B-cell activation Stephen V. Desiderio The Johns Hopkins
University
Extraordinary of
the
progress
B-cell
antigen
phosphorylation transduction, mediated
as and
School of Medicine, Baltimore,
has
been
receptor the
key
in identifying
signaling. In addition,
may rekindle
interest
Opinion
role
in
candidate
effecters
the properties
our of
USA
understanding protein-tyrosine
immunoglobulin
signal
of immunoglobulin-
of a new group of C proteins implicating
such molecules
as
in B-cell activation.
in Immunology
The structural basis of molecular recognition by the antigen receptors of B and T cells and the genetic mechanisms that underlie receptor diversity are understood, at least in broad outline. The mechanisms of signal transduction by antigen receptors, however, remain for the most part undefined. The problem of their definition has been difficult because the antigen receptors - unlike growth factor receptor kinases, for example ~ have relatively short cytoplasmic regions that are enzymatically inert. Nonetheless, in recent years the nut has begun to crack, first in the T-cell system and more recently with regard to B cells. Here, I will review progress in three interconnected areas: the structure of the B-cell antigen receptor complex; the definition of signal transductory pathways in B cells; and the identification of candidate effector components of the transduction apparatus.
receptor
refining the
intermediary
Introduction
The B-cell antigen
in
in older observations important
Current
made complex,
Maryland,
complex
An important advance toward understanding B-cell antigen-receptor-mediated signal transduction was the discovery that surface immunoglobulin (sIg), like the antigen-binding chains of the T-cell receptor, forms a complex with other transmembrane molecules. In digitonin lysates of B cells, sIgM was found in a non-covalent complex with a disulfide-linked heterodimer of 34 and 39 kD glycosylated transmembrane proteins [ 1**,2**]. Under similar conditions sIgD was found to be associated with a disulfide-linked heterodimer of 35 and 39 kD glycoproteins [ 2**,3]. The larger component of the sIgassociated heterodimer was found by peptide sequence analysis to be encoded by B29 [4,5]. The smaller components of the sIgM- and sIgD-associated heterodimers
1992, 4:252-256
were originally called IgM-cl and IgD-cl, respectively. HOWever, by amino acid sequence these proteins were both found to be encoded by mbl and are now collectively termed Ig-cz, their mobility difference arising from a difference in glycosylation [4,5,6**,7]. The other classes of sIg are also associated with Ig-cl-@ heterodimers. The Ig-ol associated with sIgE and sIgG is similar in mobility to that of sIgM; the Ig-a found with s&A comigrates with that of sIgD [6-l. and Ig-p appear to be required for efficient surface expression of all classes of transmembrane Ig. This was first seen in attempts to express sIgM in the J558L myeloma cell line [l**] . In non-lymphoid cells, expression of sIgM was found to require cotransfection of mb 1 and B29 expression vectors with DNA encoding Ig p and light chains [6-l; a portion of the p chain including the CH4 domain, the transmembrane region, and the cy toplasmic region retained dependence on Ig-associated molecules for surface expression [8]. Ig-cl and Ig-p are also required for surface transport of IKE, 1gA [6**1, and probably IgG [9]. In contrast, IgD can be expressed on the cell surface in the absence of Ig-cl or Ig-p [6**,7], in which instance it is linked to the membrane via a glycosyphosphatidylinositol anchor [lo**]. Ig-cl
The membrane-bound forms of p and 6 have cytoplas mic regions that are only three amino acids long. In contrast, Ig-cl and Ig-b have cytoplasmic regions of 61 and 48 amino acids, respectively, and could serve to physically couple sIg to intracellular effecters. Ig-a and Ig-p resemble the 6 chain of the T-cell receptor (TCR) and the y chain of some Fc receptors with respect to the Spacing of tyrosine and leucine residues in their cytoplasTic regions [ 111. The cytoplasmic portions of the 6 and y chains are able to transduce activation signals in T cells (reviewed in [ 121); it remains to be shown whether either of the @associated chains serves a similar function in B cells, although this seems likely.
Abbreviations C-stimulating C protein; EGMpidermal growth factor; C protein---GTP-binding protein; G, -phospholipase PIP*-phosphatidylinositol 4,5bisphosphate; PKC-protein kinase C; PM-phospholipase C; slg-surface immunoglobulin; TCR-T-cell receptor.
252
@ Current Biology ltd ISSN 0952-7915
B-cell activation
What features of the Ig heavy chain itself are required for surface transport and signaling? The l.t transmembrane region contains two intervals (‘ITAST and YSTTVT; in the one-letter amino acid code) rich in polar amino acids. Conversion of the former sequence to WAAV is sufficient to allow surface expression in the absence of Ig-a; this suggests that the @associated molecules promote surface expression of IgM by masking polar residues [S] Two mutant forms of u chain have been constructed that permit surface expression of IgM but abolish transduction of antigenic signals, as assessed by calcium mobilization [ 13*=]. Deletion of the u cytoplasmic tail (KVK) results in surface expression of IgM via a glycosy-phosphatidylinositol linkage; in this instance, it is perhaps not surprising that a defect in signaling is observed. From the example of IgD (see above), one might expect surface expression of this cytoplasmic deletion mutant to be in dependent of Ig-r&b, but this has not yet been proven. The more interesting mutation replaces two polar amino acids in the transmembrane region with hydrophobic residues (YS to W), resulting in loss of the intracellular calcium response to antigen or to receptor crosslinking; more subtle changes (YS to FS or YS to YA) do not impair signaling. From the example of IgD we know that surface expression of Ig and its association with the Ig-a-Ig-l3 heterodimer can be separated. It seems likely, therefore, that some Ig heavy chain mutations might allow surface expression but impair association with Ig-m and Ig-p; the YS to W transmembrane mutation may represent a member of this class. The B-cell response to antigen receptor engagement can be augmented by other ligand-receptor interactions. In T cells, signaling through the TCR is enhanced by coengagement of CD4, which is coupled to the intracellular protein-tyrosine kinase p56’@ The B-cell receptor CR2, which binds the C3dg fragment of complement component C3, interacts synergistically with sIgM in increas ing intracellular calcium. CR2 is associated with CD19, a member of the Ig superfamily that is expressed through out B-cell ontogeny; the synergistic effect of CR2 ligation on Ig signal transduction is mimicked by crosslinking of CD19 to s&M [14*]. CD19 and sIg are cocapped and comodulated, suggesting that CD19 is associated, directly or indirectly, with sIg [15]. Thus, CD19 may play a role in B cells analogous to that of CD4 in mature T cells.
