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A New Tyrosine Phosphorylation Site in PLC1: The Role of Tyrosine 775 in Immune Receptor Signaling

Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, National Institutes of Health Campus, Bethesda, MD 20892

	Abstract

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Phospholipase C (PLC) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLC is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLC1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLC1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLC1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLC2 indicated homology of PLC1 tyrosine residue 775 (Y775) with PLC2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLC1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLC1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLC1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLC1 activation and confirm the functional importance of Y783.

	Introduction

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An early consequence of ligand-induced activation of a variety of cell surface receptors is the activation of phosphoinositide-specific phospholipase C (PLC).³ The PLC isoforms 1 and 2 are downstream of receptors with intrinsic tyrosine kinase activity, such as the growth factor receptors for epidermal growth factor and plasma-derived growth factor (PDGF), and receptors without intrinsic kinase activity, such as the B and T cell Ag receptors (BCR and TCR, respectively). The latter depend on the recruitment of non-receptor tyrosine kinases for their activation (1, 2, 3, 4, 5). PLC is a cytoplasmic enzyme that requires membrane translocation and tyrosine phosphorylation for its activation. T cells predominantly express the PLC1 isoform, PLC2 is expressed at high levels in B cells (2, 6, 7). The activated enzyme hydrolyzes phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). DAG participates in the activation of the RasGRP/PKC effector pathways leading to activation of the transcription factor AP-1 (reviewed in Ref. 8). IP₃ mediates intracellular Ca²⁺ mobilization, which is required for downstream events such as activation of the transcription factor NF-AT (8, 9). Both the Ca²⁺ and DAG signaling pathways are required for T cell cytokine production and proliferation.

Four phosphorylated tyrosines (pY) have been characterized in PLC1 Y472, Y771, Y783, and Y1254 (amino acid numbering is based on the sequence of the bovine protein), after epidermal growth factor and PDGF receptor stimulation (10, 11) and in response to TCR activation (12). Studies in both receptor systems demonstrated a requirement for PLC1 Y783 phosphorylation in phosphoinositide breakdown (12) and gene induction (13). In addition, early studies indicated Y1254 was needed for maximal growth factor-induced stimulation, while Y771 was proposed as a negative regulator. However, in later studies, the role of these two tyrosines has been questioned (14).

In an effort to resolve these issues, we initiated a study that investigated what effect mutation of each site individually or in combination had on PLC1 phosphorylation and activation after Ag receptor (AgR) stimulation. Because previous work from our laboratory had indicated that activation requirements for PLC1 were similar for TCR or BCR stimulation (15), models using both receptor systems were analyzed. These studies resulted in the identification of a new phosphorylation site (Y775) that is required for AgR-induced PLC1 activation. Our data indicate that phosphorylation of Y775 and Y783 play equally important roles in the activation of PLC1 and further suggest that Y472, Y771, and Y1254 are not required for PLC1-mediated Ca²⁺ mobilization or activation of the DAG signaling pathway.

	Materials and Methods

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Cells and reagents

Jurkat E6.1 (T cell line), Jurkat TAg (16), Jurkat J1 (PLC1-deficient Jurkat) (13), P10–14 (17) (DT40-derivative B cell line deficient in PLC1 and 2) and stable PLC1 P10–14 transfectants were maintained in RPMI 1640 supplemented with 10% FBS, 25 mM HEPES,1 mM sodium pyruvate, 1x non-essential amino acids (Biosource), and 2 mM L-glutamine. The anti-TCR Ab C305 (IgM) was a gift from A. Weiss (University of California, San Francisco, CA), the anti-hemagglutinin (HA) Ab 12CA5 from A. Weissman (National Cancer Institute, Bethesda, MD) and the NF-AT and AP-1 luciferase reporter gene constructs from G. Crabtree (Stanford University, Stanford, CA). Purified goat-anti-chicken IgM was purchased from Bethyl Laboratories, anti-pY Ab 4G10 from Upstate Biotechnology, anti-pY783 from Cell Signaling Technology, and anti-HA 3F10 from Roche. The anti-pY775 rabbit serum was generated through a commercial contract with Biosource International by immunizing rabbits with the peptide Ac-YGAL(pY)EGRNPGF(Ahx)C-amide. The serum was purified on an agarose gel, eluted with low pH glycine, and quantitated spectrophotometrically at 280 nm.

