©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Syk, Activated by Cross-linking the B-cell Antigen Receptor, Localizes to the Cytosol Where It Interacts with and Phosphorylates -Tubulin on Tyrosine (*)

(Received for publication, September 11, 1995; and in revised form, December 20, 1995)

Jennifer D. Peters (1)(§) Michael T. Furlong (1)(¶) David J. Asai (2) Marietta L. Harrison (1) Robert L. Geahlen (1)(**)

From the  (1)Departments of Medicinal Chemistry and Molecular Pharmacology and (2)Biological Sciences, Purdue University, West Lafayette, Indiana 47907

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Syk (p72) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha-tubulin.


INTRODUCTION

Syk (p72) is a 72-kDa cytoplasmic protein-tyrosine kinase that is expressed in a variety of hematopoietic cells where it participates in signal transduction cascades initiated by the engagement of the cell surface receptors with which it associates. Syk is activated in B lymphocytes by aggregation of the B-cell antigen receptor (BCR)(^1)(1) ; in platelets by thrombin (2) or by integrin ligation(3) ; in mast cells by aggregation of FcRI receptors(4) ; in monocytes by cross-linking FcRI and FcRII receptors(5, 6) ; in macrophages by engagement of the FcRIIIA receptor(7) ; in T-cells by cross-linking the T-cell antigen receptor (TCR)(8, 9) ; in peripheral blood lymphocytes by interleukin-2 (10) ; and in granulocytes in response to granulocyte colony-stimulating factor(11) .

The activation of Syk in response to the engagement of multichain immune recognition receptors (12) has been studied in the greatest detail. The aggregation of these receptors leads to the phosphorylation of multiple cellular proteins on tyrosine, initiating signaling cascades resulting in increased inositol 1,4,5-trisphosphate production and calcium mobilization (for reviews, see (13, 14, 15, 16) ). That Syk is an important mediator of receptor signaling is indicated by an absence of signaling in Syk DT40 chicken B-cells(17) , an inhibition of FcRI-mediated signaling in mast cells by a Syk-selective inhibitor(18) , and a blockage in B-cell development in Syk ``knockout'' mice at stages that normally require signals to be sent through the BCR or pre-BCR(19) .

The efficient activation of Syk requires the presence on receptor components of an immunoreceptor tyrosine activation motif (ITAM), consisting of two YXXL/I cassettes separated by 6-8 amino acids (20) and the presence of at least one member of the Src family of protein-tyrosine kinases. Receptor aggregation leads to the rapid phosphorylation of the ITAM, presumably by Src family kinases, establishing a binding site for the two tandem, amino-terminal, SH2 domains of Syk. This occupancy allows the kinase to catalyze an autophosphorylation reaction, which leads to an increase in its intrinsic activity(21) .

Many of the proteins phosphorylated in response to receptor aggregation are likely to be substrates for Syk or for Syk-activated kinases. However, little is known regarding the nature of the substrates that are phosphorylated by activated Syk and where in the cell these protein-substrate interactions occur. Whether these interactions occur while Syk is associated with the receptor, for example, has not been explored in detail. To begin examining such questions, we have used cellular fractionation techniques to characterize the cytosolic and membrane-associated pools of Syk. In this report, we show that Syk activated by the aggregation of the BCR is found almost exclusively in the cytosolic fraction of activated B-cells. This activated, cytosolic Syk associates with and phosphorylates tubulin. In vitro, alpha-tubulin is an excellent substrate for Syk and becomes phosphorylated on a tyrosine residue located near the carboxyl terminus.


MATERIALS AND METHODS

Antibodies and Reagents

Rabbit polyclonal anti-Syk peptide antibodies have been described previously(22) . Additional polyclonal anti-Syk antibodies were prepared in rabbits by the Purdue University Cancer Center Antibody Production Facility against a fusion protein consisting of glutathione S-transferase (GST) linked to the carboxyl-terminal 35-kDa kinase domain of murine Syk (GST-p35). Antibodies to phosphotyrosine (1) and beta-tubulin (23) were prepared as described previously. Anti-ZAP-70 antibodies were generously provided by Dr. Lawrence Samelson, NIH. Tubulin was purified from chick brain by a reversible assembly method followed by chromatography on phosphocellulose as described elsewhere(24) . Myelin basic protein was a gift of Dr. Curt Ashendel, Purdue University, and the cytoplasmic fragment of human erythrocyte band 3 (cfb3) was obtained from Dr. Philip Low, Purdue University. Piceatannol was described previously (18) .

Preparation of GST-Syk Fusion Proteins

The cloning of murine Syk cDNA and the expression of full-length Syk in Sf9 cells will be described elsewhere. (^2)To prepare a GST-Syk fusion protein, an XhoI site was introduced by site-directed mutagenesis (25) into the Syk cDNA 15 bp upstream from the translational start site. A 3-kilobase pair XhoI/XbaI fragment containing full-length Syk cDNA was then subcloned into the XhoI/XbaI site of the baculoviral transfer vector pNTX (a gift of Dr. Harry Charbonneau, Purdue University). This vector is designed, when introduced into Sf9 cells, to direct the expression of cDNA inserts as GST fusion proteins. Sf9 cells were co-transfected with this plasmid (pNTXSyk) and BacPAK 6 viral DNA (Clontech), and plaque purification was carried out essentially as described previously(26) . Recombinant plaques were used to infect Sf9 cells, which were screened 48 h postinfection for enzymatically active GST-Syk. GST-Syk was isolated from Sf9 cell lysates by adsorption to glutathione-Sepharose.

