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* Division of Immunology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
Department of Immunology and Rheumatology, Merck Research Laboratories, Rahway, NJ 07065;
Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe, Japan;
National Institute for Basic Biology, Aichi, Japan; and
¶ Amgen, Thousand Oaks, CA 91320
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphocyte differentiation is a series of finely regulated processes whereby the coordinate regulation of cell proliferation, differentiation, and death directs the development of functional cells. Through these processes, lymphocytes reactive against self-Ags are eliminated from the developing repertoire, and sufficient numbers of functional lymphocytes are produced to guarantee the prompt and effective elaboration of immune responses. Cell-to-cell contact and soluble growth factors play important roles in the regulation of developmental processes. Self-Ags presented on stromal cells trigger Ag receptors, signals from which help to determine the fate of lymphoid precursors (7). Various growth factors, such as stem cell factor (SCF) and IL-7, assist in regulating lymphoid precursor expansion by binding to c-Kit and the IL-7R, respectively. (8)
Binding of extracellular ligands to these polypeptide receptors initiates a cascade of events through the activation of intracellular protein kinases (9, 10). The phosphorylation events catalyzed by these kinases both modulate the catalytic activity of effector enzymes and mediate protein-protein interactions that juxtapose critical signal transduction elements. Although the details of how signaling molecules are activated or recruited to receptors remain incompletely elucidated, studies in recent years have defined an array of adaptor proteins that integrate and regulate multiple signaling events (11, 12, 13). Adaptor proteins lack kinase, phosphatase, or transcriptional domains, and instead consist of multiple binding sites mediating protein-protein or protein-lipid interactions, such as Src homology (SH) 2, SH3, or pleckstrin homology (PH) domains.
Lnk is an adaptor protein expressed mainly in lymphocytes (14, 15). Together with adaptor molecule containing PH and SH2 domains (APS) and SH2-B, Lnk is part of an adaptor protein family, whose members share the presence of a homologous N-terminal domain with putative proline-rich protein interaction motifs, followed by PH and SH2 domains, and a conserved C-terminal tyrosine phosphorylation site (16, 17, 18). SH2 domains of the Lnk family proteins, whose binding specificity remains unknown, are approximately the same size and share over 90% similarity. Lnk regulates B cell production by negatively controlling pro-B cell expansion. Mutant mice lacking the lnk gene show enhanced B cell production (16, 19). This B cell overproduction is due to the hypersensitivity of B cell precursors to SCF, a c-Kit ligand (16). The absence of Lnk confers upon immature BM cells an enhanced ability to support B lymphopoiesis in adoptively transferred host animals, even in a competitive environment, such as the nonirradiated RAG2-/- host (16). In addition, the numbers of hemopoietic progenitors in the bone marrow increase in lnk-deficient mice (20). Competitive repopulation assays in irradiated host animals demonstrate that the ability of hemopoietic progenitors to generate various lineages of hemopoietic cells is greatly enhanced by the absence of Lnk.
In this study, we used a transgenic approach to define critical aspects of Lnk function in more detail. Lymphocyte production was impaired in a dose-dependent manner upon overexpression of Lnk in lymphoid cells. In addition to its importance in lymphopoiesis at the early developmental stages, Lnk also plays a role in peripheral maturing B cells. In transgenic mouse spleens, skewed B cell subpopulations and abnormalities in B cell morphology and cell cycle status were observed. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were bred and maintained at the animal facility of the Institute of Medical Science (University of Tokyo, Tokyo, Japan) under specific pathogen-free conditions. The NcoI-EcoRI cDNA fragment encompassing the entire coding region of the mouse Lnk cDNA was subcloned into the BamHI site of pcDNA3 (Stratagene, La Jolla, CA), a eukaryotic expression vector driven by the CMV enhancer and promoter, resulting in pcDNA3-Lnk as previously described (20). The BamHI fragment containing the Lnk cDNA was subcloned into the BamHI cloning site of the p1026x vector that consists of the murine lck proximal promoter, Ig intronic H chain enhancer Eµ, and a human growth hormone (hGH) gene cassette (21). A substitutional mutation at the C-terminal tyrosine residue to phenylalanine (Y536F) was introduced into the Lnk cDNA by PCR-based site-directed mutagenesis, and confirmed by DNA sequencing (20). The resulting mutated cDNA was also inserted into the p1026x vector. The lnk transgenes, purified as NotI fragments, were injected into C57BL/6J mouse zygote pronuclei as previously described (22). Transgenic founders were detected by hybridization of genomic tail DNA with a hGH probe or PCR, and stable mouse lines were generated by backcrossing founders with C57BL/6J mice.
