A Novel Cross-talk in Diacylglycerol Signaling

Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for β2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of β2-chimaerin on Ser169 located in the SH2-C1 domain linker region via protein kinase Cδ, which retained β2-chimaerin in the cytosol and prevented its C1 domain-mediated translocation to membranes. Furthermore, despite the fact that Ser169 phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable β2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts β2-chimaerin relocalization and activation.

Diacylglycerol (DAG) 2 is a key lipid second messenger generated in membranes upon receptor tyrosine-kinase or G-protein coupled receptor-mediated activation of phospholipase C enzymes. Effectors of DAG contain one or two copies of the C1 domain, a cysteine-rich motif that binds with high affinity to DAG and phorbol esters, natural mimetics of DAG (1,2). Some of the best characterized DAG receptors include the protein kinase C (PKC) isozymes, a family of at least 10 related Ser/Thr kinases that have key functions in proliferation, differentiation, apoptosis, and other processes relevant to malignant transformation (3). PKCs can be divided into three groups: classical PKCs (cPKC ␣, ␤I, ␤II, and ␥), which require DAG and calcium for activation, novel PKCs (nPKC ␦, ⑀, , and ), which are DAG-dependent but calcium-independent, and DAG/calcium unresponsive atypical PKCs (aPKC and ). C1 domains in cPKCs and nPKCs mediate membrane translocation of the protein via DAG binding, leading to a conformational change that allows for access to PKC substrates and other PKC-binding proteins (4,5).
Traditionally, PKCs were thought to be the only receptors for DAG; however, several other C1 domain-containing proteins that bind to and become activated by DAG have also been identified. One such DAG receptor is ␤2-chimaerin, a member of the chimaerin protein family comprised of ␣1-(or n-), ␣2-, ␤1-, and ␤2-chimaerin. Chimaerins contain a single C1 domain that binds DAG and phorbol esters with high affinity and mediates translocation to membranes (6,7). Chimaerins also have a C-terminal GTPase-activating protein (GAP) domain that accelerates the hydrolysis of GTP to GDP of the small G-protein Rac1, thus converting Rac from its active GTP-bound form to an inactive GDP-bound form (8). ␣2and ␤2-chimaerin also have a N-terminal SH2 domain possibly involved in protein-protein interactions. Chimaerins play important roles in neuronal processes, such as axon guidance, growth cone collapse, and dendritic morphology (9 -13), and are also important in early zebrafish development and regulation of Rac signaling in the Drosophila retina (14,15). ␤2-Chimaerin regulates cell cycle progression, actin cytoskeleton rearrangement, migration, and metastatic dissemination via Rac inactivation (16,17). Emerging evidence shows that down-regulation of the protein occurs in cancers such as glioma and breast cancer (17)(18)(19).
The mechanisms of ␤2-chimaerin activation and regulation are not completely understood. Previous work revealed that ␤2-chimaerin is a receptor tyrosine kinase effector and that epidermal growth factor receptor (EGFR) activation causes a phospholipase C␥/DAG-dependent translocation of ␤2-chimaerin to the membrane, where it associates with activated Rac and serves as a "brake" that limits the duration and intensity of Rac signaling (20). The crystal structure of ␤2-chimaerin revealed that the protein exists in a "closed" conformation that occludes the DAG-and Rac-binding sites (21). It has been postulated that translocation of ␤2-chimaerin to membranes results as a consequence of allosteric interactions with acidic phospholipids in membranes that compete with intramolecular interactions to help release the autoinhibitory conformation and allow binding of DAG to the C1 domain. This consequently allows for a full conformational rearrangement of ␤2-chimaerin that enables binding to Rac and activation of its Rac-GAP activity.
In this paper we have identified a novel mechanism of crosstalk between DAG effectors involving PKC␦-mediated phosphorylation of ␤2-chimaerin. This post-translational event prevents membrane relocalization in response to stimuli and may represent a means to down-regulate ␤2-chimaerin activity, suggesting dual roles for DAG in modulating ␤2-chimaerin relocalization and activation.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfections-COS-1 and HeLa cells (ATCC) were cultured as described (20). COS-1 cells were transfected using Lipofectamine Plus (Invitrogen). All experiments were performed at least 24 h following transfection.
