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J. Biol. Chem., Vol. 282, Issue 46, 33760-33768, November 16, 2007
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Generation of an Analog-sensitive Syk Tyrosine Kinase for the Study of Signaling Dynamics from the B Cell Antigen Receptor*
1¶2
From the
Departments of Medicinal Chemistry and Molecular Pharmacology and ¶Chemistry and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907
Received for publication, June 12, 2007 , and in revised form, September 17, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The Syk protein-tyrosine kinase is an essential component of the signaling machinery that couples the B cell receptor for antigen to multiple downstream signal transduction pathways. Syk is phosphorylated and activated rapidly and transiently following receptor engagement, but many signaling events, such as the activation of transcription factors occur over the course of several minutes or hours. To investigate a role for the continued activation of Syk in these processes, we generated an analog-sensitive mutant with an engineered ATP-binding pocket to render the kinase uniquely sensitive to an orthogonal inhibitor. Mutation of the gatekeeper residue in Syk yielded an enzyme with very low activity. Second-site mutations, selected based on structural comparisons between Syk and Src, were introduced that restored catalytic activity to the mutant Syk. Syk-deficient DT40 B cells were prepared expressing the analog-sensitive Syk (Syk-AQL). Inhibition of the activity of Syk prior to, concomitant with or shortly following receptor engagement led to the rapid inhibition of receptor-mediated tyrosine phosphorylation and blocked the activation of extracellular signal-regulated kinase, NF-
B, and NFAT. The receptor-mediated activation of NF-B required active Syk for a relatively short period of time, whereas the activation of NFAT required active kinase for a prolonged (>1 h) period. Receptor cross-linking led to the recruitment of Syk to the clustered receptor. Retention of these receptor-kinase complexes on the cell surface was dependent on the continued activity of Syk. Thus, despite the apparent transient nature of the activation of Syk, the catalytic activity of the Syk was required for sustained signaling from ligated receptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Upon engagement of the B cell antigen receptor (BCR)3 by binding polyvalent antigens, immunoreceptor tyrosine-based activation motifs in the cytoplasmic domains of Ig-
and Ig-
are phosphorylated leading to the recruitment of Syk to the receptor complex and its phosphorylation on tyrosine and subsequent activation (1). Syk is then required for coupling the BCR to the phosphorylation and activation of the downstream regulatory and adaptor molecules BLNK/SLP65, phosphoinositide 3-kinase, Btk, and phospholipase C-
2, leading to the activation of Akt and protein kinase C, the mobilization of Ca2+, activation of the Ras/Raf/ERK and p38 mitogen-activated protein kinase (MAPK) signaling cascades, and activation of the transcription factors NF-B and NFAT.
The kinetics of these BCR-initiated signaling events are of considerable interest as their magnitude and duration are thought to be critical factors in determining B cell survival, development and proliferation. The phosphorylation on tyrosine of BCR components, Syk and Btk all occur rapidly following receptor engagement (2-4). Phosphorylation is important for the activity of Syk and Btk and for the retention of BCR-signaling complexes on the cell surface (5); but these also are transient events. After immediate activation of membrane proximal signaling, there is a slower increase of negative signaling through phosphatases that had been transiently inhibited by BCR-stimulated increases in H2O2 (2, 3, 6). This dampens the wave of positive signaling and regulates the time and magnitude of the BCR response. However, other receptor-mediated responses are of longer duration. The phosphorylation of ERK, which also occurs rapidly following receptor engagement, is more sustained than the phosphorylation of Syk (2). The rapid, extensive release of calcium from intracellular stores that occurs following receptor engagement is followed by a prolonged phase of low level, extracellular calcium influx. These two phases of calcium mobilization are required for the activation of NF-B and NFAT, respectively (7). Prolonged receptor engagement is required for the induction of cell cycle arrest in immature WEHI-231 B cells (8), maintenance of B cell anergy (9), proliferation and survival of activated B cells (10), and proliferation of certain B cell lymphomas (11).