To prepare GST-p35, the GST-Syk-expressing transfer vector pNTXSyk was digested with XhoI (just upstream of the translational start site) and ApaI (codon for proline 326). The vector fragment was gel-purified, blunt-ended with mung bean nuclease, and recircularized to generate pNTXp35. Sequencing of the fusion junction confirmed that the open reading frame had been maintained. Sf9 cell co-transfection and plaque purification were carried out as described above for GST-Syk.

Cells and Cellular Fractionation

Murine Bal17 and human DG75 B lymphocytes (5-10 times 10^6 cells/sample) were incubated on ice in the presence or absence of affinity-purified anti-IgM F(ab`)(2) fragments of goat anti-mouse IgM (Cappel) at a concentration of 25 µg/ml or goat anti-human IgM antibodies (Zymed) at 37.5 µg/ml, respectively, for the times indicated. Cells were then suspended in 800 µl of 5 mM Hepes, pH 7.5, 1 mM MgCl(2), 1 mM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin and lysed by 100 strokes of a Dounce homogenizer. Nuclei and unbroken cells were removed by centrifugation at 644 times g for 5 min at 4 °C. Soluble and particulate fractions were obtained by ultracentrifugation of the cleared lysate at 150,000 times g for 30 min at 4 °C. Pellets were resuspended in the above buffer containing 1% Triton X-100 and 150 mM NaCl. The supernatant was also adjusted to a final concentration of 1% Triton X-100 and 150 mM NaCl. In some experiments, cells were pretreated with Taxol (7.5 µg/ml) for 2.5 h prior to addition of the activating antibody. For these experiments, control cells were incubated with 0.75% Me(2)SO, which serves also as the carrier for Taxol. In other experiments, Bal17 B-cells or Jurkat T-cells were pretreated with pervanadate formed by mixing sodium orthovanadate (0.1 mM) with H(2)O(2) (1 mM).

Immunoprecipitations and Immune Complex Kinase Assays

Proteins were immunoprecipitated from soluble and particulate fractions using anti-Syk or anti-phosphotyrosine antibodies coupled to protein A-Sepharose as described previously(22) . Procedures for the detection of kinases and substrates present in immune complexes by incubation with [-P]ATP, fractionation by SDS-PAGE, and transfer to poly(vinylidene fluoride) membranes (Immobilon P) have been described in detail(22) . Phosphotyrosine-containing proteins were detected by autoradiography of poly(vinylidene fluoride) membranes that had been treated with 1 N KOH at 55 °C for 2 h to reduce the levels of phosphoserine and phosphothreonine.

For some experiments, GST-Syk immobilized on glutathione-Sepharose (see above) was used in place of the antibody-protein A-Sepharose complex.

Chromatography on Colchicine-Sepharose

Colchicine-Sepharose was prepared as described by Schmitt and Littauer(27) . Lysates were prepared by Dounce homogenization of anti-IgM-activated or untreated cells as described above except the lysis buffer contained 10 mM imidazole, pH 6.8, 5 mM MgCl(2), 1 mM sodium orthovanadate, and 10 µg/ml each of aprotinin and leupeptin. In some experiments, cells were pretreated for 1 h with piceatannol (15 µg/ml) prior to activation. Lysates from 1 times 10^7 cells were incubated with 60 nmol immobilized colchicine for 1 h at 4 °C. The resin was washed 3 times with lysis buffer. Bound proteins were eluted with 1% SDS, separated by SDS-PAGE, transferred to Immobilon P membranes, and probed using antibodies against phosphotyrosine or beta-tubulin. Immunoreactive proteins were detected using the enhanced chemiluminescence (ECL) method of Amersham Corp.

Substrate Phosphorylation

In vitro kinase reactions for recombinant Syk contained 1 µM protein substrate (tubulin dimer, cfb3, or myelin basic protein), 25 mM Hepes, pH 7.5, 1 mM sodium orthovanadate, 10 µg/ml bovine serum albumin, 2 mM MnCl(2), 10 µM ATP, and 25 µCi of [-P]ATP. Reactions were carried out at 30 °C for the times indicated and terminated by the addition of SDS sample buffer. Phosphoproteins were separated by SDS-PAGE, detected by autoradiography, and excised from the gels. The extent of phosphorylation was quantified by liquid scintillation spectrometry. For some separations, electrophoresis conditions were altered to allow separation of alpha- and beta-tubulin monomers(28) .

Proteolysis of Phosphotubulin

Purified tubulin (3 µg) was phosphorylated in vitro with GST-Syk immobilized on glutathione-Sepharose for 1 min at 30 °C using the kinase reaction mixture described above. The reaction was terminated by the addition of 5 mM EDTA, and 3.7 ng of subtilisin was added and allowed to incubate for the times indicated. Alternatively, the kinase reaction was terminated by the removal of the immobilized GST-Syk by centrifugation and 0.03 µg of carboxypeptidase A was added and incubated for the indicated times. Cleavage products were separated by SDS-PAGE. Proteins were visualized by staining with Coomassie Brilliant Blue, and phosphoproteins were detected by autoradiography.