DNA plasmids, transfections, and generation of stable transfectans

The construction of vectors expressing bovine PLC1 in pBluescript II-SK (pBluSK) and HA-tagged PLC1 (PLC1-HA) in pCIneo (Promega) has been previously described (15). Tyrosine (Y) to phenylalanine (F) mutations on PLC1 were created using the QuickChange site-directed mutagenesis kit (Stratagene) or by subcloning fragments with the point mutations created with this kit. Transfections were performed by electroporation (15). Tyrosine to phenylalanine mutation constructs were named by a 5-letter code, according to the numerical sequence of tyrosines Y472, Y771, Y775, Y783, and Y1254, where "Y" indicates retained tyrosines and "F" indicates tyrosines that have been mutated to phenylalanine (e.g., WT corresponds to "YYYYY", the single mutation of 775 or 783 are YYFYY and YYYFY, respectively). Stable P10–14 lines were generated by transfection with 20 µg of the indicated pCIneo PLC1-HA construct and 2 µg of pBABEpuro (18) followed by selection with puromycin (Sigma-Aldrich).

Cell activation, immunoprecipitation, and immunoblotting

Transfected Jurkat cells were cultured at 0.5 x 10⁶/ml for 18 h and subjected to Ficoll separation. For stimulation, 10⁷ Jurkat cells were treated with medium or C305 Ab for 2 min at 37°C. P10–14 were stimulated with anti-IgM for 1 min at 37°C. Peripheral blood lymphocytes were obtained from the National Institute of Health blood bank. RBC were removed by Ficoll-Hypaque centrifugation, and the cells were stimulated overnight with 2 µg/ml PHA. PHA was then removed, and the cells were cultured for 6 days in 20 U/ml IL-2. PHA blasts (3 x 10⁷ cells/point) were incubated with the anti-TCR antibody, OKT3 (150 µg/ml; eBioscience) on ice for 20 min, washed, and then cross-linked with a goat anti-mouse IgG Ab for 3 min at 37°C. Cells were then lysed, immunoprecipitated, and resolved by SDS-PAGE followed by immunoblot analysis and chemiluminescent detection as previously described (15).

Reporter gene assays

Jurkat cells were transfected with 5 µg of the respective PLC1-HA construct and 5 µg of an NF-AT or AP-1 plasmid containing a luciferase reporter. After 18 h, the cells were distributed in duplicate wells of a 24-well plate containing either medium alone, C305 Ab (an optimized amount precoated to the plate, overnight at 4°C) or 10 nM PMA and 1 µM ionomycin (NF-AT) or PMA alone (AP-1). Luciferase activity was measured as previously described (15).

Calcium mobilization

Cells were suspended in HBSS supplemented with 1% FBS and 10 mM HEPES. They were loaded with Indo-1/AM (Molecular Probes) at 30°C for 30 min, washed and adjusted to 1 x 10⁶/ml. Calcium flux was measured on a BD Biosciences LSR flow cytometer equipped with a helium-cadmium laser (325 UV). Cell loading was assessed by stimulation with ionomycin; equal ionomycin responses were a prerequisite for the continuation of the experiments. P10–14 cells and P10–14 cells stably reconstituted with wild-type (WT) PLC1 served as negative and positive controls, respectively, in each experiment. Appropriate band-pass filters were used, and the data were analyzed using FlowJo software (Tree Star).

	Results and Discussion

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Tyrosine 775 of PLC1 is phosphorylated upon TCR and BCR activation