Although Syk is phosphorylated and activated transiently following BCR engagement and is critical for the initiation of most BCR-mediated signaling events, it is not clear whether its prolonged activation is needed for sustained signaling. We sought a model system in which the activity of Syk could be unambiguously silenced as a function of time following BCR cross-linking. For such an approach, inhibitors can be used to limit the activity of a particular kinase. However, there are often limitations regarding the specificity and selectivity of small molecule inhibitors. Conventional gene knock-out or silencing approaches do not lend themselves to an analysis of events occurring over a short time-scale. We sought instead to use a chemical genetic approach by developing an engineered form of Syk with an expanded ATP-binding site uniquely sensitive to a PP1-derived inhibitor bearing a bulky side chain (12, 13). With this approach, the activity of Syk could be specifically blocked without affecting the activities of other kinases in the cell. We found, however, that the conventional mutagenesis strategy employed for the development of an "analog-sensitive" Syk kinase resulted in a loss of catalytic activity necessitating the development of a second-site suppressor strategy for the generation of an active enzyme. Using this engineered kinase, we found that the receptor-stimulated phosphorylation of proteins on tyrosine, the activation of ERK, and the presence of active BCR-signaling complexes at the plasma membrane were rapidly reversed upon inhibition of Syk. Although the activation of NF-B required a relatively short period of Syk activity, the activation of NFAT required its prolonged activation. Thus, despite the apparent transient nature of Syk activation, its activity is actually critical for continued signaling from liganded BCR complexes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—The inhibitors 4-amino-1-tert-butyl-3-(1'naphthyl)pyrazolo[3,4-d]pyrimidine (1Na-PP1), 4-amino-1-(tert-butyl)-3-(1'-naphthylmethyl)pyrazolo [3,4-d] pyrimidine (1NM-PP1), and 4-amino-1-tert-butyl-3-(2'-naphthylmethyl) pyrazolo[3,4-d] pyrimdine (2NM-PP1) were synthesized as described (13). The antiphosphotyrosine antibody 4G10 was obtained from Upstate%20Biotechnology">Upstate Biotechnology, anti-pERK from Cell Signaling Technologies, and anti-Syk (N19) from Santa Cruz Biotechnology.
Cells, Transfections, and Stable Cell Lines—Chicken DT40 cells were cultured in RPMI 1640 media supplemented with 10% fetal calf serum, 1% chicken serum, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 IU/ml penicillin G, and 100 µg/ml streptomycin. Syk-deficient DT40 cells (14) were transfected by electroporation (300 V, 330 µF) using a Cell-Porator (Invitrogen). Cells were co-transfected with the plasmid, pBabe-puro, and selected in media containing 0.5 µg/ml puromycin to establish stable cell lines. Protein expression was confirmed by Western blot analysis and fluorescence microscopy.
Plasmids and Mutagenesis—Plasmids coding for Myc epitope-tagged Syk(M442A) or Syk(M442G) were generated with the ExSiteTM PCR-based site-directed mutagenesis kit (Strategene) using, as a template, the pCMV-Myc-Syk plasmid. Plasmids coding for Myc-tagged Syk(R428Q/M429L/M442A) (referred to here as Syk-AQL) or Syk(R428Q/M429L/M442G) (Syk-GQL) were also constructed using this PCR-based strategy from the corresponding Syk(M442A) or Syk(M442G) expression vectors. The coding sequence for Syk-AQL was amplified by PCR and inserted into the pEGFP-N2 plasmid (BD Biosciences/Clontech) to generate the Syk-AQL-EGFP expression vector.