Two-dimensional Electrophoresis-COS-1 cells expressing HA-␤2-chimaerin adenoviruses (AdV) were serum starved for 24 h and then treated with EGF (100 ng/ml, 5 min). Cell lysates were run on Novex pH 3-10 IEF gels (Invitrogen) according to the manufacturer's recommendations, then fixed in 12% trichloroacetic acid for 30 min. Following a 10-min incubation in 20% ethanol, the desired gel lane was cut and incubated in 2 ml of 2ϫ SDS sample buffer with 0.5 ml of ethanol for 5 min. The gel strip was inserted into a NuPAGE Novex 4 -12% BisTris gel (Invitrogen) and run in MOPS running buffer. Gels were transferred to nitrocellulose and subjected to Western blotting.
Cellular Radiolabeling-Serum-starved (24 h) COS-1 cells were incubated in phosphate-free Dulbecco's modified Eagle's medium for 4 h prior to the addition of 200 Ci of 32 P i . After 1 h, cells were stimulated with EGF or phorbol 12-myristate 13-acetate (PMA). Cell lysates were incubated with anti-HA antibody pre-conjugated to agarose beads (Santa Cruz) overnight at 4°C. Beads were washed three times with lysis buffer. Proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and visualized by autoradiography followed by Western blot.
Isolation of Primary Neurons-Primary mouse cerebellar granule neurons were isolated from P7 FVB mice and cultured as previously described (23).
Rac-GTP and Rho-GTP Pull-down Assays-Serum-starved HeLa cells (24 h) were infected with AdVs (multiplicity of infection ϭ 10 plaque-forming units/cell) for 16 h, washed once with serum-free medium, and 2 h later "pulldown" experiments were performed as described (8).
Migration Assays-Cells (1.5 ϫ 10 4 ) in Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin were added to the upper wells of a Boyden chamber with polycarbonate membranes (8 m pores), and FBS was added to the lower chamber. After a 17-h incubation at 37°C, membranes were fixed and stained with the DiffQuik Stain Set (Dade Behring, Deerfield, IL). Migrated cells were counted by phase microscopy at ϫ200. All samples were replicated 6 times per experiment. Five fields were counted per sample. Subcellular Fractionation-Fractionation by ultracentrifugation was carried out as previously described (24).
In Vitro GAP Assays-Assays were performed using Phosphate Sensor (Invitrogen) and purified recombinant protein as previously described (25).
Statistical Analysis-Data are expressed as mean Ϯ S.E. and analyzed using a Student's t test. A p value of Ͻ0.05 was considered statistically significant. All results shown are representative of three independent experiments unless otherwise indicated.

RESULTS
␤2-Chimaerin Is Phosphorylated on Ser 169 in Response to PMA or EGF-Emerging studies have shown that the chimaerin Rac-GAPs play important roles in development, neuritogenesis, and cancer. Although it is clear that ␤2-chimaerin and other chimaerins are regulated by phorbol esters and DAG generated by receptors, their regulation by post-translational events is less understood. To determine whether ␤2-chimaerin is phosphorylated in response to stimuli, we first analyzed if PMA could induce its phosphorylation. COS-1 cells expressing HA-tagged ␤2-chimaerin were radiolabeled with [ 32 P]orthophosphate, and HA-␤2-chimaerin was immunoprecipitated from cells that were stimulated with increasing concentrations of PMA (10 nM to 1 M). Phosphorylation of ␤2-chimaerin was robustly induced ( Fig.  1A) even at PMA concentrations below those required for translocation and activation of ␤2-chimaerin (8,22). Given that EGF also activates ␤2-chimaerin (20), we examined the effect of EGF treatment on phosphorylation. Like PMA, EGF treatment rapidly and robustly enhanced ␤2-chimaerin phosphorylation (Fig. 1B). Interestingly, EGF treatment did not induce phosphorylation of the highly homologous ␣2-chimaerin (data not shown), indicating that these two proteins may be regulated dissimilarly by phosphorylation.
To identify the site(s) of phosphorylation, we used recombinant human ␤2-chimaerin baculovirally expressed in insect Sf9 cells. Mass spectroscopy analysis of the purified protein revealed the presence of two species, one at the predicted molecular mass of the ␤2-chimaerin, 54.0 kDa, and a second less abundant species that was 80 Da larger (Fig. 1C, left panel), suggesting that a phosphorylated species was also present. In comparison, ␤2-chimaerin expressed and purified from Escherichia coli, which lack the ability to perform many posttranslational modifications, contained only one species at the predicted molecular weight for ␤2-chimaerin (Fig. 1C, right panel). Recombinant ␤2-chimaerin purified from Sf9 insect cells was treated with endoproteinase Lys-C and subjected to MALDI-TOF mass spectroscopy (Fig. 1D). Peptide fragment L9 was observed at m/z 1088.71 and a candidate phosphopeptide fragment, L9*, was detected 79.97 Da above L9 (Fig. 1E), suggesting that the L9 fragment, which comprises amino acids 164 -172 of ␤2-chimaerin, is singly phosphorylated.
