B-cell antigen receptor stimulation activates the human Bruton's

“ H E JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269,No.39,Issue of September 30,pp. 23657-23660, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication

cell stage, resulting in a dramatic decrease in mature B-cell B-cell Antigen Receptor numbers and serumimmunoglobulin levels of all isotypes (3Stimulation Activates the Human5 ) . The gene product defective in XLA has been identified as a cytoplasmic protein tyrosine kinase, named Bruton’s tyrosine Bruton’s Tyrosine Kinase, Which kinase (Btk) (6, 7). Btk has a unique N-terminal region containing a pleckstrin homology (PH) domain and a proline-rich Is Deficient in X-linked stretch, followed by Src homology (SH) 3, SH2, and kinase Agammaglobulinemia* domains (6-9). Together with Tec and Itk, Btk constitutes a unique tyrosine kinasesubfamily with homology to Src-related kinases (lo), but with unique features, including lack of an Michel de WeersS5, Gaby S. BrounsnJ, N-terminal myristoylation site anda C-terminal negative regSteve Hinshelwoodll, Christine Kinnonll, ulatory tyrosine residue, as well as the presence of a PH doRuud K. B. SchuurmanS, Rudolf W. Hendrikst**, main (8, 9). and JannieBorstlISS XLA patients display heterogeneityin mutations, which inFrom the fDepartment of Immunohaematology, clude deletions, insertions and substitutions (6, 8, 9, 11, 12). University Hospital Leiden, F! 0. Box 9600, 2300 RC Not only mutations in the kinase domain but also genetic alLeiden, The Netherlands, IDivision of Cellular terations exclusively affecting PH, SH3, or SH2 domains can Biochemistry, The Netherlands Cancer Institute, lead to the XLA phenotype (8, 9, 11, 12). Mutation of the conPlesmanlaan 121, 1066 CXAmsterdam, The Netherlands, the IlMolecular Immunology Unit, Institute of Btk hampersB-cell served Arg-28 residue in the PH domain of Child Health, 30 Guilford Street, London W C l N development and certain mature B-cell responses (13) in the IEH, United Kingdom, and the**Department of Cell xid CBA/N mouse strain (14, 15) and leads to themore severe Biology and Genetics, Erasmus University, I! 0. Box XLA phenotype in a patient (11).These observations indicate 1738, 3000 DR Rotterdam, The Netherlands that multiple protein-protein interactions are essential for Btk X-linked agammaglobulinemia (XLA) is an inherited function, butmolecules that directly or indirectly interact with human immunodeficiency disease, characterizedby an Btk in vivo have thus far not beenidentified. arrest in B-cell development, which results in a draWhereas Btk is expressed throughout myeloid as well as matic decrease in immunoglobulin production. The B-cell gene differentiation (16, 171, development of the myeloid linproduct defective in XLA has been identified as a cyto-eage appears unaffected in XLA patients, while female XLA plasmic protein tyrosine kinase, named Bruton’s tyro- carriers manifest unilateral X-chromosome inactivation only in sine kinase (Btk). Thedramatic M A phenotype indi- the B-cell lineage (18).Affected males display a marked decates a critical role for Btk in the regulation of B-cell crease in the transitionfrom pre-B-cells to matureB-cells, as is development. However, neither external stimuli leading also observed in mice with targeted deletions of membrane to Btk activation nor any ofits in vivo substrates have immunoglobulin (mIg) p (19) or X5 genes (201, which encode thus far been identified, and the mechanism of disease essential components of the B-cell antigen receptor (BCR) at inductionremainsunexplained. We reportherethat this differentiation stage. The BCR complex contains in addistimulation of the B-cell antigen receptor (membrane tion to mIg a heterodimer of transmembrane Mb-1 and B29 immunoglobulin)onmatureB-cellsinducestyrosine chains, which are essentialfor signal transduction (21,22). The phosphorylation of Btk in vivo, accompaniedbyan BCR is endowed with a tyrosine kinasefunction by associating increase in its kinase activity in vitro. These results Src-related kinases Lyn, Fyn, and Blk (231, as well as the cyplaceBtk in theB-cellreceptorsignaltransduction toplasmic protein tyrosine kinaseSyk (24). Receptor activation pathway, whichis known to beessential in driving B-cell differentiation. is accompanied by tyrosine phosphorylation of specific motifs (25) in the Mb-1 and B29 cytoplasmic tails, which either directly or indirectly form sites of association with various SH2X-linked agammaglobulinemia (XLA)l patients characterisdomain containing enzymes, initiating multiple signal transtically suffer from severe bacterial infections (1,2). In affected duction cascades (26). We examined whether Btkis involved in signal transduction males, B-lymphocyte differentiation is arrested at the pre-Bmediated by the BCR complex. Our results demonstrate that provide an expla* This work was supported by Netherlands Organizationfor Scientific BCR stimulation activates Btk, and this may Research Grants 900-504-106(toM. d. W.) and 900-509-154(to G. S. B.) nation for the mechanism of disease induction in XLA, given and by grants from the Royal Netherlands Academy of Arts and Sci- the key role of the BCR in controlling B-cell development. Loences and the Dutch Prevention Foundation (to R. W. H.). The costs of well characterized BCR-induced signalpublication of this article were defrayed inpart by the payment of page calization of Btk in the charges. This articlemust therefore be hereby marked “advertisement” ing pathway will also allow a more direct assessment of its in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. specific function in intracellular communication. P Contributed eauallvto this work. To whomcorrespbndenceshouldbe addressed. Tel.: 31-20-512EXPERIMENTALPROCEDURES 1977/1970;Fax: 31-20-512-1989. Cells-The human Burkitt lymphoma line Ramos, whichis a mature The abbreviations used are: XLA, X-linked agammaglobulinemia; Btk, Bruton’s tyrosine kinase; PH, pleckstrinhomology; SH, Src homol- B-cell line expressing mIgM complexes(27),was cultured in RPMI 1640 ogy; (m)Ig, (membrane-bound) immunoglobulin; BCR,B-cell antigen medium supplemented with 5% fetal calf serum. Tonsillar lymphocytes mAb, monoclonal antibody; IPB,im- derived from normalindividuals were isolated by density gradient cenreceptor; Tyr(P), phosphotyrosine; munoprecipitation buffer; BSA, bovine serum albumin; PBSfI’,phostrifugation andshown by flow cytometry to contain 5040% B-cells. phate-bufferedsalineheen; ECL, enhanced chemiluminescence; HRP, Reagents-Intact Ig or F(ab‘), fragments of polyclonal goat anti-huhorseradish peroxidase. man Ig antibody (TAG0 Immunologicals, Burlingame, CA) were used (Received for publication, July 19, 1994)