Cellular Activation Assays—For measurement of the activation of NF-B or NFAT, Syk-deficient DT40 cells were co-transfected with 20 µg of the indicated Syk expression plasmid or empty vector and 2 µg of an NF-B- or NFAT-driven luciferase reporter plasmid. Alternatively, DT40 cell lines stably expressing Syk or a mutant form of Syk were transfected with the reporter plasmid alone. Cells were stimulated by varying concentrations of goat anti-chicken IgM antibody (Bethyl) or with a mixture of PMA (50 ng/ml) and ionomycin (1 µM) at 37 °C for 6 h. For some experiments, cells were treated at various times before or following receptor ligation with 1-Na-PP1, 1-NM-PP1, 2-NM-PP1, or with the carrier solvent Me2SO alone. Luciferase activity was measured in cell lysates using the luciferase assay system kit (Promega).
For the analysis of protein expression or phosphorylation, DT40 cells were treated with or without anti-IgM in the presence or absence of PP1 derivatives for the indicated periods of time at 37 °C and then lysed in buffer containing 1% Nonidet P-40, 150 mM NaCl, 25 mM HEPES, pH 7.5, 1 mM EDTA, pH 7.5, 2mM sodium orthovanadate, 2 mM sodium fluoride, 100 µg/ml aprotinin, 100 µg/ml leupeptin, and 625 µM phenylmethylsulfonyl fluoride. Proteins in supernatants collected following centrifugation at 18,000 x g for 10 min were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes and analyzed by Western blotting with antiphosphotyrosine, anti-Syk, and/or anti-pERK antibodies.
In Vitro Kinase Assay—DT40 cells were collected and lysed as described above. A 10-µl aliquot of each lysate was assayed in a total volume of 50 µl containing 50 mM HEPES, pH 7.4, 2 mM MnCl2, 1 mM sodium orthovanadate, 10 µM ATP, 10 µCi [-32P]ATP and as a substrate, 3 µg of either cdb3 or tubulin for 10 min at 30 °C. Reactions were stopped by heating in SDS sample buffer. Samples were separated by SDS-PAGE and stained with Coomassie blue. Protein bands corresponding to cdb3 or tubulin were excised and analyzed by scintillation spectrometry.
Immunofluorescence Microscopy—DT40 cells expressing EGFP-tagged proteins were collected, washed twice with and resuspended in serum-free RPMI 1640 medium, and stimulated at 37 °C for the indicated times with 10 µg/ml of anti-chicken IgM antibody conjugated with Texas Red. Cells were fixed in 1 ml of 10% formaldehyde in PBS and transferred to polylysine-coated coverslips for microscopic examination. To measure receptor internalization, cells were stimulated at 37 °C with 10 µg/ml of anti-chicken IgM antibody for 30 min. 1NM-PP1 (5 µM) or Me2SO carrier was added to the cells 5 min after treatment with anti-IgM antibody. Cells on poly-L-lysine-coated coverslips were fixed for 15 min by adding 3.7% formaldehyde solution. Cells were permeabilized by incubation for 15 min with 0.5% Triton X-100 in PBS or were treated only with PBS in the case of nonpermeabilized cells. Cells were incubated at room temperature for 1 h in PBS containing 0.1 µg/ml of donkey anti-goat IgG conjugated with Alexa Fluor 594 (Molecular Probes). Cells were observed by fluorescence microscopy. Fluorescence intensity was quantified using NIH image J software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Establishing an Analog-sensitive Form of Syk—A reduction in the size of the "gatekeeper" residue in the ATP-binding site of protein kinases allows access to an additional binding pocket that can be occupied by nucleotides or inhibitors bearing bulky substituents (12, 13). Through sequence alignments, we identified the gatekeeper residue in Syk as Met-442 and then prepared site-directed mutagenesis plasmids for the expression of forms of Syk in which methionine was replaced by a smaller glycine or alanine residue.