The putative phosphopeptide identified by mass spectrometry contained three phosphorylatable residues (Ser 165 , Ser 169 , FIGURE 1. ␤2-Chimaerin is phosphorylated on Ser 169 in response to EGF or PMA. A and B, serum-starved COS-1 cells labeled with 32 P i were treated with PMA (10 min) (A) or EGF (100 ng/ml) (B). Phosphorylation was monitored by autoradiography, and HA-␤2-chimaerin expression was assessed by Western blotting. C, recombinant ␤2-chimaerin purified from Sf9 cells (left) or E. coli (right) was subjected to mass spectroscopy. D, spectrum obtained upon MALDI-TOF mass spectroscopy analysis of Lys-C-digested ␤2-chimaerin isolated from Sf9 cells. E, mass spectrum of putative phosphopeptide. Inset, localization of the putative phosphorylation sites in ␤2-chimaerin. F and G, COS-1 cells expressing HA-␤2-chimaerin Ser to Ala mutants were labeled with 32 P i and treated with EGF (100 ng/ml, 5 min) (F) or PMA (1 M, 10 min) (G). Phosphorylation was monitored by autoradiography.
and Ser 171 ), and is located in the linker region between the SH2 and C1 domain (Fig. 1E, inset), a region for which the crystal structure was unable to be resolved (21). This region is not conserved between ␣2and ␤2-chimaerin. To identify the precise site of phosphorylation, site-directed mutagenesis was performed to individually mutate each Ser to Ala. Mutation of Ser 169 completely blocked EGF-induced phosphorylation. On the other hand, phosphorylation was still detected upon mutation of Ser 165 or Ser 171 (Fig. 1F). Thus, Ser 169 of ␤2-chimaerin is the major site of EGF-induced phosphorylation. Mutation of Ser 169 also impairs PMA-induced phosphorylation of the protein (Fig. 1G).
A rabbit polyclonal antibody directed toward phospho-Ser 169 -␤2-chimaerin was generated. This antibody was highly specific and showed no cross-reactivity toward the non-phosphorylatable S169A-␤2-chimaerin mutant ( Fig. 2A). A time course analysis of ␤2-chimaerin Ser 169 phosphorylation in response to EGF or PMA stimulation revealed that phosphorylation is readily detected after 1 min, with maximal phosphorylation occurring at 3-5 min for EGF ( Fig. 2A) and 5 min for PMA (Fig. 2B). To determine the extent of EGF-induced Ser 169 phosphorylation in cells, we developed a two-dimensional gel electrophoresis approach to separate the non-phosphorylated and phosphorylated fractions of ␤2-chimaerin. From this analysis, we estimate that ϳ40% of ␤2-chimaerin in cells is phosphorylated on Ser 169 in response to EGF (Fig. 2C).
We next sought to determine whether endogenous ␤2-chimaerin is phosphorylated on Ser 169 . Due to low expression of endogenous ␤2-chimaerin in most cultured cell lines, we chose to perform this analysis on primary mouse cerebellar granule neurons, as these cells have the highest abundance of ␤2-chimaerin (26). We utilized brain-derived neurotrophic factor, a TrkB tyrosine kinase receptor ligand, as this growth factor activates Rac and causes DAG production via phospholipase C␥ (23,27). As shown in Fig. 2D, treatment of primary mouse granule neurons with brain-derived neurotrophic factor led to Ser 169 phosphorylation of endogenous ␤2-chimaerin.
PKC␦ Mediates EGF-induced Phosphorylation of ␤2-Chimaerin on Ser 169 -Because we found that phosphorylation of ␤2-chimaerin is induced by low nanomolar concentrations of PMA, we reasoned that PKC might be involved in ␤2-chimaerin phosphorylation. Moreover, analysis of the sequence surrounding Ser 169 of ␤2-chimaerin revealed that this region is a consensus site for PKC substrate recognition. To test this hypothesis, we utilized the pan-PKC inhibitor GF 109203X (GF), and found it dose dependently inhibited EGF-stimulated phosphorylation (Fig. 3A), suggesting that phosphorylation of ␤2-chimaerin is indeed PKC mediated. Top, representative Western blots. Bottom, data relative to t ϭ 0 min presented as mean Ϯ S.E. C, serum-starved COS-1 cells expressing HA-␤2chimaerin AdV were treated with EGF (100 ng/ml, 5 min). Cell lysates were subjected to two-dimensional gel electrophoresis and Western blotting. Left, representative Western blots. Right, analysis of EGF-treated samples expressed as percentage of non-phosphorylated or phosphorylated protein (relative to total protein) expressed as the mean Ϯ S.E. (n ϭ 5). D, primary mouse cerebellar granule neurons were treated with brain-derived neurotrophic factor (BDNF) (100 ng/ml) for the indicated times and assessed for Ser 169 phosphorylation of endogenous ␤2-chimaerin.