Western blotting
Single cell suspensions were prepared from lymphoid organs of 6- to 8-wk-old mice. Cells were lysed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 2 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin), and the lysates were clarified by centrifugation. Total lysates derived from 4 x 106, 2 x 106, or 1 x 106 thymocytes were separated on 8% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were probed with anti-Lnk-C-terminal Abs (15). Bound Abs were detected using HRP-conjugated secondary Abs via chemiluminescence.
Flow cytometry
Single cell suspensions were prepared from lymphoid organs of 6-
to 8-wk-old mice, and cells were stained using predetermined optimal
concentrations of the respective Abs. The stained cells were then
analyzed on a FACScan or FACSCalibur instrument (BD Biosciences, San
Jose, CA). The following mAbs were used: PE-conjugated anti-CD43
(S7), biotin-conjugated anti-BP-1/Ly-51, FITC-coupled anti-heat
stable Ag (anti-HSA, J11d), FITC-anti-CD8 (53-6.7), FITC- or
PE-anti-CD4 (RM4-5), biotin-anti-CD25 (PC61), PE-anti-CD44
(IM7), biotin-anti-CD23 (B3B4), FITC-anti-CD21/CD35 (7G6), PE-
or biotin-anti-CD3
(145-2C11), biotin-anti-TER-119,
biotin-anti-Gr-1 (RB6-8C5), (all purchased from BD PharMingen, San
Diego, CA); FITC-, PE-, or biotin-anti-B220 (RA3-6B2),
PE-anti-IgM F(ab')2, FITC-anti-Mac-1
(M1/70), (obtained from Caltag Laboratories, Burlingame, CA), and
biotin-anti-IgD (CS15, a gift from Dr. K. Miyake, University of
Tokyo). PE-streptavidin (Ancell, Bayport, MN), Tri-color-conjugated
streptavidin (Caltag Laboratories) or allophycocyanin-conjugated
streptavidin (BD PharMingen) were used for biotin-coupled Ab staining.
In some staining, 2 µg/ml 7-amino-actinomycin D (Sigma-Aldrich) were
used to gate out dead cells. For DNA staining, splenocytes were stained
with FITC-anti-CD21/CD35 and PE-anti-IgM
F(ab')2 and
CD21-IgM+ T1 or
CD21highIgMhigh T2 and MZ B
cells were purified using FACSVantage (BD Biosciences). Cells were then
fixed in ethanol and stained in 20 µg/ml propidium iodide, 0.5 mg/ml
RNase H, and 0.2% Tween 20 at room temperature for 60 min. Stained
cells were then analyzed on a FACSCalibur instrument.
Serology
Serum concentrations of each Ig isotype were determined by isotype-specific ELISA as described previously (23). To examine the Ab production against TI-2 Ags, mice were i.p. injected with 100 µg of trinitrophenyl (TNP)-Ficoll in saline and were bled 10 days after the injection. To examine the response against TD Ags, mice were immunized i.p. with 100 µg of keyhole limpet hemocyanin (KLH) in a 1:1 homogenate with CFA (Difco, Detroit, MI), and were bled on day 12. Serum serial dilutions were analyzed for TNP- or KLH-specific Ig isotypes by ELISA using dinitrophenyl-coupled BSA (cross-reacts with anti-TNP Abs) or KLH as the capture reagent.
