates in complete RPMI (10 6 cells/ml), and maintained in the absence of IL-2 for 48 h. Subsequently, cells (10 6 ) were incubated with 4 M PKH26 following the manufacturer's instructions (Sigma). A sample (10 4 cells) was taken as the start control, another sample (10 4 cells) was left untreated, and the remaining cells were incubated in the presence of IL-2 (16 units/ml). Fluorescence was measured every 24 h for 3 days to the monitor cell division rate on a FACSCalibur (BD Biosciences) flow cytometer. Data obtained were analyzed using ModFit LT 3.0.
Statistical Analysis-Student's t test for the mean of twopaired samples was used to determine the significance between data means (**, p Ͻ 0.05; ***, p Ͻ 0.001).

RESULTS
␣PIX-Rho-GEF Leads to PYGM Activation-To test if ␣PIX could be a link between IL-2 receptor and Rac 1 leading to the activation of PYGM, Kit 225 T cells were transfected with pMT2-HA or pMT2-HA-␣PIX and stimulated or not with 500 units/ml of IL-2 for 10 min, lysed, and PYGM activity was determined as described under "Experimental Procedures." As shown in Fig. 1A (first closed bar), IL-2 stimulated robust PYGM activity of empty vector-transfected Kit 225 T cells. This IL-2 stimulation of PYGM activity was already maximal and it was not further increased in ␣PIX-overexpressing cells (Fig. 1A, second closed bar compared with first closed bar). However, ␣PIX overexpression in the absence of IL-2 resulted in a significant increase of the PYGM activity when compared with control cells (Fig. 1A, second empty bar compared with first empty bar). Western blot shows ␣PIX expression levels (Fig. 1A, upper panel).
Next, we aimed to determine whether other GEFs such as Vav, Tiam-1, and/or STEF/Tiam-2 that preferentially activate Rac 1 could also modulate the activity of the PYGM in our cellular system. For that purpose, following the same procedure described above, Kit 225 T cells were transfected with pcDNA3-HA (empty vector), pEF-Vav, pcDNA3-HA-Tiam-1, and pcDNA3-HA-STEF, and 24 h post-transfection PYGM activity was determined in Kit 225 T cells unstimulated and stimulated with IL-2 for 10 min. As expected, IL-2 significantly increased PYGM activity compared with unstimulated cells (Fig. 1B, first and second bars). This maximal increase in the PYGM activity induced by IL-2 also occurred in the presence of Vav and STEF (Fig. 1B, fourth and eighth bars compared with the second bar). However, in contrast to the ␣PIX outcome, Vav or STEF overexpression per se did not modify PYGM activity (Fig. 1B, third and seventh bars). In sharp contrast, Tiam-1 overexpression dramatically blocked PYGM activity (Fig. 1B, sixth bar). Western blots show Vav, STEF, and Tiam-1 expression levels (Fig. 1B, upper panel).
Finally, to corroborate that ␣PIX was specifically regulating the glycogen phosphorylase activity in IL-2-stimulated T cells, ␣pix was knocked down in Kit 225 T cells. To this end, Kit 225 T cells were transfected with esiRNA human ARHGEF6 (␣PIX) or esiRNA targeting EGFP, as negative control. 24 h post-transfection PYGM activity was determined in Kit 225 T cells unstimulated and stimulated with IL-2 for 10 min. As shown in Fig. 1C, IL-2 stimulated robust PYGM activity in egfp(esiRNA)-transfected Kit 225 T cells. ␣pix knockdown (␣pix(esiRNA)) cells stimulated by IL-2 for 10 min did not show any PYGM activity (Fig. 1C, second closed bar compared with first closed bar). Furthermore, in the absence of IL-2 ␣pix knockdown, cells also displayed no PYGM activity, in contrast to ␣PIX overexpressing cells ( Fig. 1, C, second empty bar compared with A, second empty bar). Small aliquots of cell lysate from each condition were stored, electrophoretically resolved by SDS-PAGE, and followed by Western blot. Immunoreactive bands were visualized with specific antibodies as indicated. Fig.  1C, first and second panels show ␣PIX expression levels after esiRNA transfection and ␥ tubulin, respectively. The ␥-tubulin Western blot result shows that an equivalent amount of protein was used in each of the conditions analyzed.