23857

23858

Receptor-mediated B-cellActivation

of Btk

for BCR stimulation. Polyclonal rabbit anti-BtWglutathione S-transferase and anti-Shc sera were raised against fusion proteins of glutathione S-transferase and amino acids 163-218 of the Btk protein (28) or the SH2 domain of Shc (29),respectively. Polyclonal rabbit anti-Btk serum was raised against a peptide containing amino acids 69-88 and usedas purified Ig or a biotinylated version thereof (anti-Btk'""). Anti-phosphotyrosine (anti-Tyr(P))monoclonal antibody (mAb)4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, N Y ) , anti-CD22 mAb BL3C4 from Sanbio (Uden, The Netherlands), horseradish peroxidase (HRPbconjugated rabbit anti-mouse Ig and swine anti-rabbit Ig from DAKO A/S (Glostrup, Denmark), and streptavidine-conjugated HRP and enhanced chemiluminescence (ECL) reagents from Amersham Int. Blot s.PW mMk * (Little Chalfont, UK). Stimulation of Cells and Immunoprecipitation-Prior to BCR trig#vMkbablM gering, Ramos cells and tonsillar lymphocytes were equilibrated for 45 FIG.1. BCR triggering induces tyrosine phosphorylation of min in serum-freeRPMI 1640 a t 37 "C. Samples of 10 x IO6 Ramos cells Btk in intactRamos cells. a , anti-Btk, -Shc, and -Tyr(P) immunoprewere stimulated for the indicated times a t 37 "C with 10 pg of intact Ig cipitates from unstimulated cells (0)and cells stimulated with intact or F(ab'), fragmentsof polyclonal goat anti-human Igantibody. Tonsil- anti-Ig antibodyfor 2 min(2').b, antiBtk precipitates from cells stimular lymphocytes were stimulated with 20 pg of anti-Ig antibody/sample lated with F(ab'), fragmentsof the anti-Ig antibodyfor the times indimAb; to check for equal of 100 x lo6 cells in 1 ml of medium. Stimulation was stopped by cell cated. All samples were blotted with anti-Tyr(P) Reactivlysis in ice-cold immunoprecipitation buffer (IPB), containing1% Non- loading, Btk samples were also blotted with anti-Btk reagent. at about 50 and 25 kDaconcerns Ig chains idet P-40, 10 mM triethanolamine.HC1, pH 7.8, 150 mM NaCl, 5 mM ity in unstimulated samples EDTA, protease inhibitors, and1 mM Na,VO,. Lysates were centrifuged of the immunoprecipitating antibodies.GST, glutathione S-transferase. for 15 mina t 13,000 x g and precleared with protein A-Sepharose CUB beads (Pharmacia, Uppsala, Sweden). For specific immunoprecipitations, lysates were incubated with antibodies, followed by protein A- same experiment,BCR stimulation induced tyrosine phosphoSepharose C U B beads. Anti-BtWglutathione S-transferase antibody rylation of the 77-kDa Btk protein, as assayed by Western was in all cases usedfor Btk immunoprecipitation. blotting with anti-Tyr(P) mAb. To exclude the possibility that Immunoblotting-Immunoprecipitates werewashedthreetimes with IPB, separated on a 10% polyacrylamide gel under reducing con- Btk phosphorylation was elicited by Fc receptor stimulation, Ramos cells were activatedwith F(ab'), fragments of the ditions, and transferred to nitrocellulose. For Tyr(P) detection, blots were blocked with 1% bovine serum albumin (BSA) in phosphate-buff- anti-Ig antibody. The F(ab'), fragments effectively induced tyered saline,0.1% Tween 20 (PBSR) and incubated with first and second rosine phosphorylation of Btk, indicating that this