Tyrosine phosphorylation and B-cell activation Engagement of sIg, either by polyvalent antigen or by anti-Ig antibody, results in increased phosphatidylinosito1 4,5-bisphosphate (PIP,) hydrolysis and a subsequent rise in the concentration of intracellular free calcium, followed by activation of protein kinase C (PKC). For many years, the link between ligand binding and these intracellular responses was obscure. An important advance was the observation that crosslinking of s&M or sIgD on B cells or B cell lines results in de nouo protein-tyrosine phosphorylation [ 16**-Ip=] . ‘Iyrosine phosphorylation
Desiderio
of at least 10 different proteins was observed as early as 10 seconds after receptor crosslinking, and was sustained for at least 30 minutes to 1 hour before declining to basal levels. Two of the substrates are the Ig-associated molecules themselves, Ig-a and Ig-l3 [ 20.1, which underscores their potential importance in signal transduction; intracellular components of several other receptors, including the TCR 6 chain, are similarly phosphorylated on binding of ligand. Binding of polyvalent antigen to an antigen-specific s&M triggered tyrosine phosphory lation of a similar set of proteins (S Dymecki and S Desiderio, unpublished data). Increased tyrosine phosphotylation was not observed upon stimulation of B cells with phorbol esters or lipopolysaccharide, consistent with the idea that these agents either bypass initial stages of Ig-mediated signaling (phorbol ester) or act via a distinct pathway (lipopolysaccharide) [ 16**,17**]. The proteins phosphorylated after crosslinking of IgM or IgD are of a similar size, with an interesting exception Even though most splenic B cells express both IgM and IgD, and contain both forms of Ig-cl, the passociated form is selectively phosphorylated after liga tion of IgM and the h-associated form after ligation of IgD [ 20*]. This important observation suggests a physical association between the active kinase(s) and the receptor through which the kinase(s) was stimulated. The observation that tyrosine kinase substrates and the antigen receptor complex are colocalized after ligation of sIg [2I] reinforces this idea. What is the relationship between increased tyrosine phospholylation and PIP, hydrolysis, which leads to calcium mobilization and PKC activation? Depletion of PKC by chronic treatment of B cells with phorbol dibutyrate did not impair the tyrosine kinase response to Ig crosslinking, indicating either that PKC acts downstream of protein-tyrosine phosphotylation or that it functions in a distinct pathway [ 19**]. The former, -is probably the case, because three different, selective. inhibitors of protein-tyrosine kinases were able to suppress the generation of inositol phosphates and increased intracellular calcium in response to Ig crosslinking [ 220.1. Activation of a protein-tyrosine kinase(s) therefore seems likely to be the primary event in Ig-mediated signal transduction. One link between B-cell antigen receptor engagement and activation of the phosphoinositide hydrolysis pathway is now clear: the tyrosine phosphorylation of phospholipase C (PLC)-)I [22*=,23]. The two subtypes of PLC-y, PLC-yl and PLC-~2, are members of a group of enzymes that hydrolyze PIP,. Tyrosine phosphotylation of PLC-y by the epidermal growth factor (EGF) receptor increases its catalytic activity [24**]. Both subtypes of PLC-?I are expressed in B cells; in B-lymphoid cell lines, PLCy2 is more consistently expressed and generally more abundant than PLC-)I~ [25]. The increase in PIP, hydrolysis observed after crosslinking sIgM is accompanied by tyrosine phosphotylation of PLC-yl, and is suppressed by inhibitors of protein-tyrosine kinases, suggesting that the activation of PLC-)I by sIg is analogous to its activation by the EGF receptor [ 22**].
253
254
LvmDhocvte
activation
Candidate effecters tyrosine
and effector
functions
of B-cell activation:
protein
kinases
A definitive, functional link between sIg and a specific protein tyrosine kinase(s) has not yet been established. In growth-factor receptor kinases, such as the EGF receptor, ligand-binding and kinase domains are covalently joined. The members of the Ig receptor complex lack intrinsic tyrosine kinase activity and may therefore act via a non-covalent association with an intracellular kinase(s), as occurs between the T-cell costimulatory molecules CD4 and CD8 and the protein-tyrosine lck, a member of the src family. The tyrosine kinase blk is the only known member of the src family whose expression is restricted to B lym phoid cells [26**], but B cells express other src ki nases as well, including fyn and lyn [27*,28**]. The blk kinase is expressed at all stages of B-cell ontogeny until the differentiation of B cells to plasma cells, at which time expression ceases [29]. The restricted nature of blkexpression and its co-regulation with mbl and BZ9 during B-cell ontogeny [29.] suggest that it plays an intimate role in Ig-mediated signaling. The fyn and lyn kinases show a broader tissue distribution, al though lyn is preferentially expressed in tissues rich in B lymphocytes and in myeloid cells. All three kinases coprecipitate with the Ig receptor complex [ 27*,28**]. unlike the lckCD4/CD8 association, however, these kinase-receptor complexes are tenuous, having been observed only under conditions of mild detergent lysis, and the possibility of association after cell disruption has not been excluded. Furthermore, all src kinases (and also some other protein-tyrosine kinases: see below) possess a region of homology, the SH2 region, that interacts specifically with phosphorylated proteins, especially those phosphorylated on tyrosine. The available evidence does not provide a distinction between primary kinase-receptor complexes and associations-possibly mediated by SH2 - that are formed after tyrosine phosphorylation of the Ig-associated chains. The blk, lyn and iyn kinases all show increased tyrosine kinase activity in vitro following antigen-receptor crosslinking [ 28**,30]. Within 1 minute after ligation of sIg, the activity of blk increases five- to eight-fold, as measured by the in vitro phosphorylation of enolase by immunoprecipitated kinase; the observed increases in lyn and fyn activity are more modest (two- to four-fold). Although these results suggest that blk, lyn and ljn function in Ig-mediated signaling, they do not indicate which, if any, of these kinases is the principal effector. The issue is even more complicated. A 72 kD protein-ty rosine kinase has been purified from spleen, and a cDNA that encodes it or a related kinase has been molecularly cloned [31-l. The kinase encoded by this cDNA, called syk, is expressed preferentially in lymphoid tissues and differs from src kinases in several ways. First, all src kinases are myristoylated at their amino-termini, a modification that contributes to their membrane localization. The syk kinase lacks a myristoyl acceptor sequence.