Rac 1/PYGM Pathway Activation Depends on the Integrity of ␣PIX Serine 225 and 488 Residues-To demonstrate that ␣PIX functions as a Rac-activating molecule in Kit 225 T cells, ␣PIX was knocked-down with ␣pix(esiRNA), as we described above and the endogenous active Rac 1 was measured by the pulldown assay. As shown in Fig. 2A, in the absence of ␣PIX (knockdown), IL-2 was unable to stimulate Rac 1 activation (fourth lane compared with second lane). ␣PIX expression levels in the presence of esiRNA control (egfp) or ␣pix(esiRNA) were determined by Western blot (Fig. 2A, upper panel). Rac 1 detected in whole cell lysates shows that the total loaded proteins are equivalent in all lanes ( Fig. 2A, third panel).
Directed mutagenesis was used to investigate a putative role of serine 225 and 488 residues in activation of the Rac 1/PYGM pathway. To this end, the following single ␣PIX mutants were ␣PIX wt overexpression induced massive Rac 1 activation, which was not further increased by IL-2 stimulation (Fig. 2B, upper panel, lanes 3 and 4). In contrast, both ␣PIX single and double mutants dramatically blocked Rac 1 activation stimulated by IL-2 (Fig. 2B, upper panel, sixth,  eighth, and tenth lanes). Furthermore, overexpression of these ␣PIX mutants in unstimulated cells did not induce any Rac 1 activation as it was observed with the ␣PIX wild type form (Fig.  2B, upper panel, fifth, seventh, and ninth lanes compared with  third lane).
Next, the effect of these ␣PIX mutants on PYGM activity was also examined. As shown in Fig. 2C, IL-2 stimulated PYGM maximal activity of both empty vector and ␣PIX wt transfected Kit 225 T cells (Fig. 2C, second and fourth bars, respectively). ␣PIX overexpression without IL-2 stimulation also induced a significant increase in PYGM activity in comparison to control cells (Fig. 2C, third bar compared with first bar). In contrast, PYGM activity was completely blocked by both the single serine and double serine mutants with or without IL-2 stimulation (Fig. 2C, five right bars).
nPKCs Regulate Rac 1/PYGM Pathway Activation-To search for additional kinases also involved in IL-2 early signaling leading to PYGM activation, the possible involvement of PKA, PI3K, and/or PKCs was examined. To this end, Kit 225 T cells deprived of IL-2 for 48 h were pretreated for 1 h with vehicle, 10 M H-89 (a PKA inhibitor), 20 M LY294002 (a specific inhibitor of PI3K), 100 nM Gö6976 (an inhibitor of classic PKCs, mainly ␣ and ␤), or 5 M Rottlerin (which was initially described as a selective inhibitor of the novel PKC isoform ␦ (46)) and was subsequently described to inhibit also PKC (47,48), followed by stimulation or not with 500 units/ml of IL-2 for 10 min. PYGM activity was determined as described above. Inhibition of PKA, PI3K, or classic PKCs did not affect PYGM activity stimulated by IL-2 (Fig. 3A, fourth, sixth, and eighth bars compared with second bar); notwithstanding, Rottlerin efficiently blocked PYGM activity stimulated by IL-2 (Fig. 3A, tenth bar) in a concentration-dependent manner (Fig. 3B). 2.5 M Rottlerin was the minimal concentration leading to the maximal blockage (Fig. 3B).