To address the contribution of the previously described PLC1 tyrosine (Y) phosphorylation sites in AgR-induced PLC1 phosphorylation, PLC1 mutants were generated in which tyrosines 472, 771, 783, or 1254 were replaced by phenylalanine (F) individually or in combination, and the constructs were ectopically expressed in the Jurkat TAg cell line. The degree of AgR-induced phosphorylation of each mutant was then compared with that observed with WT PLC1 by anti-HA immunoprecipitation, and immunoblotting with the anti-pY Abs 4G10 (Fig. 1A) or PY20 (unpublished data) (16). None of the individual Y to F point mutations impaired the overall phosphorylation of PLC1 (Fig. 1A). Surprisingly, a PLC1 construct in which all four tyrosines had been mutated to phenylalanine was still found to be heavily phosphorylated upon TCR stimulation (67% of WT, Fig. 1B). The same results were observed after anti-IgM induced activation of the PLC-deficient P10–14 cell line stably reconstituted with WT PLC1 or the PLC1 Y to F mutant constructs (unpublished data). These data suggest the existence of one or more unidentified AgR-induced tyrosine phosphorylation sites within PLC1.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Identification of a new, putative tyrosine phosphorylation site in PLC1. A and B, Jurkat cells were transiently transfected with 20 µg of WT PLC1-HA or the indicated HA-PLC1 mutant construct and stimulated with C305 as indicated for 2 min at 37°C. Anti-HA immunoprecipitates were resolved by SDS-PAGE and blotted with anti-pY (4G10). The membrane was then stripped and reprobed with anti-HA as shown. Y to F mutants represent PLC1 constructs with single tyrosine to phenylalanine substitutions in the indicated tyrosine. PLC1 FFYFF has Y472, Y771, Y783, and Y1254 mutated to phenylalanine, and PLC1 FFFFF has Y472, Y771, Y775, Y783, and Y1254 mutated to phenylalanine. Phosphorylation to protein ratios were calculated for FFYFF and FFFFF normalized to WT and compared across experiments (n = 6)–FFYFF retained 67.2% SEM 7.9, FFFFF 22.6% SEM 6.2 of WT values. Black bars represent lanes that were rearranged for reasons of presentation but stem from the same experiment and film.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. AgR-induced phosphorylation of PLC1 Y775. A, Sequence alignment of human PLC2 (P16885), human PLC1 (P19174), and bovine PLC1 (P08487). B, The indicated HA-tagged PLC1 constructs were immunoprecipitated from stably transfected P10–14 cells, which were either left unstimulated or were stimulated with anti-IgM Ab for 2 min at 37°C. Anti-HA precipitates were split into three equal parts, resolved on SDS-PAGE gels, transferred and blotted with either anti-pY775, anti-pY775 plus antigenic peptide, or anti-pY775 plus non-phosphorylated antigenic peptide as indicated. Each membrane was stripped and reprobed with anti-HA. Each panel originates from one film, black bars indicate the deletion of lanes that where in between those shown. C, Endogenous PLC1 was immunoprecipitated from resting or stimulated (2 min, 37°C) E6.1 Jurkat cells, and controls of P10–14 cells stably transfected with either WT PLC1 or PLC1 Y775F were included. Peripheral blood lymphocytes were stimulated with OKT3 for 3 min, 37°C. Membranes were blotted with rabbit anti-pY775, stripped, and reprobed with anti-PLC1 Ab. Black bars represent lanes that were rearranged for reasons of presentation but stem from the same experiment and film.

The AgR-induced phosphorylation of the FFYFF construct (Y775 intact) was measured at 67% of WT as detected by 4G10, whereas the PLC1 FFFFF construct with the additional mutation of Y775 showed marked reduction to 23% of WT in transiently transfected Jurkat cells (Fig. 1B) or in a stable PLC1 FFFFF expressing P10–14 cell line (unpublished data). These data strongly suggested that Y775 is an AgR-induced PLC1 phosphorylation site.

To confirm Y775 as a true phosphorylation site, we generated a rabbit polyclonal phospho-specific Y775 Ab (anti-pY775) and tested the antibody’s reactivity on anti-HA immunoprecipitates from the stable PLC1-expressing P10–14 cell lines. Anti-pY775 showed no reactivity on PLC1 immunoprecipitated from resting cells, but yielded a single immunoreactive band at the m.w. of PLC1 after immunoprecipitation of HA-PLC1 from activated cells (Fig. 2B). No phosphorylation of the FFFFF construct was observed with anti-pY775 and little or no phosphorylation of the YYFYY construct (Y775F). Binding of anti-pY775 to PLC1 from activated cells was blocked by the phosphorylated peptide used as the immunogen for Ab production but not by its non-phosphorylated counterpart (Fig. 2B). Similar results were seen in Jurkat cells transiently transfected with the respective PLC1 constructs (unpublished data). In addition, Y775 was phosphorylated on endogenous PLC1 after anti-TCR stimulation of Jurkat cells, PHA blasts generated from human peripheral blood lymphocytes (Fig. 2C), or mouse CD4⁺ lymphocytes (unpublished data). Although the reactivity of anti-pY775 was substantially higher in PLC1 constructs with intact Y775, the Ab also exhibited a low level of reactivity with the YYFYY construct in stimulated P10–14 cells (Fig. 2B). This low level of cross-reactivity was not observed in T cells, nevertheless, we routinely included the WT and YYFYY PLC1 constructs as controls in experiments that used the anti-pY775 antibody. Phosphorylation of Y775 did not require the presence of Y783 (Fig. 2B), nor did the phosphorylation of Y783 require Y775 (unpublished data). These data indicate that PLC1 Y775 is phosphorylated upon TCR and BCR stimulation and that its phosphorylation contributes to the signal intensity observed with anti-phosphotyrosine antibodies, such as 4G10. Furthermore, Y775 phosphorylation is independent of the presence of the other four tyrosines, as there was no alteration in its level of phosphorylation in the FFYFF construct compared with WT PLC1 (Fig. 2B).