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, WT Syk) or Syk(M442G) (
, M442G) or an empty vector (
, Syk-/-) were transiently transfected into Syk-deficient DT40 cells along with a NF-
, Syk-/-) or DT40 cells stably expressing wild-type Syk (The plasmids expressing wild-type Syk, Syk(M442G), or Syk(M442A) were transiently transfected into Syk-deficient DT40 cells along with an NF-
B luciferase reporter plasmid to assess the signaling capabilities of the two mutants. Syk(M442G) was largely unable to restore BCR-stimulated signaling to the Syk-deficient cells despite a level of expression equivalent to that of the wild-type kinase (Fig. 1A). However, Syk(M442A) exhibited a partial ability to restore signaling to the Syk-deficient cells (Fig. 1B). To determine whether this level of activity would be sufficient for further analyses of receptor-mediated signaling, we established stable DT40 cell lines expressing either wild-type Syk or the engineered Syk mutant. Cells stably expressing Syk(M442G) were unable to couple BCR engagement to the activation of NF-B (Fig. 1C). In this system, Syk(M442A) also restored a low level of BCR-stimulated NF-B activity similar to that observed in the transiently transfected cells (Fig. 1C). Similarly, the BCR-stimulated phosphorylation of proteins on tyrosine was defective in cells expressing Syk(M442A) as compared with wild-type Syk and was more comparable with the pattern observed in Syk-deficient cells (Fig. 1E). The defective signaling observed in cells expressing Syk(M442G) or Syk(M442A) was likely because of a substantial decrease in their catalytic activities as lysates of cells expressing either enzyme demonstrated very low activity in an in vitro kinase assay using cdb3 as an exogenous substrate as compared with lysates from cells expressing the wild-type kinase (Fig. 1D). These data indicate that the mutation of Met-442 to a smaller amino acid residue largely abrogates the catalytic activity of Syk. Development of Second-site Suppressor Mutations to Recover the Catalytic Activity of an Analog-sensitive Form of Syk—Although the single-site mutation strategy for the generation of an analog-sensitive kinase has been successful for many enzymes, some kinases are inactivated or become unstable when the gatekeeper residue is mutated. In these cases, additional mutations often can be made to restore activity to the modified kinases (15). The loss of activity in Syk(M442A) or Syk(M442G) mutants suggests destabilization of the N-lobe structure. To identify second-site suppressor mutations that would restore catalytic activity to either the Syk(M442A) or Syk(M442G) mutant, we compared the three-dimensional structures of the catalytic domains of Syk and Src. Src was chosen because the structure of the catalytic domain is very similar to that of Syk, and mutation of the gatekeeper residue of Src yields an active, analog-sensitive kinase (12).
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, Syk-/-) was transiently transfected into Syk-deficient DT40 cells along with a NF-We compared the amino acid residues interacting with the gatekeeper residues in both ATP-binding sites (Fig. 2A). In the active Src kinase crystal structure (16), Leu-325 is within 5 Å of Thr-338 and points toward the pocket close to Thr-338. The side chain of Gln-324 is within 8 Å of Thr-338. In the Syk kinase crystal structure (17), Leu-325 is replaced by the bulkier Met-429, and Gln-324 is replaced by Arg-428. In addition to their proximity to Thr-338/Met-442, these residues are close to the C-terminal end of the important alpha helix C and may be involved in stabilizing the N-lobe structure. To determine whether we could improve the stability of the Syk gatekeeper mutants, we replaced the codons for Arg-428 and Met-429 with sequences coding for glutamine and leucine, respectively, to generate plasmids coding for Syk(R428Q/M429L/M442A) (Syk-AQL) and Syk(R428Q/M429L/M442G) (Syk-GQL).
Investigation of the Signaling Activities of Syk-AQL and Syk-GQL—The expression vectors for Syk-AQL and Syk-GQL were transiently transfected into Syk-deficient DT40 cells along with the NK-B-driven luciferase reporter plasmid as described above to determine whether the additional mutations restored activity to either kinase. Interestingly, Syk-AQL was able to signal in Syk-deficient cells as efficiently as wild-type Syk (Fig. 2B). The activity of Syk-GQL was somewhat improved from that of Syk(M442G), but was still relatively low (Fig. 2B).