Second, syk lacks the conserved SH3 homology region of src kinases and carries a duplication of the SH2 region. The syk kinase lacks an obvious transmembrane region, and is likely to be intracellular. On crosslinking of s&M, syk or a closely related kinase is phosphorylated on tyrosine, suggesting proximity to the active receptor complex, and a role in B-cell activation [32*]. As yet, however, activation of syk upon receptor crosslinking has not been demonstrated.
G-protein-mediated
signaling
in B cells: a
reassessment A role for GTP-binding proteins (G proteins) in B-cell activation is suggested by experiments in which G protein activators such as GTP-yS were shown to synergize with suboptimal doses of anti-Ig in stimulating phospholipid hydrolysis (for example, see [33]). With the exception of a provocative description of the colocalization of p21ms and sIg after antigen-receptor ligation [34*], G proteins as potential mediators of B-cell activation have received relatively little recent attention. Several important developments, however, warrant a reassessment of the issue. A group of G proteins, collectively called phospholipase C-stimulating G proteins (GP,), has been shown to specifically activate PLC-p isoforms. A subgroup of G typified by G,, is insensitive to pertussis toxin, as is tK putative G protein activity observed to synergize with sIg ligation. At least five distinct GCXsubunits of the G, class have been molecularly cloned. One of these Guts, shows preferential, but not exclusive, expression in spleen and fetal liver, and in cell lines of the B and myeloid lineages [35*]. On the basis of these findings, a reasonable explanation for the synergy between sIg ligation and GTP-yS administration is that these treatments simultaneously activate PLC-y and PLC-j3 isotypes through protein-tyrosine kinase and G protein mediators, respectively. Identification of the cell surface molecules that couple to the Gq proteins of B cells should become a goal of great interest.
Conclusions Work on the problem of Ig-mediated signaling, which had long seemed at an impasse, has seen extraordinary progress in the past year. As for other well-defined problems in immunology, the recent advances in this field have stemmed from the rigorous application of biochemistry and molecular genetics. As a result, the framework for an Ig-coupled signaling pathway in B cells, mediated by protein-tyrosine phosphorylation, has been established. Here the major challenge will be to identify, from among a variety of candidates, those kinases that function in primary Ig signaling events. In addition, the distinctive properties of a newly characterized class of G proteins promise to reopen the old debate of G protein involvement in B-cell activation.
B-cell activation
Desiderio
SHAWAC, MITCHELLRN, WEAVERYK, CAMPOS-TORRES J, ABBAS AK, LEDERP: Mutations of ImmunogIobuIin Transmembrane and Cytoplasmic Domains: Effects on IntraceUuIar SignaIing and Antigen Presentation. Cell 1990, 63:381-392. Development of a system for analysis of Ig mutations that uncouple surf face expression from signaling by antigen, and signaling from antigen presentation. 13. ..
Acknowledgements The author thanks Susan Dymecki and Doug Fearon for comments on the manuscript. Work in the author’s laboratory is supported by the Howard Hughes Medical Institute and the National Institutes of Health.
MATSUMOTO AK, KPOICKY-BURD J, CARTERRH, TV-SON DA TEDDER TF, FEARONDT: Intersection of the Complement and Immune Systems: a Signal Transduction Complex of the B Lymphocyte-containing Complement Receptor Type 2 and CD19. J Exp Med 1991, 173:55&. Clear, convincing evidence of a specific association between CR2 and CD19, and of synergy between CD19 and sIg.
14. .
References
and recommended
reading
Papers of particular interest, published within the annual period of rep view, have been highlighted as: of special interest . of outstanding interest .. HOMBACHJ, TAKESHIT, LECLERCQL, STAPPERTH, RETH M: Molecular Components of the B-cell Antigen Receptor Complex of the IgM Class. Nature 1990, 343:76G762. See [2**J. 1. ..
CAMPBEU.KS, CAMBIERJC: B Lymphocyte Antigen Receptors (mIg) are Non-covalently Associated with a DisuIfide Linked, lnducibly Phosphorylated Glycoprotein Complex. EMBO J 1990, 9:441-448. This paper and [loo] establish the existence of a B-cell antigen receptor complex consisting of membrane-bound Ig and a disuffide-linked heterodimer. These observations suggest that the antigen receptor complexes of B and T cells are structurally analogous. 2. ..
3.
WIENANDS J, HOMBACH J, RADBRUCH A, RIESTERERC, RETH M: Molecular Components of the B CeU Antigen Receptor Complex of Class IgD Differ Partly from ‘Ihose of l@l. , . EMBO J 1990, 9:449455.
4.
HOMBACHJ, ~~SPEICH F, RETH M: Identification of the Genes Encoding the IgM-a and lg-p Components of the IgM Antigen Receptor Complex by Amino-terminal Sequencing. Eur J Immunol 1990, 20~2795-2799.
5.
CAMPBELL KS, HAGER EJ, FIUEDRICH RJ, CAMBIERJC: IgM Antigen Receptor Complex Contains Phosphoprotein Products of B29 and mbl Genes. Proc Nat1 Acad Sci U S A 1991, 88:3982%3986.
6. ..
VENKITARAMAN AR, WILUAMSGT, DARIAVAC P: The B-cell Antigen Receptor of the Five ImmunogIobuIin Classes. Nature 1991, 352~777-781. The first comprehensive analysis of the receptor complexes of all Ig classes; this paper also lays the groundwork for the reconstitution of Ig complexes in non-lymphoid cells. 7.
WIENANDS J, RETH M: The B CeII Antigen Receptor of Class IgD can be Expressed on the CeII Surface in Two Different Forms. Eur J Immunol 1991, 21~2373-2378.
8.
WIL.LL~MS GT, VENKITARAMAN AR, GILMOREDJ, NEUBERGERMS: The Sequence of the c~ Transmembrane Segment Determines the Tissue Specificity of the Transport of lmmunoglobulin M to the CeU Surface. J Exp Med 1990, 171:947-952.
9.
BACHHAWAT AK, Prm S: Distinct Intracellular Fates of Membrane and Secretory ImmunogIobuUn Heavy Chains in a pre-B CeU Line. J Cell Biol 1991, 115:61$X24.
10. ..
WIENANDS J, RETH M: Glycosyl-phosphatidylinositol Linkage as a Mechanism for CeU Surface Expression of ImmunogIobuUn D. Nature 1992, 356236248. An explanation for the appearance of IgD on the cell surface in the absence of the lg~associated chains. 11.
RETH M: Antigen 338:383-384.
12.
KLAUSNER RD, SAMEL.SON L!?:
Receptor
Tail
Clue.