One-dimensional peptide mapping of phosphoproteins was performed essentially as described by Cleveland et al.(29) . Phosphopeptides excised from SDS-polyacrylamide gels were reelectrophoresed on a 15% gel in the presence of 17 ng of Staphylococcus aureus V8 protease.


RESULTS

Syk Activity in the Cytosol Increases with BCR Stimulation

Aggregation of the BCR leads to an increase in the intrinsic activity of Syk, which can be detected as an increase in the rate of Syk autophosphorylation in anti-Syk immune complexes(1, 30) . To examine the location of activated Syk, Syk was immunoprecipitated with anti-Syk peptide antibodies from the postnuclear particulate and cytosolic fractions of human DG75 B-cells that had been pretreated with anti-IgM antibodies for varying periods of time prior to Dounce homogenization. The resulting immune complexes were incubated in buffer containing [-P]ATP. As shown in Fig. 1A, receptor cross-linking resulted in only a modest increase in the intrinsic autophosphorylating activity of membrane-associated Syk (Fig. 1A). A substantial increase in Syk activity was observed in the cytosolic fractions prepared from anti-IgM-activated cells as compared to control cells (Fig. 1A). Active, soluble Syk could be detected in DG75 cells within 2 min (data not shown) and in Bal17 cells within 15 s (see Fig. 4A) of receptor aggregation. Syk phosphorylated in immune complexes isolated from the cytosol of activated DG75 cells, but not from the particulate fraction, migrated on SDS-polyacrylamide gels as a closely spaced doublet.


Figure 1: Activated Syk appears in the cytosolic fraction of anti-IgM-activated B-cells. Human DG75 B-cells were activated with anti-IgM antibodies for 0, 4, 10, or 15 min. Cells were lysed by Dounce homogenization and separated into particulate and soluble fractions by ultracentrifugation. Proteins were immunoprecipitated with polyclonal rabbit anti-Syk antiserum (lanes 2-5 and 7-10) or with preimmune serum (P), (lanes 1 and 6). The resulting immune complexes were incubated with [-P]ATP to allow Syk autophosphorylation, which was visualized by autoradiography following the separation of proteins by SDS-PAGE (Panel A). Alternatively, Syk present in immune complexes was visualized by Western blotting with anti-Syk antibodies (Panel B). The large band at 50 kDa in Panel B represents the heavy chain of rabbit IgG.




Figure 4: A 54-kDa kinase substrate co-immunoprecipitates with Syk from the cytosolic fractions of anti-IgM-activated B-cells. A, Bal17 murine B-cells were either untreated (lane 1) or were stimulated with anti-IgM antibodies for 15 s (lane 2), 45 s (lane 3), 2 min (lane 4), or 10 min (lane 5). Cytosolic fractions were prepared and Syk was immunoprecipitated with anti-Syk antibodies. The immune complexes were incubated with [-P]ATP, separated by SDS-PAGE, transferred to membranes, and subjected to autoradiography after incubation with KOH. B, particulate (P) or soluble (S) fractions from unactivated (lanes 2 and 4) or anti-IgM-activated (lanes 3 and 5) Bal17 B-cells were incubated with a GST-Syk fusion protein linked to glutathione-Sepharose. Bound proteins were incubated with [-P]ATP. Lane 1, GST-Syk alone.



Syk protein present in the anti-Syk peptide immune complexes was detected by Western blotting with anti-GST-p35 antibodies following the separation of the antigen-antibody complexes by SDS-PAGE (Fig. 1B). The cross-linking of surface IgM had no appreciable effect on the amount of Syk recovered from either the particulate or soluble fractions.

Tyrosine-phosphorylated Syk Appears Solely in the Cytosolic Fraction of Anti-IgM-activated Cells

Aggregation of the BCR leads to an increase in the phosphotyrosine content of Syk(1, 30, 31) . To examine the location of tyrosine-phosphorylated Syk, phosphotyrosine-containing proteins were immunoprecipitated with anti-phosphotyrosine antibodies from particulate and soluble fractions prepared from DG75 B-cells activated for varying periods of time with anti-IgM antibodies. As shown in Fig. 2A, the vast majority of the Syk autophosphorylating activity was recovered from the cytosolic fraction of activated cells. Very little tyrosine phosphorylated Syk was recovered from the particulate fraction. The identity of the cytosolic, tyrosine-phosphorylated protein as Syk was verified by Western blot analysis of anti-phosphotyrosine immune complexes using anti-GST-p35 antibodies (Fig. 2B).


Figure 2: Tyrosine-phosphorylated Syk appears only in the cytosolic fraction of anti-IgM-activated B-cells. DG75 human B-cells were activated with anti-IgM antibodies for 0, 4, 10, or 15 min. Cells were lysed by Dounce homogenization and separated into particulate and soluble fractions by ultracentrifugation. Phosphotyrosine-containing proteins were immunoprecipitated with anti-phosphotyrosine antibodies. Syk was visualized in the resulting immune complexes by autophosphorylation following incubation with [-P]ATP (Panel A) or by Western blotting with anti-Syk antibodies (Panel B). P = preimmune serum. The large band at 50 kDa in Panel B represents the heavy chain of rabbit IgG.