To determine which PKC isoform is involved in Ser 169 phosphorylation, we first used the inhibitors Go 6970 and rottlerin, which preferentially inhibit cPKCs and PKC␦, respectively. COS-1 cells do not express PKC␤I, -␤II, or -␥; thus, Go 6970 targets PKC␣ in these cells. Pretreatment with the PKC␦ inhibitor rottlerin dramatically reduced EGF-stimulated phosphorylation of Ser 169 on ␤2-chimaerin, whereas Go 6970 was less effective (supplemental Fig. S1). Thus, PKC␦ is likely responsible for ␤2-chimaerin phosphorylation.
Because pharmacological PKC inhibitors have limited specificity (28), we used small interfering RNA to specifically knockdown individual PKCs in HeLa cells (Fig. 3B). Although PKC␣ depletion caused partial reduction of Ser 169 phosphorylation, depletion of PKC␦ completely abolished EGF-stimulated phosphorylation. PKC⑀ depletion had no effect on Ser 169 phosphorylation (Fig. 3C). To validate these results, we also used 2 shRNA lentiviruses to create HeLa cell lines stably depleted of PKC␦. Significant PKC␦ depletion was observed in both cell lines (␦ number 1 and ␦ number 2), whereas a lentivirus expressing a non-targeting sequence (NTS) did not affect PKC␦ expression levels (Fig. 3D). EGF-induced phosphorylation was markedly reduced in PKC␦ stably depleted cells (Fig. 3E). EGF-mediated phosphorylation of ␤2-chimaerin was also significantly attenuated in H1299 lung carcinoma cells expressing PKC␦ shRNA (supplemental Fig. S2).
To further validate the involvement of PKC␦ in ␤2-chimaerin phosphorylation we overexpressed PKC␦ in COS-1 cells and found a significant increase in Ser 169 phosphorylation under basal conditions and in response to EGF (Fig. 3F). In vitro kinase assays using purified recombinant proteins also revealed that PKC␦ phosphorylates Ser 169 of ␤2-chimaerin in vitro, and that this phosphorylation can be blocked by the PKC inhibitor GF (Fig. 3G). Altogether, these results indicate that PKC␦ is involved in ␤2-chimaerin phosphorylation, probably through direct phosphorylation.
The Non-phosphorylated Form of ␤2-Chimaerin Has Enhanced Rac-GAP Activity in Cells-We next investigated the functional consequences of Ser 169 phosphorylation of ␤2-chimaerin. EGF causes translocation and activation of ␤2-chimaerin, and ectopic expression of ␤2-chimaerin inhibits EGF-mediated Rac1 activation (20). To determine whether phosphorylation had any effect on the Rac-GAP activity of ␤2-chimaerin, we used AdVs to express both wild-type (WT) and non-phosphorylatable S169A-␤2-chimaerin mutants in HeLa cells. A LacZ AdV was used as a control. Importantly, we expressed very low amounts of the proteins to express the WT ␤2-chimaerin at levels that had little to no effect on Rac1 activation. At this very low level of expression, the S169A-␤2-chimaerin mutant markedly inhibited Rac1 activation by EGF as determined by Rac-GTP pulldown assays (Fig. 4A). This indicates that S169A-␤2-chimaerin has increased EGF-stimulated Rac1-GAP activity compared with the WT ␤2-chimaerin pro- tein. Similar results were also seen in T47D breast carcinoma cells (supplemental Fig. S3). A S169E-␤2-chimaerin mutant did not accurately mimic phosphorylation (data not shown), as observed for other phosphoproteins (29,30), which precluded further functional analysis using a phosphomimetic mutant.