To verify a putative connection between nPKC and PYGM in Kit 225 T cells, PMA, a natural DAG analog, was used to directly activate PKC. Kit 225 T cells deprived of IL-2 for 48 h were stimulated with 500 units/ml of IL-2 for 10 min or 1 M PMA for 15 min. PMA was able to stimulate maximal PYGM activity; a response equivalent to the one produced by IL-2 (Fig.  3C, second and third bars). Accordingly, IL-2-and PMA-stimulated PYGM activity was blocked in Rottlerin (2.5 M, for 1 h) pretreated cells (Fig. 3C, fifth and sixth bars). Next, the impact of PKCs inhibition on Rac 1 activation stimulated by IL-2 was examined. Results presented here confirmed that IL-2 stimulation correlated with an increase of the Rac 1 active form (Fig.  3D, first panel, second lane). IL-2 stimulated Rac 1 activation was unaffected when classical PKCs were inhibited by 100 nM Gö6975 (Fig. 3D, first panel, fourth lane). However, 2.5 M Rottlerin almost completely blocked IL-2-stimulated Rac 1 activa- To further characterize the involvement of nPKCs in signaling pathways stimulated by IL-2 leading to Rac 1/PYGM activation, Kit 225 T cells were transiently transfected with pcDNA3 (empty vector) or with cDNAs encoding the ␣, ⑀, and constitutively active isoforms of PKCs for 24 h, and Rac 1 activation was analyzed. As shown in Fig. 4A, transfection of the constitutively active form of PKC activated Rac 1 to a level comparable with those obtained by IL-2 stimulation. The potent stimulating effect of PKC was specific given that neither activated PKC␣ or PKC⑀ had any effect on Rac 1 activation. In agreement with this neither PKC␣ nor PKC⑀ increased the basal level of PYGM activity, whereas constitutively active PKC induced PYGM activity to levels comparable with that stimulated by IL-2 (Fig. 4B).
To confirm the involvement of PKC in the Rac 1/PYGM pathway, Rac 1 and PYGM activation was determined in Kit 225 T cells overexpressing PKC (wt), PKC A148E (constitutively active form of PKC), and PKC K409R (dominant-negative form of PKC) with or without IL-2 stimulation. First, immunoblotting showed that all forms of PKC were equally expressed (Fig.   4, C, third panel, and D, upper panel). As shown in Fig. 4C, transfection of the PKC constitutively active form was found to activate Rac 1 to levels comparable with those stimulated by IL-2. In fact, IL-2 stimulation of cells overexpressing PKC (wt) or PKC A148E did not increase Rac 1 activation any further (Fig.  4 C, fourth and sixth lanes compared with second). In contrast, transfection with PKC K409R completely abolished Rac 1 activation either with or without IL-2 stimulation (Fig. 4C, seventh and eighth lanes). Similar results were obtained when PYGM activity was measured. PKC A148E overexpression alone was enough to stimulate maximal PYGM activity; equivalent to PYGM activity stimulated by IL-2 (Fig. 4D, fifth bar compared with second, fourth, and sixth bars). Finally, PKC K409R overexpression completely blocked PYGM activity either with or without IL-2 stimulation (Fig. 4D, seventh and eighth bars).
To confirm results obtained with PKC demonstrating that this serine-threonine kinase functions as a Rac 1/PYGM-activating molecule in Kit 225 T cells, PKC was knocked down with pkc (esiRNA), as described above. The endogenous Rac 1 active state was measured by the pulldown assay. As shown in Fig. 4E, in the absence of PKC (knockdown) IL-2 stimulation was unable to induce Rac 1 activation (fourth lane compared with second lane). The PKC expression level in cells transfected with esiRNA control (egfp) or pkc(esiRNA) was determined by Western blot (Fig. 4E, upper panel). Rac 1 detected in whole cell lysates shows that the loaded proteins were equivalent in all lanes (Fig. 4E, third panel). When PYGM activity in PKC knockdown Kit 225 T cells stimulated with IL-2 was examined, it was observed that in the absence of PKC IL-2 was unable to stimulate PYGM activation (Fig. 4F). Small aliquots of whole cell lysate from each condition were stored, electrophorectically separated on SDS-PAGE, and followed by Western blot. Immunoreactive bands were visualized with specific antibodies as indicated. Fig. 4F (first and second panels) shows PKC expression levels after esiRNA transfection and ␥-tubulin, respectively. ␥-Tubulin blot analysis indicates that equivalent amounts of protein were used in SDS-PAGE analysis.