Nature
1989,
T CeU Antigen Receptor Activation Pathways: the Tyrosine Kinase Connection. Cell 1991, 64:875-878.
15.
PESANLXJM, BOUCHAXD IS, MCMASTER BE: CD19 is FunctionaIIy and PhysicaUy Associated with Surface ImmunogIobuUn. J Exp Med 1989, 170:2159-2164.
GOLD MR, LAWDA, DEFRANCO AL: Stimulation of Protein Tyrosine Phosphorylation by the B-lymphocyte Antigen Receptor. Nature 1990, 345:81&813. See [19**]. 16. ..
CAMPBELL M-A, SEFTONBM: Protein Tyrosine Phosphorylation is Induced in Murine B Lymphocytes in Response to Stimulation with Anti-immunoglobulin. L?MBOJ 1990, 9:2125-2131. See [19**]. 17. ..
LANEPJL, MCCONNELL FM, SCHIEVENGL, CLARKF-4, LEDBETI’ER JA: The Role of Class 11 Molecules in Human B CeU Activation: Association with Phosphatidyl Inositol Turnover, Protein Tyrosine Phosphorylation, and Proliferation. J Im;.munol 1990, 144:3684-3692. See [19**1. 18. ..
BRUNSVC~CK M, SAMELSON LE, MOND JJ: Surface ImmunogIobuIin Crosslinking Activates a ‘I’yrosine Kinase Pathway in B CeUs That is Independent of Protein Kinase C. Prcc Nat1 Acad Sci USA 1991, 88:1311-1314. published within a short period, were These four papers [16**-I’?] the first to establish that ligation of surface Ig is accompanied by activa~ tion of a protein-tyrosine kinase(s). Taken together with observations of increased tyrosine, phosphorylation fobwing TCR engagement these studies provide strong evidence that the &ntigen receptors of T and B cells employ similar signaling mechanisms. 19. ..
GOLD MR, MATXJ~JCHI L, KELLY RB, DEFRANCOAL Tyrosine Phosphorylation of Components of the B-ceil Antigen Receptors Following Receptor Crosslinking. Proc Nat1 Acad Sci USA 1991, S&34363440. Emphasizes the potential importance of the Ig-associated chains in signaling; suggests a physical association between kinase(s) and the receptor molecules through which they are activated. 20. .
21.
TAKAGIS, DAIBATAM, Lks~ TJ, H~JMPHREYS RE, PARKERDC, SAIRENJI T: Intracellular Localization of Tyrosine Kinase Substrates Beneath Crosslinked Surface ImmunogIobuIins in B Cells. J Exp Med 1991, 174:381-388.
CARTERRH, PARKDJ, RHEE SG, FEXRONDT: Tyrosine Phosphorylation of Phospholipase C Induced by Membrane Immunoglobulin in B Lymphocytes. Proc Nat1 Acad Sci USA 1991, 88:2745-2749. The first demonstration of a biochemical link between Ig engagement and modification of a phosphoIipase. 22. ..
23.
PADEHS, LEVITZKI A, GAZITA, MILLSGB, ROIFMAN CM: Activation of PhosphoUpase C in Human B Cells is Dependent on Tyrosine Phosphorylation. J Clin Invest 1991, 87:111&1118.
S, WAHL MI, HEFPUNDEZ-SOTOMAYOR SMT, TONK~NK, SG, CARPENTER G: Increase of the Catalytic Activity of Phospholipase C-y 1 by Tyrosine Phosphorylation. Science 1990, 250:1253%1256. One of a series of seminal papers that have established a link between protein-tyrosine kinase activation and the phosphoinositide hydrolysis pathway.
24. ..
NISHBE RHEE
255
256
Lvmphoqte
25.
activation
HEMPEL WM,
and effector
functions
DEFRANCO AL: Expression
C Isozymes by Murine 146:3713-3720.
B Lymphocytes.
of Phospholipase J hnmunol 1991,
DYMECKISM, NIEDERHUBER JE, DESIDEFSOSV: Specific Expression of a Tyrosine Kinase Gene, blk, in B Lymphoid Cells. Science 1990, 2413322336. Molecular cloning and characterization of the first protein-tyrosine kinase expressed specifically in the B lineage. 26. ..
YAMANA~H~Y, KAKIUCHI T, MIZUGUCHI J, YAMAMOTO T, TOYOSHIMAK: Association of B Ceil Antigen Receptor with Protein Tyrosine Kinase Lyn. Science 1991, 251:192-194. First demonstration of a physical association between a src~type kinase and the Ig complex. 27. .
28. ..
BUFXHARDTAL, BRUNSWICKM, BOLEN JE, MOND JJ: Anti-immunoglobulin Stimulation of B Lymphocytes Activates srcrelated Protein-tyrosine Kinases. Proc Natl Acad Sci lrSA 1991, 88:741@7414. Coprecipitation of three different src kinases with the Ig complex; first demonstration that activity of specific kinases increases upon receptor crosslinking. 29. .
DYMECKISM, ZWOLLO P, ZELLERK, KUHAJDAFP, DESIDERIOSV:
30.
YAMANA.SHI Y, FUKUI Y, WONGSASANTB, KINOSHITAY, ICHIM~RI Y, TOYOSHIMAK, YAMAMOTOT: Activation of Src-Iike Proteintyrosine Kinase Lyn and its Association with Phosphatidylinositol 34dnase Upon B-cell Antigen Receptor-mediated Signaling. Proc Natl Acad Sci USA 1992, 89:11181122.
31. .
TANIGUCHI‘IT, KOBAYA~HIT, KONDO J, TAKAHAS to K, NAKAMURA H, SUZUKIJ, NAGAI K, YAMADAT, NAKAMURAS, YAMAMURA H:
Structure and Developmental Regulation of the B-lymphoid Tyrosine Kinase Gene blk. J Biol Cbem 1992, 267:4815-4823. Molecular and histochemical analysis of blk kinase expression in B-cell development and in lymphoid tissues showing coexpression of blk and Ig associated chains during B-cell ontogeny.
Molecular Cloning of a Porcine Gene syk That Encodes a 72-kDa Protein-tyrosine Kinase Showing High Susceptibility to Proteolysis. J Biol Cbem 1991, 266:1579&15796. Describes a protein-tyrosine kinase of unusual structure, syk, encoded by a gene that is expressed preferentially in lymphoid tissues. HUTCHCROFTJE, HAP&SON ML, GEAHLEN RL: B Lymphocyte Activation is Accompanied by Phosphorylation of a 72-kDa Protein-tyrosine Kinase. J Biol Cbem 1991, 266:1484614849. Demonstration that the syk kinase, or a closely related protein, is a major substrate for tyrosine phosphorylation following Ig crosslinking.