The Syk protein recovered from the cytosolic fraction by immunoprecipitation with anti-phosphotyrosine antibodies and detected either by autophosphorylation (Fig. 2A) or Western blotting (Fig. 2B) migrated on SDS-PAGE as a single, prominent band and co-migrated with the upper band of the doublet observed in anti-Syk immune complex kinases assays (Fig. 1A).

To determine if cytosolic Syk was also activated in murine B-cells, anti-Syk immune complexes were prepared from the particulate and soluble fractions of untreated or anti-IgM-activated Bal17 cells and incubated with buffer containing [-P]ATP. As shown in Fig. 3A, only the activity of Syk recovered from the cytosolic fraction was markedly affected by receptor cross-linking. The Ig-alpha component of the murine BCR, which migrates on SDS-polyacrylamide gels with an apparent molecular weight of 34,000, could be observed co-immunoprecipitating with Syk only in samples prepared from the particulate fraction (Fig. 3A).


Figure 3: Activated Syk appears in the cytosolic fraction of B-cells or T-cells treated with pervanadate. A, Syk was immunoprecipitated with anti-Syk antibodies from particulate (lanes 1 and 2) or soluble (lanes 3 and 4) fractions prepared from untreated(-) or anti-IgM-activated (10 min) (+) murine Bal17 B-cells. The resulting immune complexes were incubated in the presence of [-P]ATP. Phosphoproteins were separated by SDS-PAGE, transferred to poly(vinylidene fluoride) membranes, treated with 1 N KOH, and visualized by autoradiography. The migration position of the Ig-alpha component of the mouse BCR is indicated by the open arrow. B, Syk was immunoprecipitated with anti-Syk antibodies from particulate (P) (lanes 1 and 2) or soluble (S) (lanes 3 and 4) fractions prepared from untreated(-) or pervanadate (PV)-treated (+) Bal17 murine B-cells and visualized by autophosphorylation with [-P]ATP. C, Syk (lanes 1-4) or ZAP-70 (lanes 5 and 6) was immunoprecipitated from the soluble fractions of untreated(-) or pervanadate-treated (+) Bal17 B-cells (lanes 1 and 2) or Jurkat T cells (lanes 3-6) with anti-Syk (lanes 1-4) or anti-ZAP-70 (lanes 5 and 6) antibodies. Kinases were visualized in the resulting immune complexes by autophosphorylation.



Activation of Syk Kinases by Pervanadate in B- and T-cells

The treatment of hematopoietic cells with oxidizing agents, such as H(2)O(2)(32) or pervanadate(33, 34) , leads to the tyrosine phosphorylation and activation of Syk, presumably by inhibiting the activities of phosphotyrosine phosphatases. To determine if pervanadate-stimulated Syk also appeared in the cytosolic fraction, anti-Syk immune complexes were prepared from the particulate or cytosolic fractions of untreated or pervanadate-treated Bal17 B-cells and incubated with [-P]ATP. As shown in Fig. 3B, addition of pervanadate primarily affected the activity of Syk recovered from the cytosolic fraction. Cytosolic Syk was also activated in Jurkat T-cells following treatment with pervanadate, as shown in Fig. 3C. The activity of ZAP-70, which is a Syk-related kinase expressed predominantly in T-cells, was also increased dramatically in the cytosolic fraction of pervanadate-treated Jurkat T-cells (Fig. 3C).

Association of Syk with a 54-kDa Substrate

A 54-kDa protein co-immunoprecipitated with Syk from the cytosolic fractions of B-cells and became phosphorylated during immune complex kinase assays (Fig. 1A and Fig. 3A), suggesting that this protein might represent a Syk-associated substrate. In murine Bal17 B-cells, this protein appeared in anti-Syk immune complexes shortly following receptor cross-linking and then decreased as a function of time (Fig. 4A). To further explore the possibility that this was a Syk-associated protein, particulate and cytosolic extracts of anti-IgM-activated or unactivated cells were incubated with a glutathione-Sepharose resin containing bound GST-Syk. After washing, the resin was incubated with [-P]ATP to identify Syk-associated substrates, which were detected by autoradiography following separation by SDS-PAGE. As shown in Fig. 4B, a major radiolabeled protein of 54 kDa was observed in the cytosolic extracts. This protein co-migrated on SDS-polyacrylamide gels with the 54-kDa phosphoprotein detected in the anti-Syk immune complexes.

Identification of the 54-kDa Syk Substrate as Tubulin

Based on the apparent molecular weight of the Syk-associated substrate and recent reports of the tyrosine-phosphorylation of tubulin in human Jurkat T-cells(35) , we examined the possibility that the 54-kDa phosphoprotein was, in fact, tubulin. To determine if tubulin was a substrate for Syk, purified chick brain tubulin was incubated with recombinant Syk in an in vitro kinase assay. As shown in Fig. 5A, tubulin was readily phosphorylated by Syk. Phosphoamino acid analysis identified the phosphorylated amino acid in tubulin as phosphotyrosine (data not shown). Purified tubulin alone contained no endogenous kinase activity and the preparation of recombinant Syk was devoid of phosphoproteins of 54 kDa.