PKC Controls ␣PIX Phosphorylation in Vivo and in Vitro-To test whether or not nPKCs could phosphorylate ␣PIX serine residues when Kit 225 T cells were stimulated by IL-2, HA-␣PIX overexpressing cells were pretreated or not with 2.5 M Rottlerin for 1 h, and stimulated or not with 500 units/ml of IL-2 for 10 min. HA-␣PIX present in cell lysates was immunoprecipitated using anti-HA antibody, as described under "Experimental Procedures." As shown in Fig. 5A (first left panel, fourth lane), IL-2 stimulated ␣PIX serine phosphorylation, which was blocked by Rottlerin (Fig. 5A, first right panel, fourth  lane). Given that these experiments were performed in ␣PIX overexpressing cells, endogenous ␣PIX phosphorylation was also examined. Kit 225 T cells were transfected with pkc (esiRNA) to knockdown PKC and cell lysates were immunoprecipitated using anti-␣PIX antibody, as described under "Experimental Procedures." As shown in Fig. 5B (fourth lane 4  compared with second lane), in the absence of PKC expression, IL-2 was unable to induce ␣PIX serine phosphorylation.
To determine the amount of immunoprecipitated ␣PIX, membranes that were used to examine the ␣PIX serine phosphorylation were stripped and reblotted with anti-␣PIX antibody (Fig. 5B, second panel). PKC expression levels in the presence of esiRNA control (egfp) or pkc (esiRNA) present in whole cell lysates were also examined by SDS-PAGE and followed by Western blot. Immunoreactive bands were visualized using anti-PKC antibody (Fig. 5B, third panel). ␥ Tubulin shows that , and E, cell extracts were used to measure Rac 1 activation by affinity precipitation assays. Precipitated active Rac 1 (Rac 1-GTP) and total Rac 1 from cell lysates were analyzed by Western blot (W.B.) using anti-Rac 1 specific antibody. Results are representative of three independent experiments. The expression levels of PKC␣ A25E , PKC⑀ A159E , and PKC A148E were visualized using anti-PKC␣ antibody to detect PKC␣ A25E , and anti-HA antibody to detect both PKC⑀ A159E and PKC A148E , and endogenous PKC (E) was visualized using anti-PKC antibody. B, D, and F, cell lysates were used to examine glycogen phosphorylase activity. Results show the mean of three independent experiments Ϯ S.D., and statistical analysis shows a significant difference (***, p Ͻ 0.001) in histograms B and D and (**, p Ͻ 0.05) in histogram E. Immunoreactive bands in B and D corresponding to the expression levels of PKC␣ A25E , PKC⑀ A159E , and PKC A148E were visualized using anti-PKC␣ antibody to detect PKC␣ A25E , and anti-HA antibody to detect both PKC⑀ A159E and PKC A148E . Endogenous PKC (E) and ␥-tubulin (E) were visualized using anti-PKC and anti-␥-tubulin antibodies total protein was equivalent in all assay conditions (Fig. 5B,  fourth panel). These results suggest that ␣PIX serine phosphorylation stimulated by IL-2 requires PKC protein expression.
As shown in Fig. 2, ␣PIX serine residues 225 and 488 are required for Rac 1 and PYGM activation in IL-2-stimulated Kit 225 T cells. Therefore, to determine whether or not PKC could phosphorylate the ␣PIX region comprising both serines, an in vitro kinase assay was performed using a GST-␣PIX 204 -532 fusion protein as an exogenous substrate for PKC. The GST moiety was fused to the ␣PIX region spanning from 204 to 532 residues, as described under "Experimental Procedures." Briefly, to carry out the in vitro kinase assay PKC␣ A25E , PKC⑀ A159E , and PKC A148E were immunoprecipitated from cell lysates derived from stimulated or unstimulated Kit 225 T cells and immunocomplexes were incubated with ATP and GST-␣PIX 204 -532 . Subsequently, proteins were resolved by SDS-PAGE followed by Western blot and GST-␣PIX 204 -532 serine phosphorylation was visualized using an anti-phosphoserine antibody. As illustrated in Fig. 5C, PKC A148E induced robust GST-␣PIX 204 -532 serine phosphorylation and it was not modified by IL-2 stimulation (fifth and sixth lanes). However, serine phosphorylation was not detectable either in the presence of PKC␣ A25E or PKC⑀ A159E (Fig. 5C, from first to fourth  lanes).