Y775 and Y783 phosphorylation is required for PLC1-dependent calcium mobilization

PLC1 Y783, and its counterpart Y759 in PLC2, has been shown by several groups to be important for receptor-mediated activation of PLC1 (14, 19, 20, 22, 23). The role of other tyrosines is less clear. Data from Kim et al. (14) suggested that after PDGF receptor (PDGFR) stimulation, mutation of Y1254 inhibited, and mutation of Y771 enhanced PLC1 activity. Recent work from this group, however, raises questions about the significance of Y771 and Y1254 phosphorylation in terms of PLC1 activity after both AgR and PDGFR stimulation (23). For PLC2 activation there is also contradictory data on the role of Y1197 and Y1217 (20, 22) which may be analogous to Y1254 in PLC1. To delineate the functional importance of the tyrosine sites for in vivo AgR-induced PLC1 activation, we first investigated the requirement for each tyrosine in BCR-induced Ca²⁺ mobilization in stable transfectants of the P10–14 cell line. This B cell line was chosen because it is devoid of PLC1 and PLC2 and thus has no Ca²⁺ mobilization, while the PLC1-deficient Jurkat T cell line, J1, has residual detectable calcium responses due to endogenous PLC2 (13). Clones with matching levels of HA-PLC1 and BCR expression were used. All clones responded equally well to stimulation with ionomycin (unpublished data). The positive control cell line expressing WT PLC1 showed a characteristic pattern of Ca²⁺ signaling upon BCR activation (Fig. 3A). As expected, neither the parental PLC-deficient P10–14 cell line (Null), nor the PLC1 FFFFF expressing cell line mobilized Ca²⁺ after BCR cross-linking (Fig. 3A). Significantly, a single mutation in either Y775 or Y783 resulted in a dramatic decrease in BCR-induced Ca²⁺ mobilization, whereas the single mutation of Y771 had no effect on the Ca²⁺ response (Fig. 3B). Mutation of both Y775 and Y783 to phenylalanine in the context of intact Y472, Y771 and Y1254 (PLC 1 YYFFY) produced a PLC1 construct that was incapable of mediating a BCR-induced Ca²⁺ flux (Fig. 3C). Conversely, a PLC1 construct that had Y472, Y771 and Y1254 mutated to phenylalanine, but had intact Y775 and Y783 residues (PLC1 FFYYF) mediated a BCR-induced Ca²⁺ response that was comparable to WT PLC1 (Fig. 3D). These data confirm the importance of Y783 in PLC1 activation and indicate that Y775 plays an equally important role in AgR-induced PLC1 activation. Data from the PLC1 FFYYF and YYFFY constructs suggest that, of the known tyrosine phosphorylation sites in PLC1, Y775 and Y783 are both required and sufficient for full AgR-induced PLC1 activation.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. PLC1 Y775 and Y783 are required for BCR-induced calcium mobilization. P10–14 stable cell lines expressing the indicated PLC1 phosphorylation mutant were loaded with Indo-1/AM and stimulated with anti-IgM Ab. The ratio of fluorescence emission at 405 and 495 nm was plotted as a function of time. In total, three stable clones per construct were tested at least three times. The representative kinetics in A, B, and D are from the same experiment. WT PLC1 and parental P10–14 cells (null) served as positive and negative controls, respectively, in each experiment.