Figure 5: Syk associates with and phosphorylates tubulin. A, Syk was immunoprecipitated with anti-Syk antibodies from the cytosolic fraction of anti-IgM-activated Bal17 B-cells and incubated with [-P]ATP to detect the 54 kDa Syk-associated protein (lane 1). Recombinant Syk and purified tubulin (lane 2), purified tubulin alone (lane 3) or recombinant Syk alone (lane 4) were incubated in vitro with [-P]ATP, separated by SDS-PAGE and subjected to autoradiography. B, phosphopeptide map generated by the partial proteolysis of the 54-kDa, Syk-associated substrate (lane 1) and of in vitro phosphorylated tubulin (lane 2). C, Syk was immunoprecipitated with anti-Syk antibodies from the cytosolic fractions of anti-IgM-activated (lanes 2 and 4) or unactivated (lanes 1 and 3) Bal17 B-cells that had been pretreated with (lanes 3 and 4) or without (lanes 1 and 2) Taxol. Syk and associated substrates were visualized by autophosphorylation with [-P]ATP. The migration position of tubulin is indicated by the arrow.



Tubulin phosphorylated in vitro by Syk co-migrated by SDS-PAGE with the 54-kDa Syk-associated substrate (Fig. 5A). Phosphopeptide mapping by partial proteolysis (29) of each protein gave rise to a single, prominent phosphopeptide that co-migrated on 15% SDS-polyacrylamide gels (Fig. 5B), suggesting that they represented the same or highly related proteins.

To support the identification of the 54-kDa substrate as tubulin, we examined the effect of Taxol on its recovery from cytosolic fractions of activated Bal17 B-cells. Taxol stabilizes microtubules and promotes the polymerization of unassembled tubulin(36, 37, 38) . Syk was immunoprecipitated with anti-Syk antibodies from the cytosolic fractions of anti-IgM-activated and unactivated Bal17 cells or Taxol-pretreated Bal17 cells and detected by autophosphorylation. As shown in Fig. 5C, the 54-kDa protein co-immunoprecipitated with Syk from the cytosolic fraction of anti-IgM-activated Bal17 cells, but not from activated cells that had been pretreated with Taxol.

Phosphorylation of alpha-Tubulin by Syk in Vitro

Syk exhibits a very restricted substrate specificity in vitro. Therefore, we examined the ability of recombinant Syk to phosphorylate tubulin as compared to cfb3, an excellent Syk substrate(34, 39, 40) , and myelin basic protein, another substrate sometimes used to detect Syk activity (41, 42) . Equimolar amounts of tubulin dimer, cfb3, and myelin basic protein were phosphorylated in identical in vitro kinase reactions. The results, illustrated in Fig. 6, A and B, indicate that tubulin, like cfb3, is an excellent substrate for Syk. In contrast, myelin basic protein is only poorly recognized as a substrate.


Figure 6: Phosphorylation of tubulin in vitro by Syk. Equimolar amounts of tubulin dimer (down triangle), the cytoplasmic domain of band 3 (cfb3) (bullet), and myelin basic protein (MBP) () were phosphorylated in vitro without (lane 1) or with (lanes 2-5) recombinant Syk for 0.5, 2, 5, or 10 min. In Panel A, phosphoproteins were detected by exposure of SDS-polyacrylamide gels to x-ray film for 15 min except for the lower right panel, which was exposed for 12 h. For Panel B, radiolabeled proteins were excised from the gel and the amount of P incorporated was quantified by liquid scintillation spectrometry.



Tubulin exists as a heterodimer of tightly associated alpha and beta subunits that exhibit about 40% sequence similarity(43) . To further examine the substrate specificity of Syk, phosphotubulin was separated by SDS-PAGE under conditions that allow separation of the distinct alpha and beta subunits(28) . As shown in Fig. 7A, Syk selectively catalyzed the phosphorylation of the alpha-tubulin subunit.


Figure 7: Syk phosphorylates alpha-tubulin on a tyrosine located near the carboxyl terminus. A, purified chick brain tubulin was phosphorylated in vitro with [-P]ATP using recombinant Syk and was separated by SDS-PAGE under conditions that resolve the alpha and beta monomers. Phosphotubulin detected by autoradiography is shown in lane 1, tubulin protein in lane 2. B, tubulin was phosphorylated in vitro by GST-Syk and then treated with carboxypeptidase A for 10 (lane 1), 20 (lane 2), or 50 (lane 3) min to remove the carboxyl-terminal tyrosine. Phosphotubulin was detected by autoradiography following SDS-PAGE. C, In vitro phosphorylated tubulin was treated with subtilisin for 0 (lane 1), 5 (lane 2), 10 (lane 3), 20 (lane 4), or 30 (lane 5) min to remove 4 kDa of the carboxyl terminus. The resulting 50-kDa amino-terminal fragment detected by staining with Coomassie Blue is indicated by the arrow. The resulting autoradiogram is shown in the lower panel.



Cleavage of alpha- and beta-tubulin with subtilisin generates 50,000-kDa intermediates that lack the acidic carboxyl termini(44) . To explore the location of the site of phosphorylation on alpha-tubulin, purified tubulin was phosphorylated in vitro with recombinant GST-Syk and then incubated with subtilisin for varying periods of time. As illustrated in Fig. 7C, no radiolabel was associated with the 50-kDa fragments of alpha- and beta-tubulin generated by digestion with subtilisin.