32. .
33.
HAFZVETT MM, Kt~us GGB: G Protein Coupling of Antigen Receptor-stimulated Polyphosphoinositide Hydrolysis in B Cells. J Immund 1988, 140:31353139. GRAUDEI L, RLABOWOL K, BAR-SAGID: Co-capping
of ras Proteins with Surface Immunoglobtdins in B Lymphocytes. Natwe 1990, 347:39&400. A provocative paper that establishes co-localization of sIg and p2lra upon antigen receptor engagement. 34. .
WILKIE TM, SCHERLEPA, STRATHMANNMP, SIEPAK VZ, SIMON MI: Characterization of G-protein a Subunits in the Gq Class: Expression in Murine Tissues and in Stromal and Hematopoietic Cell Lines. Proc Natl Acad Sci Cl S A 1991, 88:1004910053. A description of pertussis-toxin-insensitive phospholipase-stimulatory G-proteins that are expressed preferentiaky in hematopoietic cells. 35. .
SV Desiderio, Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
University
Extraordinary of
the
progress
B-cell
antigen
phosphorylation transduction, mediated
as and
School of Medicine, Baltimore,
has
been
receptor the
key
in identifying
signaling. In addition,
may rekindle
interest
Opinion
role
in
candidate
effecters
the properties
our of
USA
understanding protein-tyrosine
immunoglobulin
signal
of immunoglobulin-
of a new group of C proteins implicating
such molecules
as
in B-cell activation.
in Immunology
The structural basis of molecular recognition by the antigen receptors of B and T cells and the genetic mechanisms that underlie receptor diversity are understood, at least in broad outline. The mechanisms of signal transduction by antigen receptors, however, remain for the most part undefined. The problem of their definition has been difficult because the antigen receptors - unlike growth factor receptor kinases, for example ~ have relatively short cytoplasmic regions that are enzymatically inert. Nonetheless, in recent years the nut has begun to crack, first in the T-cell system and more recently with regard to B cells. Here, I will review progress in three interconnected areas: the structure of the B-cell antigen receptor complex; the definition of signal transductory pathways in B cells; and the identification of candidate effector components of the transduction apparatus.
receptor
refining the
intermediary
Introduction
The B-cell antigen
in
in older observations important
Current
made complex,
Maryland,
complex
An important advance toward understanding B-cell antigen-receptor-mediated signal transduction was the discovery that surface immunoglobulin (sIg), like the antigen-binding chains of the T-cell receptor, forms a complex with other transmembrane molecules. In digitonin lysates of B cells, sIgM was found in a non-covalent complex with a disulfide-linked heterodimer of 34 and 39 kD glycosylated transmembrane proteins [ 1**,2**]. Under similar conditions sIgD was found to be associated with a disulfide-linked heterodimer of 35 and 39 kD glycoproteins [ 2**,3]. The larger component of the sIgassociated heterodimer was found by peptide sequence analysis to be encoded by B29 [4,5]. The smaller components of the sIgM- and sIgD-associated heterodimers
1992, 4:252-256
were originally called IgM-cl and IgD-cl, respectively. HOWever, by amino acid sequence these proteins were both found to be encoded by mbl and are now collectively termed Ig-cz, their mobility difference arising from a difference in glycosylation [4,5,6**,7]. The other classes of sIg are also associated with Ig-cl-@ heterodimers. The Ig-ol associated with sIgE and sIgG is similar in mobility to that of sIgM; the Ig-a found with s&A comigrates with that of sIgD [6-l. and Ig-p appear to be required for efficient surface expression of all classes of transmembrane Ig. This was first seen in attempts to express sIgM in the J558L myeloma cell line [l**] . In non-lymphoid cells, expression of sIgM was found to require cotransfection of mb 1 and B29 expression vectors with DNA encoding Ig p and light chains [6-l; a portion of the p chain including the CH4 domain, the transmembrane region, and the cy toplasmic region retained dependence on Ig-associated molecules for surface expression [8]. Ig-cl and Ig-p are also required for surface transport of IKE, 1gA [6**1, and probably IgG [9]. In contrast, IgD can be expressed on the cell surface in the absence of Ig-cl or Ig-p [6**,7], in which instance it is linked to the membrane via a glycosyphosphatidylinositol anchor [lo**]. Ig-cl
The membrane-bound forms of p and 6 have cytoplas mic regions that are only three amino acids long. In contrast, Ig-cl and Ig-b have cytoplasmic regions of 61 and 48 amino acids, respectively, and could serve to physically couple sIg to intracellular effecters. Ig-a and Ig-p resemble the 6 chain of the T-cell receptor (TCR) and the y chain of some Fc receptors with respect to the Spacing of tyrosine and leucine residues in their cytoplasTic regions [ 111. The cytoplasmic portions of the 6 and y chains are able to transduce activation signals in T cells (reviewed in [ 121); it remains to be shown whether either of the @associated chains serves a similar function in B cells, although this seems likely.
Abbreviations C-stimulating C protein; EGMpidermal growth factor; C protein---GTP-binding protein; G, -phospholipase PIP*-phosphatidylinositol 4,5bisphosphate; PKC-protein kinase C; PM-phospholipase C; slg-surface immunoglobulin; TCR-T-cell receptor.
252
@ Current Biology ltd ISSN 0952-7915
B-cell activation
What features of the Ig heavy chain itself are required for surface transport and signaling? The l.t transmembrane region contains two intervals (‘ITAST and YSTTVT; in the one-letter amino acid code) rich in polar amino acids. Conversion of the former sequence to WAAV is sufficient to allow surface expression in the absence of Ig-a; this suggests that the @associated molecules promote surface expression of IgM by masking polar residues [S] Two mutant forms of u chain have been constructed that permit surface expression of IgM but abolish transduction of antigenic signals, as assessed by calcium mobilization [ 13*=]. Deletion of the u cytoplasmic tail (KVK) results in surface expression of IgM via a glycosy-phosphatidylinositol linkage; in this instance, it is perhaps not surprising that a defect in signaling is observed. From the example of IgD (see above), one might expect surface expression of this cytoplasmic deletion mutant to be in dependent of Ig-r&b, but this has not yet been proven. The more interesting mutation replaces two polar amino acids in the transmembrane region with hydrophobic residues (YS to W), resulting in loss of the intracellular calcium response to antigen or to receptor crosslinking; more subtle changes (YS to FS or YS to YA) do not impair signaling. From the example of IgD we know that surface expression of Ig and its association with the Ig-a-Ig-l3 heterodimer can be separated. It seems likely, therefore, that some Ig heavy chain mutations might allow surface expression but impair association with Ig-m and Ig-p; the YS to W transmembrane mutation may represent a member of this class. The B-cell response to antigen receptor engagement can be augmented by other ligand-receptor interactions. In T cells, signaling through the TCR is enhanced by coengagement of CD4, which is coupled to the intracellular protein-tyrosine kinase p56’@ The B-cell receptor CR2, which binds the C3dg fragment of complement component C3, interacts synergistically with sIgM in increas ing intracellular calcium. CR2 is associated with CD19, a member of the Ig superfamily that is expressed through out B-cell ontogeny; the synergistic effect of CR2 ligation on Ig signal transduction is mimicked by crosslinking of CD19 to s&M [14*]. CD19 and sIg are cocapped and comodulated, suggesting that CD19 is associated, directly or indirectly, with sIg [15]. Thus, CD19 may play a role in B cells analogous to that of CD4 in mature T cells.