Cell lines stably transfected to express Syk-AQL were generated for further analyses. BCR-stimulated NF-B activity was restored in cells expressing Syk-AQL to the levels observed in cells containing wild-type Syk (Fig. 2C). Similarly, the extent of receptor-induced tyrosine phosphorylation in cells expressing Syk-AQL was comparable with that of cells expressing the wild-type kinase (Fig. 2D). Cells stably expressing Syk-GQL were unable to signal, most likely because of the lower level of expression of the mutant in stable cell lines as compared with transiently transfected cells. These results indicate that the substitution of glutamine for Arg-428 and leucine for Met-429 within the ATP-binding site can restore activity to a Syk(M442A) mutant.
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Sensitivity of Syk-AQL to Engineered PP1 Derivatives—To be useful, the engineered kinase needs to exhibit the desired selective sensitivity to bulky, PP1-derived inhibitors. To test this, we incubated cells expressing wild-type Syk or Syk-AQL with varying concentrations of 1Na-PP1, 1NM-PP1, 2NM-PP1, or Me2SO carrier for 15 min prior to the addition of anti-IgM antibody. The activation of NF-
B was assessed using the NF-B-driven luciferase reporter assay. 1Na-PP1 and 1NM-PP1 both inhibited the ability of Syk-AQL to couple the BCR to the activation of NF-B in a dose-dependent manner, but neither affected signaling in cells expressing wild-type Syk (Fig. 3, A and B). Neither kinase was inhibited by 2NM-PP1 (Fig. 3C), illustrating the specificity of the kinase-inhibitor interaction. Investigations of the Kinetics of BCR Signaling—Many of the antigen receptor-mediated phosphorylation that occur following BCR cross-linking are dependent on the expression of a functional Syk kinase. To examine the effects of the specific inhibition of Syk on BCR signaling to protein-tyrosine phosphorylation, we treated cells expressing Syk-AQL or wild-type Syk with 1NM-PP1 either 5 min prior to or simultaneously with the addition of activating anti-IgM antibodies. After 5 min of activation, cells were harvested and analyzed for content of tyrosine-phosphorylated proteins by Western blotting. Treatment with 1NM-PP1 dramatically reduced the level of phosphorylation both in the pretreated cells and in cells in which 1NM-PP1 and an anti-IgM antibody were added simultaneously (Fig. 4A). The inhibitor had little effect on tyrosine phosphorylation in cells expressing wild-type Syk. Similar results were observed if 1Na-PP1 was substituted for 1NM-PP1 (data not shown). These data indicate that the inhibitor can penetrate cells quickly to inhibit tyrosine phosphorylation mediated by Syk-AQL after anti-IgM stimulation.
Because Syk is phosphorylated rapidly and transiently following receptor aggregation, we were interested in determining if the continued activity of Syk was necessary to sustain the phosphorylation of downstream substrates. To investigate this, we treated Syk-AQL-expressing cells with or without 1NM-PP1 at various times prior to or following BCR engagement and examined the profile of tyrosine-phosphorylated proteins by Western blotting. When cells were treated with inhibitor 1 min prior to the addition of anti-IgM, receptor-stimulated protein-tyrosine phosphorylation was blocked when analyzed at 5, 10, or 15 min after activation (Fig. 4B). Interestingly, addition of the inhibitor even at 5 or 10 min following receptor engagement resulted in a rapid reduction of global protein-tyrosine phosphorylation to near basal levels. Thus, the continued activity of Syk is required for maintaining the state of tyrosine phosphorylation of essentially all of the readily detectable substrates that are modified following BCR engagement.
To determine whether the continued activity of Syk was required for the prolonged activation of ERK, the membrane from the above experiment was stripped and reprobed with anti-pERK antibodies. The inhibition of Syk-AQL resulted in a loss of ERK phosphorylation regardless of the time of addition of the inhibitor (Fig. 4B).