To ensure that adenoviral expression of WT or S169A-␤2chimaerin was not interfering with EGFR activation, we looked at levels of phospho-EGFR using site-specific phospho-EGFR antibodies and found no significant differences between cells expressing control, WT, or S169A-␤2-chimaerin AdVs (supplemental Fig. S4). We also wanted to determine whether the enhanced GAP activity of S169A-␤2-chimaerin was specific for Rac1. We have previously shown that ␤2-chimaerin has GAP activity toward Rac, but not other Rho GTPases (8). Expression of S169A-␤2-chimaerin showed no difference in EGF-stimulated RhoA activation when compared with control or WT-␤2-chimaerin expressing cells, indicating that the GAP activity of S169A-␤2-chimaerin is specific toward Rac1 (Fig. 4B).
To translate these findings into a functional setting, we assessed cell motility, a Rac-dependent process, using a Boyden chamber. Chemotactic migration toward FBS of HeLa cells infected with LacZ, WT, or S169A-␤2-chimaerin AdVs was compared. We found that although expression of WT ␤2-chimaerin significantly attenuated migration, expression of S169A-␤2-chimaerin had an even greater effect and nearly suppressed migration (Fig. 4C). To verify that the effects of S169A-␤2chimaerin on migration were due specifically to Rac inactivation, we utilized a V12Rac1 mutant, a constitutively active form of Rac1. Expression of V12Rac1 in cells reversed the inhibitory effect of both WT and S169A-␤2-chimaerin on migration and enhanced the FBS-directed migration of cells to similar levels as LacZ controls (Fig. 4D). Together, these findings demonstrate that the unphosphorylated form of ␤2-chimaerin has enhanced Rac-GAP activity in cellular models, arguing for a negative role for Ser 169 phosphorylation in ␤2-chimaerin activation.
Phosphorylation of Ser 169 Impedes ␤2-Chimaerin Translocation to Membranes-One of the hallmarks of ␤2-chimaerin activation is translocation to membranes upon PMA stimulation or DAG generation (20,22). To begin to account for the enhanced GAP activity of the S169A-␤2-chimaerin mutant, we first determined if Ser 169 phosphorylation of the ␤2chimaerin could impact its subcellular localization. Cells expressing ␤2-chimaerin and growing in serum were subjected to subcellular fractionation. We observed that ␤2-chimaerin is roughly equally distributed in the soluble (cytosolic) and insoluble fractions (Fig. 5A). Remarkably, Ser 169 -phosphorylated ␤2-chimaerin was almost exclusively localized in the soluble fraction. This suggests that phosphor- ylated ␤2-chimaerin is predominantly cytosolic, whereas the activated form of ␤2-chimaerin found at membranes is not phosphorylated on Ser 169 .
We reasoned that if phosphorylated ␤2-chimaerin is found in the cytosol, a mutant of ␤2-chimaerin that is unable to translocate to membranes would have enhanced phosphorylation, whereas a mutant with enhanced membrane association would have reduced levels of phosphorylation. We took advantage of the C1 domain mutant P223A-␤2-chimaerin (Pro 11 in the C1 domain consensus (31)), which has impaired ability to bind DAG or phorbol esters, whereas still preserving the overall C1 domain structure. This mutant was unable to translocate to membranes upon PMA stimulation (Fig. 5B). We also used the hyperactive mutant I130A-␤2-chimaerin. Ile 130 stabilizes the closed conformation of ␤2-chimaerin by interacting with the C1 domain. Mutation of Ile 130 "exposes" the C1 domain and enhances association to membranes (21,25). Striking differences in Ser 169 phosphorylation levels of these proteins were seen in COS-1 cells. While P223A-␤2-chimaerin had significantly higher phosphorylation levels than WT-␤2-chimaerin under basal conditions and in response to EGF, phosphorylation of I130A-␤2chimaerin was barely detectable (Fig. 5C). Thus, subcellular protein localization of ␤2-chimaerin is key for determining its phosphorylation status.
To further explore whether phosphorylation of Ser 169 affects ␤2chimaerin relocalization, we analyzed the effect of PKC inhibition on PMA-induced translocation. We predicted that inhibition of Ser 169 phosphorylation would favor ␤2chimaerin translocation. Interestingly, when cells were treated with PMA in the absence of the PKC inhibitor GF, ␤2-chimaerin translocation to the insoluble fraction was not detected (Fig. 6A, left). Under this experimental condition a robust increase in phospho-Ser 169 levels was also observed. In contrast, GF treatment blocked ␤2-chimaerin phosphorylation and allowed for PMA-induced translocation, arguing that phosphorylation of Ser 169 prevents translocation of the protein.  We reasoned that if translocation of ␤2-chimaerin depends on its phosphorylation status, then the non-phosphorylatable S169A mutant should translocate in response to PMA regardless of whether or not GF is present. Indeed, we found that, unlike WT-␤2-chimaerin, S169A-␤2-chimaerin translocates upon PMA stimulation both in the absence and presence of GF (Fig. 6A, center), further suggesting that phosphorylation of Ser 169 negatively regulates translocation to membranes. To ensure that the ability of the S169A mutant to translocate was driven by specific binding of PMA to the C1 domain of S169A-␤2-chimaerin, we also created the S169A/P223A-␤2-chimaerin phorbol ester/DAG unresponsive double mutant. Like P223A-␤2-chimaerin (Fig. 5B), S169A/P223A-␤2-chimaerin also failed to translocate in response to PMA, regardless of the presence or absence of GF (Fig. 6A, right). This indicates that S169A-␤2-chimaerin translocation is dependent upon a functional C1 domain.