Tyrosine phosphorylation and B-cell activation Engagement of sIg, either by polyvalent antigen or by anti-Ig antibody, results in increased phosphatidylinosito1 4,5-bisphosphate (PIP,) hydrolysis and a subsequent rise in the concentration of intracellular free calcium, followed by activation of protein kinase C (PKC). For many years, the link between ligand binding and these intracellular responses was obscure. An important advance was the observation that crosslinking of s&M or sIgD on B cells or B cell lines results in de nouo protein-tyrosine phosphorylation [ 16**-Ip=] . ‘Iyrosine phosphorylation
Desiderio
of at least 10 different proteins was observed as early as 10 seconds after receptor crosslinking, and was sustained for at least 30 minutes to 1 hour before declining to basal levels. Two of the substrates are the Ig-associated molecules themselves, Ig-a and Ig-l3 [ 20.1, which underscores their potential importance in signal transduction; intracellular components of several other receptors, including the TCR 6 chain, are similarly phosphorylated on binding of ligand. Binding of polyvalent antigen to an antigen-specific s&M triggered tyrosine phosphory lation of a similar set of proteins (S Dymecki and S Desiderio, unpublished data). Increased tyrosine phosphotylation was not observed upon stimulation of B cells with phorbol esters or lipopolysaccharide, consistent with the idea that these agents either bypass initial stages of Ig-mediated signaling (phorbol ester) or act via a distinct pathway (lipopolysaccharide) [ 16**,17**]. The proteins phosphorylated after crosslinking of IgM or IgD are of a similar size, with an interesting exception Even though most splenic B cells express both IgM and IgD, and contain both forms of Ig-cl, the passociated form is selectively phosphorylated after liga tion of IgM and the h-associated form after ligation of IgD [ 20*]. This important observation suggests a physical association between the active kinase(s) and the receptor through which the kinase(s) was stimulated. The observation that tyrosine kinase substrates and the antigen receptor complex are colocalized after ligation of sIg [2I] reinforces this idea. What is the relationship between increased tyrosine phospholylation and PIP, hydrolysis, which leads to calcium mobilization and PKC activation? Depletion of PKC by chronic treatment of B cells with phorbol dibutyrate did not impair the tyrosine kinase response to Ig crosslinking, indicating either that PKC acts downstream of protein-tyrosine phosphotylation or that it functions in a distinct pathway [ 19**]. The former, -is probably the case, because three different, selective. inhibitors of protein-tyrosine kinases were able to suppress the generation of inositol phosphates and increased intracellular calcium in response to Ig crosslinking [ 220.1. Activation of a protein-tyrosine kinase(s) therefore seems likely to be the primary event in Ig-mediated signal transduction. One link between B-cell antigen receptor engagement and activation of the phosphoinositide hydrolysis pathway is now clear: the tyrosine phosphorylation of phospholipase C (PLC)-)I [22*=,23]. The two subtypes of PLC-y, PLC-yl and PLC-~2, are members of a group of enzymes that hydrolyze PIP,. Tyrosine phosphotylation of PLC-y by the epidermal growth factor (EGF) receptor increases its catalytic activity [24**]. Both subtypes of PLC-?I are expressed in B cells; in B-lymphoid cell lines, PLCy2 is more consistently expressed and generally more abundant than PLC-)I~ [25]. The increase in PIP, hydrolysis observed after crosslinking sIgM is accompanied by tyrosine phosphotylation of PLC-yl, and is suppressed by inhibitors of protein-tyrosine kinases, suggesting that the activation of PLC-)I by sIg is analogous to its activation by the EGF receptor [ 22**].
253
254
LvmDhocvte
activation
Candidate effecters tyrosine
and effector
functions
of B-cell activation:
protein
kinases
A definitive, functional link between sIg and a specific protein tyrosine kinase(s) has not yet been established. In growth-factor receptor kinases, such as the EGF receptor, ligand-binding and kinase domains are covalently joined. The members of the Ig receptor complex lack intrinsic tyrosine kinase activity and may therefore act via a non-covalent association with an intracellular kinase(s), as occurs between the T-cell costimulatory molecules CD4 and CD8 and the protein-tyrosine lck, a member of the src family. The tyrosine kinase blk is the only known member of the src family whose expression is restricted to B lym phoid cells [26**], but B cells express other src ki nases as well, including fyn and lyn [27*,28**]. The blk kinase is expressed at all stages of B-cell ontogeny until the differentiation of B cells to plasma cells, at which time expression ceases [29]. The restricted nature of blkexpression and its co-regulation with mbl and BZ9 during B-cell ontogeny [29.] suggest that it plays an intimate role in Ig-mediated signaling. The fyn and lyn kinases show a broader tissue distribution, al though lyn is preferentially expressed in tissues rich in B lymphocytes and in myeloid cells. All three kinases coprecipitate with the Ig receptor complex [ 27*,28**]. unlike the lckCD4/CD8 association, however, these kinase-receptor complexes are tenuous, having been observed only under conditions of mild detergent lysis, and the possibility of association after cell disruption has not been excluded. Furthermore, all src kinases (and also some other protein-tyrosine kinases: see below) possess a region of homology, the SH2 region, that interacts specifically with phosphorylated proteins, especially those phosphorylated on tyrosine. The available evidence does not provide a distinction between primary kinase-receptor complexes and associations-possibly mediated by SH2 - that are formed after tyrosine phosphorylation of the Ig-associated chains. The blk, lyn and iyn kinases all show increased tyrosine kinase activity in vitro following antigen-receptor crosslinking [ 28**,30]. Within 1 minute after ligation of sIg, the activity of blk increases five- to eight-fold, as measured by the in vitro phosphorylation of enolase by immunoprecipitated kinase; the observed increases in lyn and fyn activity are more modest (two- to four-fold). Although these results suggest that blk, lyn and ljn function in Ig-mediated signaling, they do not indicate which, if any, of these kinases is the principal effector. The issue is even more complicated. A 72 kD protein-ty rosine kinase has been purified from spleen, and a cDNA that encodes it or a related kinase has been molecularly cloned [31-l. The kinase encoded by this cDNA, called syk, is expressed preferentially in lymphoid tissues and differs from src kinases in several ways. First, all src kinases are myristoylated at their amino-termini, a modification that contributes to their membrane localization. The syk kinase lacks a myristoyl acceptor sequence.