aisspecific step antibodies in 0.1%BSA, PBSR. For Btk detection, blots were result of BCR triggering (Fig. l b ) . Additionally, stimulation of blocked with 5% nonfat dry milk in 20 mM Tris.HC1, pH 7.6, 137 mM Ramos cells with various irrelevant antibodies did not affect NaCl, 0.05% Tween 20 and incubated withfirst and second step reagent the in vivo tyrosine phosphorylation status of Btk (data not in the same buffer with1% nonfat dry milk. Anti-Btk peptide serum was shown). in all cases used for Btk immunoblotting. Blots were incubated with HRP-conjugated rabbit anti-mouse Ig to detect 4G10, with HRP-conjuKinetic analysis revealed thatBtkwas phosphorylated gated swine anti-rabbit Ig to detect anti-Btk, or with streptavidineRamos cells (Fig. within 30 s after addition of anti-Ig reagent to conjugated HRPto detect anti-Btkbi", and developed by ECL. Blots were 2). Phosphorylationreached a maximum within 2 min and stripped by incubation in 67 mM Tris.HC1, pH 6.8, 2% SDS, 100 mM declined only after 120 min (data not shown). BCR-induced P-ME for 30 min at 50"C. In Vitro Kinase Assay-Btk immunoprecipitates preparedfrom 10 x tyrosine phosphorylation of Btk was observed not only in the IO6Ramos cells were washedin IPB lacking EDTA and incubatedfor 10 Burkitt lymphoma line Ramos, but also in tonsillar lymphomin a t room temperature in 50 mM HEPES, 3.7 mM MgCl,,4.6 mM cytes isolated from normal individuals (Fig. 21, indicating the MnCl,, containing 3 pCi of [y-"PlATP/sample. After washing two times general relevance of this finding. As in Ramos cells, induction of in IPB, samples were resolved on a 10% SDS-polyacrylamide gel and tyrosine phosphorylation of Btk occurred within 30 s, but in transferred tonitrocellulose. "P-containing proteins were visualized by tonsillar lymphocytes the decrease was more rapid. autoradiography. BCR DiggeringEnhances Kinase Activityof Btk-The increPhosphoamino Acid Analysis-In vitro phosphorylated Btk protein was extracted from a SDS-polyacrylamide gel and hydrolyzed, and re- ment inTyr(P)content of Btk in vivocould reflect its enhanced sulting amino acids were resolved by two-dimensional electrophoresis kinase activity. Alternatively, Btk mightmerely be substrate to on thin layer plates as described (27). another tyrosine kinase, such as Src-related kinases or Syk, Cell Fractionation-Cells were resuspended in hypotonic buffer, con- which are known to be activated by BCR stimulation (23, 24). taining 10mM Tris.HC1, pH 7.8,l mM Na,VO,, and protease inhibitors, To evaluate the functional status of Btk following B-cell actiand disrupted using a Potter homogenizer. Nuclei were removed by centrifugation for 10 minat 2000 x g, and the supernatant was centri- vation, an in vitro kinase assay was performed on Btk immuisolated from unstimufuged for 60 min a t 100,000 x g. After this spin, the supernatant was noprecipitates. The Btk preparation considered as the cytosolic fraction and the pellet as the crude memlated Ramos cells displayed detectable kinase activity in vitro brane fraction. The membrane fraction was solubilized in IPB and pre- (Fig. 3a). The enzyme was phosphorylated primarily on tyrocleared by centrifugation for 15 min at 13,000 x g. sine residues and tosome extent on serine and threonineresi"