Coupling the BCR to the Regulation of Transcription Factors—We were interested in examining the relationship between the duration of the activation of Syk and the kinetics of NF-B- or NFAT-induced gene expression because of their different responses to changes in the magnitude and period of calcium fluxes. Cells expressing Syk-AQL were transfected with either an NF-B- or an NFAT-driven luciferase plasmid and were treated with 1NM-PP1 at different time points following stimulation with anti-IgM. The level of luciferase was measured 6 h later. The inhibition of Syk immediately following receptor engagement decreased the extent to which NF-B was activated (Fig. 5A). However, a substantial recovery of NF-B activity was observed when the inhibitor was added 30 min or longer following stimulation, suggesting that a relatively short-term activation of Syk was enough to induce the activation of NF-B. The inhibition of Syk-AQL also blocked the receptor-mediated activation of NFAT. Interestingly, however, the activation of NFAT was blocked even when the inhibitor was added 1 h after the initial stimulation (Fig. 5B). This result indicates that the full activation of NFAT requires that Syk be continuously active for a prolonged period of time following BCR stimulation.
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Recruitment of Syk-AQL to the Antigen Receptor—To monitor the interactions of Syk-AQL with the antigen receptor, we generated an expression vector coding for Syk-AQL fused at the C terminus with enhanced green fluorescent protein (Syk-AQL-EGFP). A Syk-deficient cell line stably expressing Syk-AQL-EGFP was generated. Antigen receptors were engaged using an anti-IgM antibody conjugated with Texas Red, and the localization of both the BCR and Syk-AQL-EGFP were examined by fluorescence microscopy. Ten minutes following addition of anti-IgM antibody, the cross-linked BCR complex formed a cap at one pole of the cell that co-localized with Syk-AQL-EGFP (Fig. 6A), indicating that Syk-AQL-EGFP could be recruited normally to the clustered BCR. Similar results were seen 6 min following receptor aggregation (not shown).
After 30 min of stimulation, a fraction of the clustered BCR complexes are internalized, whereas others are retained at the cell surface (18). Consistent with this, clusters of surface IgM in association with the kinase were readily visible at the plasma membrane of cells expressing Syk-AQL-EGFP that had been treated with anti-IgM antibody for 30 min in the absence of inhibitor (Fig. 6B). However, the addition of 1NM-PP1 to cells with preformed caps enhanced the internalization of receptor complexes and decreased the retention of BCR-Syk complexes at the membrane (Fig. 6B). The internalized receptors were not associated with Syk-AQL-EGFP, whereas those at the membrane retained the interaction. To confirm changes in receptor location, we stimulated cells expressing Syk-AQL-EGFP as above, but using unlabeled anti-IgM antibodies. The resulting anti-IgM-BCR complexes were then detected in either detergent-permeabilized or intact, nonpermeabilized cells using a labeled secondary antibody. The treatment of cells with 1NM-PP1 resulted in a substantial decrease in the level of cell surface receptor with a corresponding increase in the level of internalized receptor (Fig. 6C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this study, we describe a chemical genetic approach to the study of Syk and its role in BCR signaling. Shokat and co-workers (19) have shown that mutations can be made to expand the ATP-binding sites of kinases to engineer in a sensitivity to inhibitors specifically designed to occupy the newly enlarged binding pocket. Although this strategy works for many kinases, an estimated 30% are destabilized by a reduction in size of the gatekeeper residue and are rendered largely inactive. We find that the activity of Syk also is greatly reduced by the substitution of the gatekeeper methionine with either glycine or alanine. A random mutagenesis approach has been described to identify second-site suppressor mutations that can be introduced into the