Second, syk lacks the conserved SH3 homology region of src kinases and carries a duplication of the SH2 region. The syk kinase lacks an obvious transmembrane region, and is likely to be intracellular. On crosslinking of s&M, syk or a closely related kinase is phosphorylated on tyrosine, suggesting proximity to the active receptor complex, and a role in B-cell activation [32*]. As yet, however, activation of syk upon receptor crosslinking has not been demonstrated.
G-protein-mediated
signaling
in B cells: a
reassessment A role for GTP-binding proteins (G proteins) in B-cell activation is suggested by experiments in which G protein activators such as GTP-yS were shown to synergize with suboptimal doses of anti-Ig in stimulating phospholipid hydrolysis (for example, see [33]). With the exception of a provocative description of the colocalization of p21ms and sIg after antigen-receptor ligation [34*], G proteins as potential mediators of B-cell activation have received relatively little recent attention. Several important developments, however, warrant a reassessment of the issue. A group of G proteins, collectively called phospholipase C-stimulating G proteins (GP,), has been shown to specifically activate PLC-p isoforms. A subgroup of G typified by G,, is insensitive to pertussis toxin, as is tK putative G protein activity observed to synergize with sIg ligation. At least five distinct GCXsubunits of the G, class have been molecularly cloned. One of these Guts, shows preferential, but not exclusive, expression in spleen and fetal liver, and in cell lines of the B and myeloid lineages [35*]. On the basis of these findings, a reasonable explanation for the synergy between sIg ligation and GTP-yS administration is that these treatments simultaneously activate PLC-y and PLC-j3 isotypes through protein-tyrosine kinase and G protein mediators, respectively. Identification of the cell surface molecules that couple to the Gq proteins of B cells should become a goal of great interest.
Conclusions Work on the problem of Ig-mediated signaling, which had long seemed at an impasse, has seen extraordinary progress in the past year. As for other well-defined problems in immunology, the recent advances in this field have stemmed from the rigorous application of biochemistry and molecular genetics. As a result, the framework for an Ig-coupled signaling pathway in B cells, mediated by protein-tyrosine phosphorylation, has been established. Here the major challenge will be to identify, from among a variety of candidates, those kinases that function in primary Ig signaling events. In addition, the distinctive properties of a newly characterized class of G proteins promise to reopen the old debate of G protein involvement in B-cell activation.
B-cell activation
Desiderio
SHAWAC, MITCHELLRN, WEAVERYK, CAMPOS-TORRES J, ABBAS AK, LEDERP: Mutations of ImmunogIobuIin Transmembrane and Cytoplasmic Domains: Effects on IntraceUuIar SignaIing and Antigen Presentation. Cell 1990, 63:381-392. Development of a system for analysis of Ig mutations that uncouple surf face expression from signaling by antigen, and signaling from antigen presentation. 13. ..
Acknowledgements The author thanks Susan Dymecki and Doug Fearon for comments on the manuscript. Work in the author’s laboratory is supported by the Howard Hughes Medical Institute and the National Institutes of Health.
MATSUMOTO AK, KPOICKY-BURD J, CARTERRH, TV-SON DA TEDDER TF, FEARONDT: Intersection of the Complement and Immune Systems: a Signal Transduction Complex of the B Lymphocyte-containing Complement Receptor Type 2 and CD19. J Exp Med 1991, 173:55&. Clear, convincing evidence of a specific association between CR2 and CD19, and of synergy between CD19 and sIg.
14. .
References
and recommended
reading
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CAMPBEU.KS, CAMBIERJC: B Lymphocyte Antigen Receptors (mIg) are Non-covalently Associated with a DisuIfide Linked, lnducibly Phosphorylated Glycoprotein Complex. EMBO J 1990, 9:441-448. This paper and [loo] establish the existence of a B-cell antigen receptor complex consisting of membrane-bound Ig and a disuffide-linked heterodimer. These observations suggest that the antigen receptor complexes of B and T cells are structurally analogous. 2. ..
3.
WIENANDS J, HOMBACH J, RADBRUCH A, RIESTERERC, RETH M: Molecular Components of the B CeU Antigen Receptor Complex of Class IgD Differ Partly from ‘Ihose of l@l. , . EMBO J 1990, 9:449455.
4.
HOMBACHJ, ~~SPEICH F, RETH M: Identification of the Genes Encoding the IgM-a and lg-p Components of the IgM Antigen Receptor Complex by Amino-terminal Sequencing. Eur J Immunol 1990, 20~2795-2799.
5.
CAMPBELL KS, HAGER EJ, FIUEDRICH RJ, CAMBIERJC: IgM Antigen Receptor Complex Contains Phosphoprotein Products of B29 and mbl Genes. Proc Nat1 Acad Sci U S A 1991, 88:3982%3986.
6. ..
VENKITARAMAN AR, WILUAMSGT, DARIAVAC P: The B-cell Antigen Receptor of the Five ImmunogIobuIin Classes. Nature 1991, 352~777-781. The first comprehensive analysis of the receptor complexes of all Ig classes; this paper also lays the groundwork for the reconstitution of Ig complexes in non-lymphoid cells. 7.
WIENANDS J, RETH M: The B CeII Antigen Receptor of Class IgD can be Expressed on the CeII Surface in Two Different Forms. Eur J Immunol 1991, 21~2373-2378.
8.
WIL.LL~MS GT, VENKITARAMAN AR, GILMOREDJ, NEUBERGERMS: The Sequence of the c~ Transmembrane Segment Determines the Tissue Specificity of the Transport of lmmunoglobulin M to the CeU Surface. J Exp Med 1990, 171:947-952.