Proliferation assay
Splenic B cells were purified using a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) after incubation with biotin-conjugated anti-CD43 and streptavidin-coupled microbeads. Resulting purified B cells (1 x 105) were cultivated in 200 µl of medium in 96-well plates. Cells were stimulated with various concentrations of anti-IgM F(ab')2 (The Jackson Laboratory, Bar Harbor, ME), anti-CD40 mAb (BD PharMingen), IL-4 (PeproTech, London, U.K.), or LPS. Cells were pulse-labeled with [3H]thymidine (0.2 µCi per well) during the last 16 h of the 72-h culture period, and incorporated [3H]thymidine was measured using a Matrix 96 direct beta counter (Packard Instrument, Meriden, CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In mice lacking the lnk gene, an accumulation of B lineage cells caused by overproduction of pro-B cells was observed (16). To characterize further the importance of Lnk, and to reveal its roles in lymphopoiesis and lymphocyte function, we generated transgenic mice that overexpress Lnk under the control of the lck proximal promoter in combination with the Eµ enhancer (Fig. 1A). The promoter drives expression of the inserted cDNA in T and B lineage cells from their early developmental stages (21). Seven lines of transgenic mice were obtained from independent founders, and five lines that overexpress Lnk at various levels were further analyzed. Lnk protein expression levels in thymocytes from each transgenic line were measured by immunoblotting a 2-fold serial dilution of the lysates using anti-Lnk Ab in combination with densitometric quantification (Fig. 1B). Transgene expression in peripheral T or B cells was well correlated with that in thymocytes in low or medium expressers (data not shown). Severe reduction of peripheral B cells and altered distribution of mature B cell fractions in a high expressing line (see below) made it difficult to directly measure the Lnk protein expression in B cells. In contrast, T cell development assessed on the basis of CD4 and CD8 expression was unaffected in the transgenic lines (see below). Thus, expression levels in the thymus were used to compare the Lnk levels in each transgenic line. The Lnk no. 4 line expressed Lnk at the highest level, 23-fold over endogenous Lnk protein levels in normal C57BL/6 thymocytes. The Lnk no. 99 line expressed Lnk at the lowest level, 2.5-fold greater than the endogenous level.
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B cell development in each transgenic mouse line was analyzed. The highest expresser, Lnk no. 4, showed severe reduction of B lineage cells as shown in Fig. 2. The BM contained very few B220+ cells, many of which were CD43+ pro-B cells. Most of the pro-B cells were HSA- and BP-1- (Hardy et al.’s fraction A; Ref. 1), indicating that B cell development was blocked at a very early stage. Other transgenic lines showed mild reduction of B lineage cells that correlated in a dose-dependent manner with the Lnk expression level (Fig. 3). Interestingly, the correlation between reduction of B lineage cells and Lnk expression levels was not a linear correlation. Instead, the reduction correlated with the logarithmic value of Lnk expression levels. The reduction of pre-B and immature B cells was more severe than that of pro-B cells. This may suggest the existence of Lnk-dependent regulation in the transition from pro-B to pre-B cell stages, in addition to the known role for Lnk in pro-B cell expansion (16). However, our pro-B cell discrimination based on the method of Hardy and colleagues (24) contains non-B lineage cells, especially in Fraction A (B220+CD43+HSA-BP-1-). Thus, an alternative possibility could be that those non-B lineage cells in the pro-B cell fraction not expressing the transgene were maintained at relatively normal levels, leading to an underestimation of the pro-B cell reduction. As expected, no cell number reduction was observed in myeloid cells, which should not express the transgenes.
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We next examined consequences of Lnk overexpression in T lineage cell development. Total thymocyte numbers declined as Lnk expression in thymocytes increased (Fig. 3). However, thymocyte development, evaluated on the basis of CD4 and CD8 expression, was grossly normal (Fig. 2), except for a slight reduction of CD4+CD8- and CD4-CD8+ mature thymocytes. The reduction in number was slightly more severe in CD4-CD8+ thymocytes than that of CD4+CD8- mature thymocytes in Lnk transgenic lines. Within the CD4-CD8- immature thymocyte compartment, a proportion of CD44-CD25+ or CD44-CD25- cells was significantly reduced in transgenic mice compared with normal littermates (Fig. 2). This led to the overrepresentation of CD44+CD25+ cells indicating inefficient transition of CD44+CD25+ to CD44-CD25+ cells or impaired expansion of CD44-CD25+ cells.