To confirm PKC involvement in the ␣PIX 204 -532 region serine phosphorylation, Kit 225 T cells were transiently transfected with pcDNA3 (empty vector) or with cDNAs encoding PKC (wt), PKC A148E (constitutively active form), and PKC K409R (dominant-negative form). Equal expression levels of these PKC forms in Kit 225 T cells were confirmed by immunoblotting (Fig. 5D). As shown in Fig. 5D (second lane), IL-2 stimulates GST-␣PIX 204 -532 serine phosphorylation in Kit 225 T cells overexpressing PKC (wt). When the effect of PKC A148E overexpression on GST-␣PIX 204 -532 phosphorylation was examined, a stronger level of exogenous substrate phosphorylation was observed than that found in cells overexpressing PKC (wt) and stimulated by IL-2 (Fig. 5D, third lane compared with second lane). IL-2 stimulation of PKC A148E overexpressing cells did not increase the phophorylation level further than that of unstimulated PKC A148E overexpressing cells (Fig. 5D, third and fourth lanes). In contrast, transfection with PKC K409R (PKC, dominant-negative) completely blocked GST-␣PIX 204 -532 phosphorylation independently of IL-2 stimulation (Fig. 5D, fifth and sixth lanes).
PKC and ␣PIX Are Needed for IL-2-induced Chemotaxis and Proliferation of Kit 225 T Cells-To investigate the role of PKC and ␣PIX in IL-2-stimulated Kit 225 T cell migration, cells were transfected with egfp (as control), pkc, or ␣pix (esiRNAs) and their effects on the IL-2-induced T cell chemotaxis through polyethylene terephthalate membranes were examined. As shown in Fig. 6A, IL-2 stimulated robust migration of Kit 225 T cells (first solid bar). On the other hand, lack of either PKC or ␣PIX expression abolished Kit 225 T cells migration stimulated by IL-2 (Fig. 6A, second and third solid  bars). At the end of the experiment, cells in the upper and lower chambers from each transfected condition were mixed. Cells were lysed and whole cell lysates were separated by SDS-PAGE followed by Western blot. The expression levels of ␣PIX and PKC were visualized using specific antibodies as indicated in Fig. 6A (first and second panels). ␥-Tubulin Western blot analysis shows that equal amounts of protein were used (Fig. 6A,  third panel). To evaluate the role of PKC and ␣PIX in IL-2-stimulated cell proliferation, esiRNAs for egfp, pkc, and ␣pix were used. Cell proliferation was analyzed after flow cytometry by monitoring the decrease in fluorescence of dye PKH6 incorporated in cell membrane, which is diluted approximately 2-fold with each cell division. PKH26-labeled cells were treated with 16 units/ml of IL-2 every 24 h for 3 days. In control cells (egfp (esiRNA) transfected cells) cultured for 3 days, IL-2 stimulation resulted in approximately a 2-fold increase in cell number (Fig.  6B, closed circles) compared with IL-2-unstimulated cells (Fig.  6B, open circles). Remarkably, IL-2-stimulated cell proliferation was significantly reduced with either pkc (esiRNA) (Fig. 6B, closed triangles) or with ␣pix(esiRNA) (Fig. 6B, closed diamonds) knockdown. At the beginning of the experiment, 3 ϫ 10 5 cells from each transfected condition were taken and lysed. Total protein in lysates was separated by SDS-PAGE followed by Western blot. The expression level of ␣PIX and PKC was visualized using specific antibodies as indicated in Fig. 6B (first  and second panels). ␥-Tubulin Western blot analysis indicates that equal amounts of protein were used in the analysis (Fig. 6B,  third panel).

DISCUSSION
Small GTPases of the Rho family actively participate in the immune response after antigenic stimulation, allowing for appropriate actin cytoskeleton reorganization and regulation of transcription factors activity (53). Cooperation between transcription factors such as NFAT, NF-B, and JNK is key to guarantee an adequate T cell clonal proliferation; in part by regulating transcription of IL-2 and the ␣ chain of the IL-2R␣ (49). Recently, we reported that upon IL-2R activation in Kit 225 T cells, the Rac 1 GTPase active form binds to and activates the metabolic enzyme PYGM, leading to cell proliferation (13). In the present study, we show that the IL-2/IL2-R engagement signals to the Rac 1/PYGM pathway through PKC and the GEF ␣PIX. More importantly, we identify ␣PIX as a Rac 1-specific GEF in IL-2-stimulated T cells and provide novel evidence demonstrating that ␣PIX requires serine phosphorylation by PKC to control the Rac 1/PYGM pathway, and thereby regulate T cell migration and proliferation.