To recapitulate and verify the fractionation results, we also performed confocal microscopy studies using GFP-tagged chimaerins. As previously shown (22), GFP-␤2-chimaerin is predominantly cytoplasmic. Translocation of GFP-␤2-chimaerin by PMA in the absence of GF was barely detected (Fig. 6B). Remarkably, GF restored the ability of PMA to translocate GFP-␤2-chimaerin. On the other hand, a pronounced translocation of GFP-S169A-␤2-chimaerin to the plasma membrane was observed in the absence of GF. These results further establish a negative role for Ser 169 phosphorylation in membrane translocation of ␤2-chimaerin.
The Non-phosphorylatable Form of ␤2-Chimaerin Has Enhanced Binding to Activated Rac1-To determine whether the enhanced GAP activity of S169A-␤2-chimaerin in cells was due to changes in the intrinsic Rac-GAP activity, we performed in vitro GAP assays using purified recombinant Rac1 and ␤2-chimaerin proteins. GTP hydrolysis was measured using a fluorescent phosphate sensor that detects inorganic phosphate (32). Recombinant S169A-␤2-chimaerin had a nearly equal level of GAP activity when compared with WT protein (Fig. 7). As a control, we used recombinant ␤1-chimaerin, a related chimaerin isoform that lacks the N-terminal autoinhibitory domain. In agreement with previous results (25), ␤1-chimaerin has markedly higher in vitro GAP activity. These results indicate that the enhanced Rac-GAP activity of S169A-␤2-chimaerin in cells is not due to enhanced intrinsic GAP activity of the protein.
␤2-Chimaerin has previously been shown to bind to Rac1 in its active form in response to serum or EGF (8,20). Given the enhanced GAP activity of the S169A-␤2-chimaerin mutant in cells, we hypothesized that this mutant may differentially bind to Rac1. To address this issue we assessed binding to GST-V12Rac1 in COS-1 cells using a GST pulldown assay. Fig. 8A shows that the association of S169A-␤2-chimaerin to V12Rac1 in cells is 3-fold higher than WT-␤2-chimaerin. We hypothesized that binding of S169A-␤2chimaerin to Rac1 may be due to its enhanced ability to translocate to membranes and hence, enhanced access to the membrane-localized V12Rac1. To test this, we compared V12Rac1 binding of S169A-␤2-chimaerin to that of the P223A/S169A-␤2-chimaerin double mutant that cannot translocate to membranes. Indeed, we found that binding of S169A/P223A-␤2chimaerin to V12Rac1 is much lower than that of S169A-␤2-chimaerin (Fig. 8B). This suggests that the enhanced ability of the S169A mutant to bind to active Rac1 in cells requires a functional C1 domain and is therefore due largely to enhanced association to membranes.

DISCUSSION
Chimaerins are the only known Rac-GAPs regulated by the lipid second messenger DAG in response to tyrosine kinase receptor activation. Here we have provided the first evidence that ␤2-chimaerin is phosphorylated in a novel mechanism of DAG receptor cross-talk. EGF and PMA rapidly induce phosphorylation of Ser 169 of ␤2-chimaerin, and PKC␦ is implicated in this phosphorylation. Phosphorylation of Ser 169 of ␤2-chimaerin appears to be a means of limiting its access to Rac at the plasma membrane, thereby resulting in reduced activation of ␤2-chimaerin in cells. A model for this paradigm is presented in Fig. 9. We postulate that rapid EGF-induced phosphorylation of ␤2-chimaerin may be a mechanism of limiting the strength of and/or spatially restricting the extent of ␤2-chimaerin activation.