RESULTS AND DISCUSSION

BCR Diggering Induces Drosine Phosphorylation of Btk in Vivo-TheBCR complex on the mature B-cell line Ramos is composed of mIg p chains, h light chains, and theCD79 (Mb1/B29) heterodimer, which is essential for transmembrane signaling (21,221. In accordance with published results (301, triggering of the BCR on intact Ramos cells for 2 min a t 37 "C effectively induced tyrosine phosphorylation of a number of target proteins, as determined by Western blotting of antiTyr(P) immunoprecipitates (Fig. l a ) . BCR stimulation also gave rise totyrosine phosphorylationof the Shc adaptor protein and associated molecules, as described recently (31). In the

dues, as determined by phosphoamino acid analysis (Fig. 3b). The Btk sample isolated from anti-Ig-stimulated Ramos cells had significantly increased kinase activity, again with primary specificity for tyrosine residues(Fig. 3). Since no specific additional tyrosine-phosphorylated proteins were detected in the Btk precipitate afterin vitro kinase reaction, we conclude that BCR triggering enhances Btk activity resulting in autophosphorylation. The minor serinekhreonine phosphorylation may be due to an associated kinase(s). Btk Is Found Primarily in the Cytosol, BothBefore and After B-cell Activation-Although the primary structureof Btk suggests a cytosolic localization (6, 71, the multiple functional domains may mediate direct interactions with membrane-local-

of Btk

Receptor-mediated B-cellActivation Cell type:

Ramos

23859

Tonsillar lymphocytes

ized proteins. To investigate this possibility, the kinase was recovered from cytosolic and membrane fractions of Ramos cells, before and after BCR triggering. The efficacy of cellular fractionation, as well as that ofBCR stimulation, was confirmed by the exclusive detection of the tyrosine-phosphorylated CD22 transmembrane receptor (32) in the crude mema-pTyr blol o.pTyr bbl brane fraction of activated cells (Fig. 4c). In unstimulated b[k"---"" " d " Ramos cells, the majority of Btk protein was found in the cyeblxbwbbl mbl?wbbl tosolic fraction and a small amount in the membrane fraction. FIG.2. Kinetics of BCR-induced tyrosine phosphorylation of This distribution did not change appreciably after cellular acBtk in intact Ramos cells and tonsillar lymphocytes. Cells were stimulated for the indicated time periods, and Btk was isolated by tivation (Fig. 4b).The large cytosolic Btk pool, as well as the immunoprecipitation. Phosphotyrosine content of Btk was determined small membrane-associated pool were subject to BCR-induced by Western blotting with4G10 mAb (upper panel),and the same blots tyrosine phosphorylation (Fig. 4u). The predominance of tyrowere stripped and reprobed with anti-Btk"'" to check for equal loading (lower panel).The efficacy of BCR stimulation was confirmed by 4G10 sine-phosphorylated Btkin thecytosolic fraction and thelack of significant membrane translocation suggest that the kinase immunoblotting of total cell lysates. exerts a function in thecytosol. Our resultalso indicates that if a Btk interacts during its activation with plasma membranelocalized components such as the BCR, its associated kinases, Prec#p#lat#on: n.blWGST or accessory transmembrane molecules, this must be a short I3 06 0 I02 0 I M, Tme 1 0 1 0 ' 30' 3 lived event. - 97 Given the pre-B-cell differentiation arrest in XLA, it is of blk "interest to know whether ligation of the BCR on pre-B-cells also -e8 activates Btk. However, these studies are hampered by the extremely low plasma membrane expression levels of the preBCR (27). The issue whether the various Btk mutations found - 43 in XLA patients affect its activation induced by BCR stimulaIn v8tlo k m l e array tion was addressed using in vitro Epstein-Barr virus transformed B-cell lines derived from XLA patients and healthy blk, - ----A "" donors. Our results thus far indicate that such cell lines are not n.blkb" blot appropriate tools to study BCR-mediated signal transduction. The BCR is unique in that controls it not only the transitions b between successive B-cell differentiation stages (reviewed in unstlmulalea 1' sllmulaled Ref.221, but also mature B-cell responses to antigen. It has been observed in XLA patients that the few B-lymphocytes, - P.Ser - P-Thr which escape from the pre-B-cell differentiation arrest andbe- P-Tyr 0 come mature, mIgM-bearing cells, do not differentiate into Igsecreting plasma cells upon antigenic challenge (reviewed in Ref. 33). Mature B-cells, which develop in greater numbers in xid mice, also show defects in plasma cell differentiation as FIG.3. BCR stimulation enhances tyrosine kinase activity of induced by specific antigens. The finding that Btk islocalized Btk as assayed by phosphorylationin uitro. a,SDS-polyacrylamide gel electrophoresis of in vitro phosphorylated Btk immunoprecipitates in theBCR-induced signal transduction cascade offers a potenisolated from Ramos cells stimulated for the indicated timeperiods and tial explanation for the manifestation of the Btk defect not only Btk quantification by Western blotting. After SDS-polyacrylamide gel in B-lymphocyte development to the mature B-cell stage, but electrophoresis, samples were transferred nitrocellulose; to the blot was also in antigen-driven differentiation into functional plasma exposed to x-rayfilm (upperpanel)and subsequentlyprobed with anticells. Btk reagent (lower panel).b, phosphoamino acid analysis of in vitro Time:

1 o 30' 2'

10' 30' 60'

'

M,

lo

10' 30' 90.

s

IS 4 ~ 1 2 0 . ~

-

I

:w

phosphorylated Btk protein isolated from unstimulated or stimulated Ramos cells. The positionsof phosphoserine (P-Ser),phosphothreonine (P-Thr),and phosphotyrosine ( P - Q r )are indicated. GST, glutathione S-transferase.

Acknowledgments- We thank M. Kraakman and E. de Vries for excellent technical assistance and J. W. Drijfhout andF. UytdeHaag for help in the generationof anti-peptide reagents.

0 Prectpralm: Tune

FIG.4. Btk is primarily foundin the cytosol both before and after BCR stimulation. Btk recovered from cytoso-

Fracton

lic ( C y ) and membrane ( M )fractions of Ramos cells, stimulated for the indicated time periods, was detected by immunoblotting with a n t i - w ( P ) mAb ( a )or antiBtk antibody( b ) .c, CD22 immunoprecipitate served a s control for cellular fractionation and was blotted with antilj~(P)mAb. GST, glutathioneS-transferase.

btk

- 200 - 97 -

C

, -

'

n.pTyl blot

b

blk

68 43

, 4C022

n.pTyr Mol

23860

Receptor-mediated B-cellActivation REFERENCES

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