Consequence of Lnk overexpression in peripheral lymphocytes
The total cell number in the spleen was also reduced as more Lnk transgenes were expressed. Only a residual number of B cells remained in the spleen of the highest expresser, Lnk no. 4 (Fig. 2). T cell numbers also declined as Lnk expression increased, although B cell reduction was more prominent than that of T cells (Fig. 3). We then examined splenic B cell subfractions by staining for CD21, CD23, and IgM. This analysis demonstrated that the CD21+CD23-IgMhigh MZ B cells were relatively maintained in most transgenic lines, except in the most potent expresser, Lnk no. 4. In contrast, all other peripheral B cell fractions in spleen, transitional T1 and T2 cells, and FO B cells decreased in a dose-dependent manner (Fig. 3). CD23-IgMhighCD5+ B1a and CD23-IgMhighCD5- B1b cells in the peritoneal cavity were also relatively maintained in most transgenic lines, except in Lnk no. 4, whereas B2 cells and T cells in the cavity were reduced (Fig. 3 and data not shown).
Next, we examined whether Lnk overexpression affects the function of
peripheral mature B cells. Lnk no. 98 moderately expresses Lnk protein
(13-fold greater expression than endogenous Lnk in thymocytes) and was
able to produce substantial numbers of splenic lymphocytes (
40% of
normal C57BL/6 control mice). Histological and immunohistochemical
analysis demonstrated that splenic architecture was grossly maintained
in these transgenic mice, although the white pulp regions were reduced
(data not shown). However,
CD21+CD23-IgMhigh
MZ B cells occupied a major compartment of splenic B cell populations
(Fig. 4A). CD23 expression
levels on CD23+IgMlow FO B
cells in transgenic mice were slightly higher than those observed on
normal FO B cells.
CD21-CD23-IgM+
T1 B cells showed slightly decreased IgM levels. In addition, T1, as
well as
CD21highCD23+IgMhigh
T2, cells in transgenic mice were larger in size. In contrast, cells
from other B lineage fractions, pro-B and pre-B cells, immature B cells
in the BM, and FO and MZ B cells in the spleen were all similar in size
to those of normal C57BL/6 mice (Fig. 4B). Interestingly,
larger transgenic T1 cells were not actively proliferating (Fig. 4C). The cycling fraction of T1 B cells was severely reduced
as assessed via DNA content analysis. In addition, expression levels of
B cell activation markers, such as MHC class II and CD86 or CD25, on
T1 cells were unchanged (data not shown). Thus, Lnk
overexpression in peripheral splenic B cells compromised B cell
maturation and proliferation, especially in the T1 fraction.
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Negative regulation of lymphocyte development by Lnk is independent of its phosphorylation
Lnk contains a tyrosine phosphorylation motif involving Y536 at its C terminus that is conserved among all Lnk family adaptor proteins, including APS, SH2-B, and the Lnk-like protein in Drosophila. Y536 is the major site phosphorylated by c-Kit (20) and by various tyrosine kinases when simultaneously overexpressed in COS7 cells (S. Takaki, unpublished observation). We examined a potential role for the conserved tyrosine phosphorylation motif in the negative regulatory functions of Lnk in lymphocyte development. Y536 was substituted with a phenylalanine residue and the resulting mutant form of Lnk (Y536F) was inserted downstream of the lck proximal promoter and the Eµ enhancer, and expressed in lymphoid cells in transgenic mice. The Y536F transgenic mice expressed comparable amounts of Lnk protein with the mice expressing the wild-type Lnk, Lnk no. 4 (Fig. 5B). As in the case of wild-type Lnk overexpression, B cell development in Y536F transgenic mice was also inhibited at the pro-B cell stage. The BM contained very few B220+ cells, which were mostly CD43+ pro-B cells, similar to the BM of Lnk no. 4 transgenic mice (Figs. 5 and 1). The numbers of thymocytes and B1 cells in the peritoneal cavity were also decreased in the Y536F transgenic mice (data not shown). These results indicate that the Lnk C-terminal tyrosine phosphorylation site is dispensable for the negative regulatory effects of overexpressed Lnk in lymphocyte development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results document a potent regulatory mechanism in lymphopoiesis mediated by the adaptor protein, Lnk. Lnk overexpression in lymphoid precursors resulted in both B and T cell reduction. Expansion of pro-B cells in bone marrow, and of pro-T cells in thymus was impaired as Lnk expression increased. We previously reported that mutant mice lacking the lnk gene showed enhanced B cell production due to the hypersensitivity of B cell precursors to SCF, a c-Kit ligand. However, the lnk-/- mice do not show any abnormality in T cell