2, 3, and 5 strands of the N-lobe sheet of the kinase domains of "intolerant" kinases to stabilize their structures and restore activity (15). Wild-type Syk, however, already contains the residues at these positions that would be expected to stabilize the N-lobe sheet. Consequently, we sought an alternative approach to the generation of second-site suppressor mutations. In this approach, we focused on the amino acids in closest contact with the gatekeeper methionine in Syk reasoning that these might be the most affected by a gatekeeper mutation. We substituted two of these amino acids, Arg-428 and Met-429, with glutamine and leucine, respectively, based on the fact that Gln-324 and Leu-325 are found at the same relative positions in Src and are in close contact with the Src gatekeeper Thr-338. Moreover, Src is stable to mutagenesis of its gatekeeper residue (12). Our studies indicate that this approach is successful for restoring catalytic function to a Syk(M442A), but not a Syk(M442G) gatekeeper mutant. The resulting kinase, designated Syk-AQL, is effectively recruited to the aggregated BCR complex and restores signaling from the receptor to the activation of protein-tyrosine phosphorylation, ERK, NFAT, and NF-B to an extent similar to that of the wild-type kinase, indicating that the mutation does not disrupt the normal biological function of the enzyme. The engineered kinase also retains its selective sensitivity to the bulky PP1 derivatives, 1NM-PP1 and 1Na-PP1, indicating that the additional mutations do not reduce the size of the enlarged binding pocket. The engineered kinase can also discriminate between different isomers of the inhibitor, as 2NM-PP1 does not affect the activity of Syk-AQL. Thus, this strategy provides a new tool to aid in the development of active, analog-sensitive kinases in cases where the gatekeeper mutation compromises the activity of the enzyme.
Chemical genetic approaches are well suited to the study of dynamic processes that cannot be studied by conventional knock-out or knock-down strategies. Our examination of the roles of Syk in propagating signals from the BCR was aided by the availability of Syk-deficient cells with signaling restored by the expression of either the engineered kinase or the wild-type enzyme. Signaling from the antigen receptor to the phosphorylation of proteins on tyrosine and the activation of ERK, NF-B, and NFAT are all inhibited by 1NM-PP1 in cells expressing Syk-AQL, but not in cells expressing wild-type Syk, indicating that the effects of the inhibitor are confined to the inhibition of the engineered kinase.
Inhibition of Syk-AQL activity prior to receptor engagement blocks mostly all receptor-stimulated tyrosine phosphorylation. A similar effect is seen if the inhibitor is added simultaneously with the activating antibody, suggesting that cells are highly permeable to 1NM-PP1 and that inhibition is rapid. It is interesting to note, however, that addition of inhibitor at various times after receptor engagement also leads to a substantial loss of tyrosine phosphorylation to the point where levels are nearly as low as those observed in inhibitor-pretreated cells. This is most certainly because of the actions of protein-tyrosine phosphatases that rapidly dephosphorylate proteins once the activity of Syk is terminated. Thus, the extent of tyrosine phosphorylation that is observed following receptor engagement represents a dynamic balance between protein phosphorylation and dephosphorylation. Despite the fact that additional tyrosine kinases such as Btk and Lyn are activated following receptor aggregation, the continued activity of Syk is essential for maintaining the level of tyrosine phosphorylation at detectable levels in anti-IgM-stimulated cells.
The BCR-dependent activation of ERK, as measured by its phosphorylation, is more prolonged than the phosphorylation and activation of Syk, suggesting the additional contribution of BCR-dependent, but Syk-independent, regulatory mechanisms (2, 20). Despite this difference in activation kinetics, our data indicate that the continued activity of Syk is, in fact, required for maintaining ERK in its activated state as the inhibition of Syk-AQL at varying times following BCR engagement decreases the amount of phosphorylated, activated ERK to basal levels.