The 4-kDa carboxyl-terminal region of alpha-tubulin contains two potential sites of tyrosine phosphorylation, one internal site and a second at the extreme carboxyl terminus. To differentiate between these possible sites, tubulin phosphorylated in vitro by recombinant GST-Syk was incubated with carboxypeptidase A for varying periods of time. As shown in Fig. 7B, while carboxypeptidase A digestion generated slightly faster migrating forms of phosphotubulin, it did not rapidly catalyze removal of the radiolabeled amino acid (Fig. 7B). This indicates that the principal site of phosphorylation on alpha-tubulin is not at the extreme carboxyl terminus.

Phosphorylation of Tubulin by BCR Cross-linking

To determine if tubulin was phosphorylated in anti-IgM-activated cells under conditions in which Syk was activated, cytosolic extracts of anti-IgM-activated Bal17 B-cells were adsorbed to colchicine-Sepharose and immunoblotted with anti-beta-tubulin and anti-phosphotyrosine antibodies. As shown in Fig. 8, much of the tubulin present in cytosolic extracts was adsorbed to the colchicine resin (Panel B). Tubulin was rapidly and transiently phosphorylated on tyrosine following receptor cross-linking (Panel A).


Figure 8: Tubulin is phosphorylated on tyrosine in intact cells following activation. A and B, Bal17 B-cells were stimulated with anti-IgM antibodies for 0 (lanes 1, 4, and 7), 0.75 (lanes 2, 5, and 8) or 10 (lanes 3, 6, and 9) min. Soluble lysates were adsorbed to colchicine-Sepharose. Phosphotyrosine-containing proteins were identified by Western blotting with anti-phosphotyrosine antibodies (Panel A). Tubulin was identified by Western blotting with anti-beta-tubulin antibodies (Panel B). Lanes 1-3 represent crude soluble extracts; lanes 4-6, unadsorbed proteins; and lanes 7-9, proteins bound to colchicine-Sepharose. C, DG75 B cells were stimulated for 0 (lane 1), 2 (lane 2), 10 (lane 3), or 15 (lane 4) min with anti-IgM antibodies. Cytosolic extracts were adsorbed to colchicine-Sepharose. Bound proteins were separated by SDS-PAGE and those containing phosphotyrosine were detected by Western blotting with anti-phosphotyrosine antibodies. Lane 5 shows the migration position of beta-tubulin as detected by Western blotting with anti-beta-tubulin. D, Bal17 B-cells were pretreated for 60 min without (lanes 1-3) or with (lanes 4-6) 15 µg/ml piceatannol and then activated for 0 (lanes 1 and 4), 0.75 (lanes 2 and 5), or 10 (lanes 3 and 6) min with anti-IgM antibodies. Phosphotyrosine-containing proteins in the soluble fraction that bound to colchicine-agarose were detected by Western blotting with anti-phosphotyrosine antibodies.



Similar results were observed using the human DG75 B-cells. In this experiment, the alpha and beta monomers were separated during electrophoresis. Receptor cross-linking primarily led to increases in the phosphorylation of the alpha subunit.

To further establish a role for Syk in the phosphorylation of tubulin in intact cells, we examined the ability of piceatannol to block anti-IgM-stimulated tubulin phosphorylation. At low concentrations, piceatannol selectively inhibits the receptor-mediated activation of Syk as compared to the Src family kinases in mast cells (18) and B-cells. (^3)Bal17 B-cells were pretreated for 1 h with 15 µg/ml piceatannol or with Me(2)SO carrier alone (final concentration, 0.15%) prior to activation with anti-IgM antibodies. As shown in Fig. 8D, the time-dependent phosphorylation of tubulin in Bal17 B-cells in response to receptor cross-linking was inhibited by pretreatment with piceatannol.


DISCUSSION

In lymphoid cells, the Src family kinases are localized to the particulate fraction where they are anchored by protein-protein and protein-membrane interactions mediated, in part, by post- and co-translational acylations and the presence of SH3 domains (for review, see (45) ). Syk, which lacks both an SH3 domain and a consensus sequence for N-myristoylation, is distributed more evenly between the particulate and soluble fractions in B-cells (Fig. 1). The distribution observed in B-cells is similar to that reported previously for peripheral blood lymphocytes(46) . Both Syk and Src family kinases are activated when transmembrane antigen receptors are aggregated by extracellular polyvalent ligands or antireceptor antibodies. The activated Src family kinases remain associated with membrane or cytoskeletal components present in the particulate fraction where they are presumably poised to phosphorylate physiologically relevant substrates also localized to these regions (e.g. components of the antigen receptor complex). Activated Syk, on the other hand, appears predominantly in the soluble fraction of B-cell lysates (Fig. 2). Syk, activated in peripheral blood lymphocytes in response to interleukin-2, is also localized predominantly to the cytosolic fraction(46) . Thus, Syk may be positioned within the cell to interact with and phosphorylate a distinct subset of proteins distinguished from those of the Src family kinases, in part, by their subcellular location.