Both Y775 and Y783 are required for PLC1-dependent gene transcription events

To further delineate the impact of PLC1 phosphorylation on downstream signaling events, we measured the transcriptional activation of NF-AT and AP-1 using luciferase reporter gene assays. For this purpose, the PLC1-deficient Jurkat cell line, J1 (13), was transiently cotransfected with either an NF-AT- or AP-1-driven luciferase reporter construct, and the respective PLC1 Y to F mutant. The cells were subsequently activated through the TCR with C305, and the luciferase activity measured (Fig. 4, A and B). J1 cells are able to generate small but detectable NF-AT and AP-1 responses, which are probably mediated by low levels of PLC2 in these cells (13). PLC1 FFFFF was therefore included in all experiments to control for this background activity. Cells transfected with WT PLC1 and PLC1 FFYYF showed robust NF-AT activation when stimulated with C305 compared with non-stimulated cells (Fig. 4A). No activity was observed in the double Y775 and Y783 mutant (PLC1 YYFFY). The single Y775F construct (YYFYY) had reduced TCR-induced NF-AT activation whereas the response of PLC1 Y783F (YYYFY) was comparable with that observed with PLC1 FFFFF, suggesting that it was incapable of inducing NF-AT activation. Conversely, PLC1 FFFYF retained a marginal level of activity, whereas PLC1 FFYFF had baseline NF-AT activation. Similar results were obtained in transiently transfected P10–14 cells (Fig. 4C). These data suggest that phosphorylation of both tyrosines is required for PLC1 to mediate full NF-AT activation.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. PLC1 Y775 and Y783 are required for PLC1-dependent AgR-induced transcriptional events. A, PLC1-deficient J1 Jurkat cells were transiently transfected with NF-AT luciferase reporter plasmid and the indicated PLC1 expression plasmid. Cells were cultured in medium and stimulated with C305 Ab or PMA plus ionomycin. NF-AT activation was normalized to the maximal activity obtained with PMA plus ionomycin in each sample for intra- and interexperimental comparison. B, J1 cells were transiently transfected with AP-1-luciferase reporter plasmid and the indicated PLC1 expression plasmid. Cells were cultured in medium and stimulated with C305 mAb or PMA. AP-1 activation was normalized to the maximal activity obtained with PMA in each sample. C, PLC1-deficient P10–14 cells were transiently transfected with NF-AT luciferase reporter plasmid and the indicated PLC1 expression plasmid. Cells were cultured in medium, stimulated with anti-IgM Ab, or PMA plus ionomycin. NF-AT activation was normalized to the maximal activity obtained with PMA plus ionomycin for each construct and normalized to WT for cross-experimental comparison. All panels show the mean and SEM of three experiments, and in all cases, cells were transfected with 5 µg of reporter plasmid and 5 µg of the respective PLC1 construct.

These results definitively identify Y775 as a new, functionally important phosphorylation site on PLC1. They further indicate that phosphorylation of both Y775 and Y783 is required for PLC1 activation as measured by AgR-induced Ca²⁺ flux, NF-AT and AP-1 activation. Y775 and Y783 are therefore identified as the primary regulatory tyrosines for both the IP₃-mediated Ca²⁺ flux and the DAG-mediated activation of the PKC/RasGRP pathway. No role for AgR-induced Y472, Y771, and Y1254 phosphorylation is apparent from our data, suggesting that phosphorylation of these sites is not required for AgR-induced PLC1 activation. In addition, our data suggest that the phosphorylation of Y775 occurs independently of the other tyrosine residues and that it does not affect the phosphorylation of Y783.

These results underscore the importance of tyrosine phosphorylation for the activation of PLC1. Additional work is required to distinguish whether phosphorylation alters the intramolecular conformation of PLC1 (24), its substrate recognition (12) or protein/protein interactions that are required for activation (25).

Furthermore, although we have shown that Y775 is phosphorylated after TCR engagement in human peripheral T cells and mouse CD4⁺ T cells, the role that Y775 plays in PLC1 activation during T and B cell development and lymphocyte activation in vivo remains to be confirmed. Our data emphasize the importance of using site-specific anti-pY Abs for the analysis of proteins with multiple tyrosine phosphorylation sites and delineate the critical roles of Y775 and Y783 for PLC1 function.

	Acknowledgments

 
We thank Drs. P. Schwartzberg and W. J. Wu for critically reading the manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ Current address: MacroGenics, 1500 East Gude Drive, Rockville, MD 20850.

² Address correspondence and reprint requests to Dr. Barbara L. Rellahan, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, National Institutes of Health Campus, Building 29B, HFD-123, 29 Lincoln Drive, MSC-4555, Bethesda, MD 20892-4555. E-mail address: rellahan{at}cber.fda.gov

³ Abbreviations used in this paper: PLC, phospholipase C; PDGF, platelet-derived growth factor; IP₃, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; HA, hemagglutinin; pY, phosphotyrosine; WT, wild type.

Received for publication December 15, 2004. Accepted for publication March 3, 2005.

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Top Abstract Introduction Materials and Methods Results and Discussion Disclosures References

 

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