BCR clustering eventually leads to the activation of transcription factors and changes in gene expression that determine the final physiological outcome of receptor engagement. Two important transcription factors that are activated through the BCR are NF-B and NFAT. The activation of both are inhibited by the preincubation of Syk-AQL-expressing cells with 1NM-PP1 reflecting the need for a functional Syk kinase to initiate the signaling cascades leading to their stimulation. However, the length of time that Syk needs to be active following receptor engagement is markedly different for regulation of the two factors. Substantial activation of NF-B requires Syk to remain active for a relatively short period of time following BCR ligation. This likely reflects the fact that NF-B is activated following an immediate and pronounced increase in intracellular Ca2+ (7), an event that is known to be regulated by Syk (21). In contrast, the activation of NFAT, which requires a prolonged residual Ca2+ flux, also requires a prolonged period of time, in excess of 1 h, during which Syk must remain active. Thus, despite the apparent rapid and transient nature of the phosphorylation of Syk and activation following receptor engagement, its activity is still required for an extended period of time for the effective activation of NFAT. Because prolonged Ca2+ influx in B cells is regulated by Btk (21), these data suggest that the continued activity of Syk is necessary for maintaining the required level of activity of Btk needed to regulate this process.
Interactions of the BCR with polyvalent antigens or anti-receptor antibodies lead both to the generation of downstream signals and the internalization of receptor-ligand complexes. Aggregated receptors form clustered patches on the cell surface, which then migrate to a single pole of the cell to form a cap where most are internalized. The extent of capping varies between B cell lines but is quite pronounced in DT40 cells and is enhanced by the expression of Syk (18). Syk-AQL-EGFP, like Syk-EGFP (18) is associated with the capped receptors but is not associated with the internalized receptors.
A subset of the BCR/Syk-AQL-EGFP complexes that form at the plasma membrane is retained for at least 30 min following receptor engagement. This is consistent with our previous studies on BCR-Syk interactions in cells expressing Syk-EGFP, which demonstrated the persistence of small clusters or patches of receptor-associated Syk on the cell surface for periods of time in excess of 1 h and after most receptors have been internalized (18). An interesting recent study has shown that receptor complexes that are active for signaling remain on the cell surface because of their phosphorylation on the same tyrosine residues that also mediate signaling; the internalized receptors are not phosphorylated (5). Therefore, it is likely that these Syk-AQL-EGFP-containing BCR complexes are retained because of receptor phosphorylation. It has been proposed that Syk, associated with phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs), is active to phosphorylate neighboring BCR complexes to amplify signals initiated by BCR engagement (5). An analogous process may also allow the long-term, dynamic turnover of phosphate on the cytoplasmic chains of the clustered BCR complexes that contain active Syk to maintain these at the plasma membrane. Consequently, the subsequent treatment of anti-IgM-activated cells with 1NM-PP1, which leads to the enhanced internalization of antigen receptors, is most likely because of receptor dephosphorylation in the absence of active Syk. These dephosphorylated, internalized receptors lack associated Syk. These observations suggest that prolonged BCR-dependent signaling can continue from cell surface receptors that are clustered and contain the catalytically active Syk, which is needed to maintain the phosphorylation required to retain them at the cell surface.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA37372 awarded by the National Cancer Institute (to R. L. G. and M. L. H.). 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: Dept. of Chemical Engineering, Bogazici University, Bebek 34342, Istanbul, Turkey.
2 To whom correspondence should be addressed: Dept. of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Hansen Life Sciences Research Bldg., 201 S. University St., West Lafayette, IN 47907-2064. E-mail: geahlen{at}purdue.edu.
3 The abbreviations used are: BCR, B cell receptor for antigen; BLNK/SLP65, B cell linker protein/SH2 domain-containing leukocyte protein of 65 kDa; Btk, Bruton's tyrosine kinase; NF-B, nuclear factor B; NFAT, nuclear factor of activated T cells; ERK, extracellular signal-regulated kinase; 1Na-PP1, 4-amino-1-tert-butyl-3-(1'naphthyl)pyrazolo[3,4-d]pyrimidine; 1NM-PP1, 4-amino-1-(tert-butyl)-3-(1'-naphthylmethyl)pyrazolo [3,4-d] pyrimidine; 2NM-PP1, 4-amino-1-tert-butyl)-3-(2'-naphthylmethyl) pyrazolo[3,4-d] pyrimdine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; IgM, immunoglobulin M.
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