Biochemical and genetic evidence indicates that the BCR-mediated activation of Syk proceeds via the association of its tandem, amino-terminal, SH2 domains with the tyrosine-phosphorylated ITAM motifs of Ig-alpha or Ig-beta(21, 22, 47) . Thus, Syk would be expected to be recruited to the antigen receptor complex following its aggregation and phosphorylation. This recruitment of Syk to the receptor, however, does not measurably alter the overall subcellular distribution of the kinase between particulate and soluble fractions (Fig. 1). Following the interaction of Syk with the phosphorylated ITAM or with dually phosphorylated peptides modeled on the ITAMs of Ig-alpha or FcRI-, Syk is activated as it becomes phosphorylated on tyrosine(21, 48) . In the intact B-cell, subcellular fractionation studies indicate that this tyrosine-phosphorylated Syk is almost exclusively confined to the soluble fraction (Fig. 2). This is most easily seen in extracts prepared from human B-cells where enhanced tyrosine phosphorylation leads to a shift in the electrophoretic mobility of Syk to a slower migrating species (Fig. 1). This slower migrating form of Syk is the major form that is immunoprecipitated with anti-phosphotyrosine antibodies (Fig. 2). Immunoprecipitates of soluble, tyrosine-phosphorylated Syk do not contain the co-immunoprecipitated Ig-alpha that is visible in anti-Syk immune complexes isolated from the membrane fraction of activated B-cells (Fig. 3A). These observations suggest a model whereby Syk interacts only transiently with the aggregated receptor and is released from the receptor when the kinase becomes phosphorylated on tyrosine. This phosphorylation could occur as a consequence of the actions of Src-family kinases or from Syk autophosphorylation. Increases in the intrinsic activity of Syk accompany this phosphorylation such that the activated Syk is also found predominantly in the soluble fraction ( Fig. 1and Fig. 3).

Syk is also activated in B-cells treated with pervanadate, a potent protein tyrosine phosphatase inhibitor (Fig. 3). In B-cells, vanadate, which also inhibits protein tyrosine phosphatases, induces the hyperphosphorylation of the Ig-alpha and Ig-beta components of the BCR (49) and pervanadate likely functions in a similar fashion. This pervanadate-activated Syk does not remain associated with the phosphorylated antigen receptor complex and is found primarily in the soluble fraction (Fig. 3). ZAP-70, a Syk homolog that is expressed predominantly in T-cells, also appears in a soluble, activated form when a Jurkat T-cell line that expresses both Syk and ZAP-70 is treated with pervanadate (Fig. 3). This activation of ZAP-70 is blocked in J.CaM1 cells, (^4)a Jurkat-derived cell line that lacks the functional Lck needed to phosphorylate components of the TCR/CD3 complex(50) . Thus, ZAP-70 is likely released from the T-cell receptor following its activation and phosphorylation on tyrosine in a manner similar to that observed for Syk and the BCR.

Soluble, activated Syk would be poised to interact with and phosphorylate a subset of protein-tyrosine kinase target proteins present in the cytosol of activated cells. A key to the nature of one of these potential substrates was the observation that a 54-kDa protein co-immunoprecipitated with Syk and became phosphorylated on tyrosine in anti-Syk immune complexes. This protein was found in both anti-Syk immune complexes and among proteins adsorbed to an immobilized GST-Syk fusion protein (Fig. 4). The 54-kDa protein was identified as tubulin by peptide mapping and by its loss from the cytosolic fraction of activated cells in which the polymerization of tubulin into microtubules was forced by pretreatment with Taxol (Fig. 5).

Tubulin is an excellent in vitro substrate for Syk. Syk preferentially catalyzes the phosphorylation of the alpha-tubulin monomer on a single tyrosine residue localized to a region of the protein near the carboxyl terminus. This site lies within a carboxyl-terminal peptide that is released from alpha-tubulin following limited proteolytic digestion with subtilisin that has the sequence FSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEY(44) (sequence given for the major brain forms of mammalian and chicken alpha-tubulin)(43, 51) . This peptide contains two tyrosine residues, one of which is located at the extreme carboxyl terminus and has been reported to be an in vitro site of phosphorylation catalyzed by the insulin receptor based on its rapid release in response to carboxypeptidase A(52) . Treatment of Syk-phosphorylated tubulin with carboxypeptidase A, however, has little effect on its state of phosphorylation (Fig. 7B), indicating that the tyrosine 20 residues upstream from the carboxyl terminus, Tyr, rather than the carboxyl-terminal tyrosine, is the major site of phosphorylation. Other protein-tyrosine kinases have been reported to phosphorylate tubulin, but the pattern of phosphorylation generally differs from that observed with Syk. For example, Src phosphorylates alpha- and beta-tubulin with nearly equal efficiency in vitro(53) and in nerve growth cone membranes(54) , while the epidermal growth factor receptor preferentially phosphorylates beta-tubulin(53) .