Signals emanating from membrane receptors such as TCR, BCR, and IL-2R leading not only to the activation of proteintyrosine kinases, but also of serine/threonine kinases, can positively regulate downstream effector molecules, including small GTPases of the Ras superfamily (18,50). However, little is known about which Rho-GEF activates Rho GTPases after IL-2 receptor activation. Kit 225 T cells express IL-2R constitutively and depend exclusively on IL-2 for cellular proliferation (41). This feature represents an important advantage for IL-2-stimulated signaling studies and this cellular system has emphasized the importance of RhoA (51) and Rac 1 (12,13) in T cell biology regulated by IL-2.
Given that tyrosine phosphorylation of cellular proteins is one of the most important and characteristic events in early cell signaling stimulated by IL-2, Osinalde et al. (35) used high resolution mass spectrometry, combined with phosphotyrosine immunoprecipitation and stable isotope labeling by amino acids in cell culture (SILAC) to identify 172 tyrosine-phosphorylated target proteins; among which is ␣PIX-RhoGEF. This result prompted us to examine the potential participation of ␣PIX in PYGM activation. Overexpression experiments with ␣PIX demonstrated a significant increase in PYGM activity in the absence of any stimuli. ␣PIX overexpression promotes its own spontaneous dimerization in the absence of any stimuli, and in this configuration ␣PIX activates Rac 1 (52). Furthermore, in the presence of IL-2, ␣PIX-transfected cells reached a maximum level of PYGM activity. However, these results do not exclude the possibility that other GEFs capable of undergoing tyrosine phosphorylation may be involved in this signaling FIGURE 6. PKC/␣PIX pathway mediates IL-2-stimulated Kit 225 chemotaxis and proliferation. A, esiRNA (egfp, pkc, ␣pix) transfected Kit 225 T cells migration was studied in a Transwell assay. Data representing the percentage of migrating cells are expressed as the mean of three independent experiments Ϯ S.D. and statistical analysis shows a significant difference (***, p Ͻ 0.001). At the end of the assay, cells were recovered, lysed, and cell lysates were analyzed by Western blot (W.B.) using specific antibodies as indicated. B, esiRNA (egfp, pkc, ␣pix)-transfected Kit 225 T cells were stained with PKH26. Fluorescence was analyzed before IL-2 stimulation (ϪIL-2 (0 h) and after every 24-h incubation with IL-2 (ϩIL-2) for 3 consecutive days. Results represent the mean of three independent experiments Ϯ S.D. and the statistical analysis showed a significant difference (**, p Ͻ 0.05 and ***, p Ͻ 0.001). 3 ϫ 10 5 Kit 225 T cells were taken for each transfection condition, lysed, and cell lysates were analyzed by Western blot using specific antibodies as indicated.
pathway stimulated by IL-2. In fact, following this hypothesis we proposed that Rac 1 might also require Vav Rho-GEF to bind to and activate PYGM. Vav is the main GEF for Rac 1 in the hematopoietic system and its GEF function targeting the Rho family of GTPases is modulated by tyrosine phosphorylation at residue 174 (26,27). Evans et al. (53) also reported than in peripheral blood lymphocytes, Vav is tyrosine phosphorylated after IL-2 stimulation. However, in Vav overexpressing Kit 225 T cells there is no increase of PYGM activity. In our view, this was quite an expected result. This is because specifically Vav Tyr 174 must be phosphorylated (26,27) to release its DH domain from its inhibitory configuration and thus gain access to and activate Rac 1 (27). When the effect of IL-2 on Tyr 174 phosphorylation was examined, it was observed that this cytokine was not able to stimulate Tyr 174 phosphorylation (data not shown). These results do not contradict the observations reported by Evans et al. (53), given that this group described general tyrosine phosphorylation of Vav occurring mostly in its SH2 domain and located at Vav carboxyl-terminal region; whereas, Tyr 174 is located between Vav CH and DH domains at the amino-terminal region (26,27).