The current mechanism of activation for ␤2-chimaerin is derived from the solved crystal structure of the protein (21). ␤2-Chimaerin exists in an inactive conformation in which extensive intramolecular interactions occlude the DAG/phorbol ester binding site of the C1 domain as well as the Rac interacting site of the GAP domain. Upon DAG generation or PMA treatment, the inactive conformation of ␤2-chimaerin must undergo an extensive conformational rearrangement that exposes large hydrophobic patches along the length of the protein that are thought to align with the membrane to avoid interactions with polar solvent. This activates the protein by positioning ␤2-chimaerin at the membrane for interaction with DAG and its effector Rac.
Given that non-phosphorylated ␤2-chimaerin is the predominant form of the protein found translocated to membranes, we propose that this is the main species that is susceptible to the allosteric activation and conformational rearrangement that serves to activate the protein. Indeed, most of the previous studies of PMA-induced translocation of ␤2-chimaerin to membranes were done in the presence of the pan-PKC inhibitor GF to avoid phorbol ester ligand binding competition with PKC isoforms. Based on the present studies, we know that treatment with GF blocks PMA-induced phosphorylation of Ser 169 , and thus most of the protein remains in the non-phosphorylated form and is able to translocate. Furthermore, we have shown here that PMA treatment in the absence of GF is unable to cause translocation of the WT protein, whereas the S169A-␤2-chimaerin mutant readily translocates to membranes upon PMA stimulation even in the absence of GF. This is not due to a change in the overall intrinsic ability of the mutant protein to translocate, as S169A-␤2-chimaerin translocates to membranes with a similar EC 50 of phorbol ester-induced translocation, and as WT-␤2-chimaerin in the presence of GF (data not shown). These results strongly suggest that when phosphorylated on Ser 169 , ␤2-chimaerin is unable to undergo the allosteric activation and conformational rearrangement needed for full activation. Indeed, this is most likely the reason why S169A-␤2-chimaerin is more active in cells: it is more readily available for translocation and hence activation due to a lack of Ser 169 phosphorylation. Some Rac inactivation is observed with wild-type ␤2-chimaerin, as more than 50% of the protein remains non-phosphorylated (Fig. 2C) and therefore available for activation.
One important question is, what is the physiological role of Ser 169 phosphorylation of ␤2-chimaerin. One potential model we envision is that upon receptor tyrosine kinase stimulation, a fraction of ␤2-chimaerin becomes rapidly phosphorylated on Ser 169 , and this phosphorylated fraction is unable to translocate to membranes and become activated, thus allowing for Rac1 activation to occur. By limiting the amount of ␤2-chimaerin in the cell that is available for activation, the strength of ␤2-chimaerin activation can be controlled; hence, the magnitude of Rac1 activation can also be regulated. Furthermore, this may serve as a mechanism to spatially restrict ␤2-chimaerin activation to cell compartments where inactivation of Rac is crucial, whereas simultaneously limiting the amount of activated ␤2-chimaerin available in compartments where Rac activation is essential. This may be especially important for regulating processes in which spatial restriction of Rac activation is important, such as directional migration (33).
The mechanism through which Ser 169 phosphorylation precludes translocation has not been identified thus far, but one hypothesis is that phosphorylation of Ser 169 may alter interactions with a binding partner that serves to sequester ␤2-chimaerin away from sites of DAG production. Recent studies have shown that chimaerins interact with Nck1 and -2, multidomain adaptor proteins that may play an important role in linking signaling by the related ␣2-chimaerin isoform with Ephrin A2 signaling to control neuronal processes (11). The mechanism through which Nck regulates the signaling of chimaerins is not well studied as yet, but preliminary evidence suggests that Nck may bind inactive ␤2-chimaerin and position it close to membranes, thus bringing it into proximity with sites of DAG generation and facilitating activation. 3 If this were the case, ␤2-chimaerin would also be positioned near potential sites of PKC␦ activation, which would in turn preclude activation of ␤2-chimaerin through phosphorylation. Studies to determine the effect of Ser 169 phosphorylation on Nck binding to ␤2-chimaerin are underway.
Although this study is the first to report Ser phosphorylation of ␤2-chimaerin, two alternate papers have recently reported Tyr phosphorylation of ␤2-chimaerin. Phosphorylation at Tyr 21 of ␤2-chimaerin by Src family kinases was reported in response to EGF (34). Like Ser 169 phosphorylation, Tyr 21 phosphorylation negatively regulates the GAP activity of ␤2-chimaerin, although the mechanism through which this occurs was not reported. Interestingly, the kinetics of EGF-induced Tyr 21 phosphorylation are markedly different from those of Ser 169 phosphorylation, with Tyr 21 phosphorylation being detectable 5 min post-EGF treatment and increasing up to 30 min following stimulation. This indicates that ␤2-chimaerin may be regulated in two phases. Initial Ser 169 phosphorylation may play a role in limiting the magnitude of ␤2-chimaerin activation, whereas subsequent Tyr 21 phosphorylation may be more important for down-regulation of the activated protein. It is important to note, however, that EGF-induced Tyr 21 phosphorylation of ␤2-chimaerin was only seen in the presence of tyrosine phosphatase inhibitors. Indeed, we did not detect induced Tyr or Thr phosphorylation of ␤2-chimaerin upon EGF stimulation in the absence of tyrosine phosphatase inhibitors (supplemental Fig. S5), thus suggesting that EGF-induced Tyr 21 phosphorylation is probably transient and limited in extent.