In our hands, Syk demonstrates a highly restricted substrate specificity in vitro(55) . The site of Syk phosphorylation on alpha-tubulin is similar in sequence to the sites of phosphorylation previously identified on the erythrocyte anion transport channel (band 3) (56) and glycogen synthase(57) . An alignment of the phosphorylation sites identified on these three substrates indicate a strong preference for acidic amino acid residues located both proximal and distal to the site of tyrosine phosphorylation: alpha-tubulin, Met-Ala-Ala-Leu-Glu-Lys-Asp-Tyr-GluGlu-Val-Gly; band 3, Met-Glu-Glu-Leu-Asn-Asp-Asp-Tyr-Glu-Asp-Asp-Met-; glycogen synthase, Pro-Glu-Glu-Asp-Gly-Glu-Arg-Tyr-Glu-Asp-Glu-Glu-. beta-Tubulin, which also contains a conserved tyrosine residue in an analogous position(43) , lacks the surrounding acidic residues that are important for substrate recognition by Syk and is not readily phosphorylated by Syk in vitro (Fig. 7). Sites of Syk phosphorylation that have been sequenced for histone H2B (58) and myelin basic protein (41) do not fit this general motif. However, myelin basic protein is a comparatively poor substrate for Syk (Fig. 6) and is phosphorylated at a rate much slower than that observed for the phosphorylation of band 3 or alpha-tubulin.

Tubulin is also phosphorylated on tyrosine in intact B-cells following aggregation of the BCR (Fig. 8). This phosphorylation occurs under conditions in which Syk is activated and occurs primarily on the alpha-subunit. Preferential phosphorylation of alpha-tubulin is consistent with the demonstrated substrate specificity of the kinase in vitro (Fig. 7). The preincubation of B-cells with piceatannol at concentrations in which the activation of Syk is preferentially inhibited blocks receptor-mediated phosphorylation of tubulin, suggesting that Syk is the kinase primarily responsible for the tyrosine phosphorylation of tubulin following receptor aggregation. The observation that tubulin is phosphorylated in activated lymphoid cells is similar to that reported previously by Ley et al.(59) who found that alpha-tubulin is tyrosine-phosphorylated in human T lymphocytes in response to engagement of the T-cell antigen receptor. The phosphorylated alpha-tubulin was restricted to the 45% of tubulin that is not polymerized in Jurkat T-cells(59) . TCR engagement leads to the activation of ZAP-70, a Syk homolog found in T-cells that has a substrate specificity similar to that of Syk. When Jurkat T-cells are treated with pervanadate, activated ZAP-70 appears in the soluble fraction (Fig. 3) where the unpolymerized alpha-tubulin would be expected to be located. Thus, ZAP-70 would be a good candidate for the T-cell alpha-tubulin kinase.

Determinations of the effect that tyrosine-phosphorylation has on the properties of tubulin and the role that this plays in B-cell function must await further experimentation. Tubulin phosphorylated on the carboxyl-terminal tyrosine by the insulin receptor fails to polymerize into microtubules(52) . Similarly, alpha- and beta-tubulin phosphorylated near the carboxyl terminus by a Ca/calmodulin-dependent protein kinase fails to polymerize(60) . An inhibitory effect of phosphorylation on the incorporation of tubulin into microtubules is also consistent with the observation that alpha-tubulin, phosphorylated in response to ligation of the TCR, is found exclusively in the cytosolic fractions of Jurkat T-cells(59) . Thus, tyrosine phosphorylation may play a role in altering the microtubule/tubulin monomer equilibrium by lowering the pool of tubulin monomers available for polymerization. The acidic carboxyl-terminal region of tubulin contains the sites of interaction with the microtubule-associated proteins (MAPs) that regulate microtubule assembly and function(61, 62) . Through the use of synthetic peptides, the site of interaction of MAP2 and tau with alpha-tubulin has been narrowed to a 12 amino acid region, KDYEEVGVDSVE, that encompasses Tyr(61) . Antibodies prepared against a peptide corresponding in sequence to this region block MAP-induced microtubule assembly and depolymerize preformed microtubules(62) . Thus, phosphorylation in this region of alpha-tubulin might reasonably be expected to alter its association with one or more MAPs. The binding of the acidic amino terminus of erythrocyte band 3 to glycolytic enzymes is inhibited by tyrosine phosphorylation(56) . By analogy to band 3, the phosphorylation of alpha-tubulin may likewise block its ability to interact with MAPs. Alternatively, tyrosine-phosphorylated tubulin could serve as a binding site for signaling molecules that possess SH2 domains and could serve a role in mediating their redistribution within the cell following receptor aggregation. Experiments to explore these possibilities are currently under way.


FOOTNOTES

*
This research was supported in part by United States Public Health Service, National Institutes of Health Grants CA37372 (to R. L. G. and M. L. H.) and GM49889 (to D. L. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by United States Public Health Service Training Grant GM08298.

Supported by a fellowship from the American Heart Association, Indiana Affiliate, Inc.

**
To whom correspondence should be addressed: Dept. of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Sciences Research Bldg., Purdue University, West Lafayette, IN 47907. Tel.: 317-494-1457; Fax: 317-494-9193.

(^1)
The abbreviations used are: BCR, B-cell antigen receptor; TCR, T-cell antigen receptor; ITAM, immunoreceptor tyrosine activation motif; GST, glutathione S-transferase; cfb3, cytoplasmic fragment of erythrocyte band 3; PAGE, polyacrylamide gel electrophoresis; MAP, microtubule-associated protein.

(^2)
M. T. Furlong and R. L. Geahlen, manuscript in preparation.

(^3)
M. T. Samson and R. L. Geahlen, manuscript in preparation.

(^4)
J. D. Peters and R. L. Geahlen, unpublished observations.


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