From the start our results showed that ␣PIX, and not Vav, mediated PYGM activation in IL-2-stimulated Kit 225 T cells. Although, additional GEFs from the Dbl family of exchange factors such as Tiam-1 and STEF are capable of activating Rac 1 (28,54), these GEFs have not been reported as being activated by tyrosine phosphorylation, but rather Tiam-1 was reported being activated by threonine phosphorylation (30), whereas STEF was reported being activated by serine/threonine phosphorylation (31). Even so, their effects on PYGM activity were evaluated. When these GEFs were individually overexpressed in Kit 225 T cells, it was observed that neither of them increased PYGM activity. Moreover, overexpression of Tiam-1 seems to have a dominant-negative effect on PYGM activity. Furthermore, when we examined the effects of ␣PIX knockdown with ␣pix(esiRNA) on either PYGM activity or Rac 1 activation, it was observed that the absence of the ␣PIX-Rho-GEF expression was enough to block activation of the Rac 1/PYGM pathway. Collectively, these results strongly suggest that ␣PIX is the only GEF responsible for activation of the Rac 1/PYGM pathway in Kit 225 T cells stimulated by IL-2.
Even if the ␣PIX predicted phosphorylation sites were the serines phosphorylated at residues 225 and 488 rather tyrosine residues (35), this prediction does not contradict experimental evidence obtained from SILAC-based quantitative phosphoproteomics data (35), because a protein enriched by immunoprecipitation with antiphosphotyrosine antibody upon IL-2 stimulation does not necessarily mean that it has been tyrosine phosphorylated in response to that cytokine. Any non-tyrosine-phosphorylated proteins may also be enriched if it is bound to a tyrosine-phosphorylated protein. The prediction that ␣PIX serine residues 225 and 488 may be susceptible to being phosphorylated in Kit 225 T cells (35) is consistent with the phosphoproteomic data obtained from ES and iPS cells (36), KG1 AML cells (37), and colorectal cancer cells (38).
From a functional point of view, phosphorylation/dephosphorylation cycles are major early events in intracellular signaling cascades. Therefore, the importance of these two ␣PIX-RhoGEF residues (Ser 225 and Ser 488 ) on Rac 1 and the subsequent PYGM activation was examined. The results obtained are compelling; both residues are essential to control Rac 1 activation and PYGM enzymatic activity. In addition, by pharmacological and genetic (either by PKC loss or gain of function) approaches we were able to find out that PKC was in control of ␣PIX phosphorylation and therefore activation of the Rac 1/PYGM pathway in Kit 225 T cells. The involvement of PKC in this cellular model is a novel finding although not a surprising one; given that its expression is restricted to certain tissues and cell types, including T cells (55). Since the relevance of this serine/threonine kinase in regulating the dynamics of the immunological synapse was described (56 -58), many additional functions have been discovered, such as the control of NF-B, AP-1, and NFAT transcription factors activation, which regulate the expression of proinflammatory cytokines and anti-proapoptotic molecules Bcl-XL (59). Moreover, the adhesive capacities of T lymphocytes (60) is the mechanism through which stable adhesion between T cells and antigen presenting cells is achieved (55,61,62). More recently, a new role for PKC in T cell physiology was described; i.e. PKC participation in the CCR7 downstream signaling driving T cell migration (63). Therefore, the present findings are in agreement with those reported by Cannon et al. (63) that not only stimulated IL-2 T cell proliferation but also T cell migration is regulated by the PKC/␣PIX axis upstream of Rac 1/PYGM.
In conclusion, our findings reveal the mechanism through which IL-2 stimulates Rac 1 activation in Kit 225 T cells. We identified ␣PIX as the GEF that specifically activates Rac 1 and consequently regulates PYGM activation, as well as the molecular mechanism of ␣PIX activation of the Rac1/PYGM pathway. Regulation of this novel metabolic pathway requires that PKC phosphorylates ␣PIX serine residues participating in the control of T cell migration and/or proliferation. Mechanistically, the specific molecular players connecting the activated IL-2R and the PKC/␣PIX/Rac 1/PYGM pathway, and also PYGM downstream signaling targets are still unknown. Future studies will allow us to characterize the signaling molecules upstream of PKC and effector molecules that participate in this signal transduction pathway.