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BACHHAWAT AK, Prm S: Distinct Intracellular Fates of Membrane and Secretory ImmunogIobuUn Heavy Chains in a pre-B CeU Line. J Cell Biol 1991, 115:61$X24.
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WIENANDS J, RETH M: Glycosyl-phosphatidylinositol Linkage as a Mechanism for CeU Surface Expression of ImmunogIobuUn D. Nature 1992, 356236248. An explanation for the appearance of IgD on the cell surface in the absence of the lg~associated chains. 11.
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GOLD MR, LAWDA, DEFRANCO AL: Stimulation of Protein Tyrosine Phosphorylation by the B-lymphocyte Antigen Receptor. Nature 1990, 345:81&813. See [19**]. 16. ..
CAMPBELL M-A, SEFTONBM: Protein Tyrosine Phosphorylation is Induced in Murine B Lymphocytes in Response to Stimulation with Anti-immunoglobulin. L?MBOJ 1990, 9:2125-2131. See [19**]. 17. ..
LANEPJL, MCCONNELL FM, SCHIEVENGL, CLARKF-4, LEDBETI’ER JA: The Role of Class 11 Molecules in Human B CeU Activation: Association with Phosphatidyl Inositol Turnover, Protein Tyrosine Phosphorylation, and Proliferation. J Im;.munol 1990, 144:3684-3692. See [19**1. 18. ..
BRUNSVC~CK M, SAMELSON LE, MOND JJ: Surface ImmunogIobuIin Crosslinking Activates a ‘I’yrosine Kinase Pathway in B CeUs That is Independent of Protein Kinase C. Prcc Nat1 Acad Sci USA 1991, 88:1311-1314. published within a short period, were These four papers [16**-I’?] the first to establish that ligation of surface Ig is accompanied by activa~ tion of a protein-tyrosine kinase(s). Taken together with observations of increased tyrosine, phosphorylation fobwing TCR engagement these studies provide strong evidence that the &ntigen receptors of T and B cells employ similar signaling mechanisms. 19. ..
GOLD MR, MATXJ~JCHI L, KELLY RB, DEFRANCOAL Tyrosine Phosphorylation of Components of the B-ceil Antigen Receptors Following Receptor Crosslinking. Proc Nat1 Acad Sci USA 1991, S&34363440. Emphasizes the potential importance of the Ig-associated chains in signaling; suggests a physical association between kinase(s) and the receptor molecules through which they are activated. 20. .
21.
TAKAGIS, DAIBATAM, Lks~ TJ, H~JMPHREYS RE, PARKERDC, SAIRENJI T: Intracellular Localization of Tyrosine Kinase Substrates Beneath Crosslinked Surface ImmunogIobuIins in B Cells. J Exp Med 1991, 174:381-388.
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23.
PADEHS, LEVITZKI A, GAZITA, MILLSGB, ROIFMAN CM: Activation of PhosphoUpase C in Human B Cells is Dependent on Tyrosine Phosphorylation. J Clin Invest 1991, 87:111&1118.
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NISHBE RHEE
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25.
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HEMPEL WM,
and effector
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YAMANA~H~Y, KAKIUCHI T, MIZUGUCHI J, YAMAMOTO T, TOYOSHIMAK: Association of B Ceil Antigen Receptor with Protein Tyrosine Kinase Lyn. Science 1991, 251:192-194. First demonstration of a physical association between a src~type kinase and the Ig complex. 27. .
28. ..
BUFXHARDTAL, BRUNSWICKM, BOLEN JE, MOND JJ: Anti-immunoglobulin Stimulation of B Lymphocytes Activates srcrelated Protein-tyrosine Kinases. Proc Natl Acad Sci lrSA 1991, 88:741@7414. Coprecipitation of three different src kinases with the Ig complex; first demonstration that activity of specific kinases increases upon receptor crosslinking. 29. .
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30.
YAMANA.SHI Y, FUKUI Y, WONGSASANTB, KINOSHITAY, ICHIM~RI Y, TOYOSHIMAK, YAMAMOTOT: Activation of Src-Iike Proteintyrosine Kinase Lyn and its Association with Phosphatidylinositol 34dnase Upon B-cell Antigen Receptor-mediated Signaling. Proc Natl Acad Sci USA 1992, 89:11181122.
31. .
TANIGUCHI‘IT, KOBAYA~HIT, KONDO J, TAKAHAS to K, NAKAMURA H, SUZUKIJ, NAGAI K, YAMADAT, NAKAMURAS, YAMAMURA H:
Structure and Developmental Regulation of the B-lymphoid Tyrosine Kinase Gene blk. J Biol Cbem 1992, 267:4815-4823. Molecular and histochemical analysis of blk kinase expression in B-cell development and in lymphoid tissues showing coexpression of blk and Ig associated chains during B-cell ontogeny.
Molecular Cloning of a Porcine Gene syk That Encodes a 72-kDa Protein-tyrosine Kinase Showing High Susceptibility to Proteolysis. J Biol Cbem 1991, 266:1579&15796. Describes a protein-tyrosine kinase of unusual structure, syk, encoded by a gene that is expressed preferentially in lymphoid tissues. HUTCHCROFTJE, HAP&SON ML, GEAHLEN RL: B Lymphocyte Activation is Accompanied by Phosphorylation of a 72-kDa Protein-tyrosine Kinase. J Biol Cbem 1991, 266:1484614849. Demonstration that the syk kinase, or a closely related protein, is a major substrate for tyrosine phosphorylation following Ig crosslinking.
32. .
33.
HAFZVETT MM, Kt~us GGB: G Protein Coupling of Antigen Receptor-stimulated Polyphosphoinositide Hydrolysis in B Cells. J Immund 1988, 140:31353139. GRAUDEI L, RLABOWOL K, BAR-SAGID: Co-capping
of ras Proteins with Surface Immunoglobtdins in B Lymphocytes. Natwe 1990, 347:39&400. A provocative paper that establishes co-localization of sIg and p2lra upon antigen receptor engagement. 34. .
WILKIE TM, SCHERLEPA, STRATHMANNMP, SIEPAK VZ, SIMON MI: Characterization of G-protein a Subunits in the Gq Class: Expression in Murine Tissues and in Stromal and Hematopoietic Cell Lines. Proc Natl Acad Sci Cl S A 1991, 88:1004910053. A description of pertussis-toxin-insensitive phospholipase-stimulatory G-proteins that are expressed preferentiaky in hematopoietic cells. 35. .
SV Desiderio, Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