Phosphorylation of a second Tyr residue, Tyr 153 , was recently reported to occur in response to T-cell receptor stimulation via the tyrosine kinase Lck (35). Like Ser 169 phosphorylation, Tyr 153 phosphorylation negatively regulates Rac-GAP activity of ␤2-chimaerin, as a non-phosphorylatable mutant enhanced Rac-GAP activity and correspondingly decreased such Rac-mediated T-cell events as actin reorganization in response to immune synapse formation. Interestingly, under the same conditions of T-cell receptor stimulation, Ser 169 phosphorylation of ␤2-chimaerin was not observed (supplemental Fig. S6), indicating that regulation of ␤2-chimaerin by phosphorylation is largely cell-type and stimulus specific. The authors hypothesize that Tyr 153 phosphorylation in T-cells is a mechanism for down-regulation of the protein by promoting dissociation of the activated protein from membranes, although they do not explore the mechanisms through which this dissociation may occur. However, mutation of Tyr 153 to Phe leads to PMA-induced translocation of ␤2-chimaerin to membranes in a manner analogous to that seen for the I130A-␤2-chimaerin hyperactive mutant. We would sug-gest that the T153F-␤2-chimaerin mutation also destabilizes intramolecular contacts within the protein, and that this mutant functions differently from the S169A mutation, which we believe does not destabilize the inactive closed conformation of ␤2-chimaerin, but still has an enhanced ability to translocate to membranes. It has become clear from our study and others that regulation of ␤2-chimaerin via phosphorylation is a complex process that is most likely cell-type and stimulus-specific, and further experiments will be necessary to determine the relative contributions of each identified phosphorylation site to the activation and/or down-regulation of ␤2-chimaerin.
The concept of cross-talk between DAG receptors is one that has not been fully appreciated to date. There are currently two known examples of one DAG receptor regulating another. First, RasGRP3, a Ras guanine nucleotide exchange factor expressed in B cells, is phosphorylated by PKCs on Thr 133 upon B-cell receptor activation (36,37). This phosphorylation is essential for activation of RasGRP3 and consequent activation of Ras in response to B-cell receptor stimulation. Second, PKD1-3, Ser/Thr kinases important for mediating such cellular processes as membrane trafficking, migration, and proliferation are also phosphorylated by PKCs (38). Indeed, PKD1 must be phosphorylated by PKC on both Ser 744 and Ser 748 to relieve autoinhibition and activate the protein (39). In these two cases DAG serves as a dual role in regulating these proteins, first through direct binding to their C1 domains, and second through activation via PKC-mediated phosphorylation. Interestingly, DAG also regulates the localization and hence activation of ␤2-chimaerin in a reciprocal manner: whereas phosphorylation through PKC␦ leads to negative regulation of the protein through restricted localization to the cytosol, ligand binding to DAG is required for translocation of unphosphorylated ␤2-chimaerin to membranes and activation. The relative concentrations of DAG and PKC␦ in any one location of the cell will likely greatly influence the activation status of ␤2-chimaerin. Furthermore, enhanced PKC␦ activation will in turn lead to decreased ␤2-chimaerin translocation/activation. Thus, it is likely that very tightly regulated mechanisms for controlling relative PKC␦ and ␤2-chimaerin activation exist in cells.
This work identifies for the first time a regulatory mechanism for ␤2-chimaerin via Ser phosphorylation. In addition to revealing a novel phosphorylation site on ␤2-chimaerin and determining that phosphorylation on Ser 169 negatively regulates activity and localization, we also provide the first evidence for a paradigm of cross-talk between DAG receptors ␤2-chimaerin and PKC␦. The fact that Rac-GAPs can be regulated by PKCs suggests that the lipid second messenger DAG plays an important role in controlling the activation status of the small GTPase Rac and highlights the complexity of DAG signaling via multiple C1 domain-containing proteins.