development or altered thymocyte expansion as seen in lnk-transgenic mice (16). B lineage cells express more Lnk than T lineage cells (15). Thus, different levels of endogenous Lnk expression in B lineage cells from those found in T lineage cells probably account for this discrepancy. In other words, Lnk could function in both B and T precursor cells, however, B precursors that express higher amounts of Lnk are more stringently controlled by Lnk-mediated regulatory signals. The physiological importance of this more stringent restriction of conventional B cell production by Lnk remains to be elucidated. In the C57BL/6 background, loss of the lnk gene alone does not cause autoimmunity, malignant transformation of B lineage cells, nor exhaustive loss of hemopoietic progenitor cells. Although it is possible that other Lnk family proteins might compensate for some Lnk function in lnk-deficient T cells, T lineage cells do not express APS (25). Moreover, despite the homology between Lnk and SH2-B, many studies have demonstrated positive effects of SH2-B on cell growth and differentiation (26, 27, 28, 29, 30, 31, 32), while Lnk functions as a negative regulator of lymphopoiesis as clearly shown in this study and in the analysis of the lnk-deficient mice. We recently reported that male and female gonad maturation is impaired in SH2-B-/- mice, while both B and T cell development are normal (33). Thus, in lnk-/- T lineage cells, compensation by other Lnk-family members does not appear likely. The generation of lnk, SH2-B double-deficient mice will help to elucidate the special regulatory characteristics of thymic progenitors.
Both B and T cell numbers were reduced as the amount of Lnk exponentially increased. This contrasts with that of the perturbed lymphocyte development by the increasing activity of many effector enzymes, such as p56lck, by overexpression (34) or by the decreasing activity of p56lck, Erk1, and ras by their dominant negative mutants (21, 35, 36). All these examples show a linear correlation between phenotype and protein expression levels. SH2-B forms a pentameric complex through homotypic association via its N-terminal domain (30). Because the N-terminal domains of Lnk-family adaptors are conserved, Lnk may also manifest its function as a multimeric complex. In transfected COS7 cells, Lnk indeed exists as a multimer (S. M. Kwon and S. Takaki, unpublished observation). Lnk multimerization could account for the semilogarithmic relation between lymphocyte reduction and Lnk expression levels. The stoichiometries between Lnk complexes, membrane receptors, and signaling molecules may also contribute to this unique correlation between phenotype and Lnk expression levels.
Lnk function in peripheral lymphocytes
Many unrelated transgenic and gene-targeted models show an enlarged splenic MZ compartment (3). In several cases, the enlarged MZ phenotype is linked to compromised peripheral B cell generation. For example, IL-7-/- (37) and conditional Rag-knockout mice (38) show reduced B lymphopoiesis at precursor levels and develop a larger MZ B subset. The peripheral B cells in these mice with impaired B lymphopoiesis show an activated phenotype: increased expression of CD25, class II, and CD86, and augmented entrance into the cell cycle (37, 38). These properties could result from compensatory mechanisms which allow enhanced generation of the mature B cell compartment. The peripheral phenotype observed in our lnk-transgenic mice was similar to that observed in IL-7-/- or conditional Rag-knockout mice in terms of increased MZ B cells accompanied by reduced B lymphopoiesis, but not fully consistent with the phenotype due to limited BM B lymphopoiesis. Immature splenic T1 and T2 B cells in lnk-transgenic mice were enlarged, but were not entered in the G2/M phase of the cell cycle. These results imply that Lnk also constrains the proliferating ability of peripheral B cells, as well as BM B cell precursors. Apart from FO or MZ B cells, most B cells in IL-7-/- and conditional Rag-knockout mice are enlarged, express activation markers, and show an increased proportion of proliferating cells (37, 38). In contrast, in lnk-transgenic mice, enlarged cells were only seen in newly generated T1 and T2 cell compartments. Hence, maturation or compensatory proliferation of peripheral B cells might be perturbed by Lnk overload.
Another possibility is that the phenotype observed in peripheral B
cells could be a consequence of unusual selection of B cells that are
relatively resistant to Lnk overexpression. Cells which have an
advantage in maturation or proliferation might selectively develop in
the BM and expand into the periphery in lnk-transgenic mice,
where they could preferentially differentiate into the B1 and MZ B cell
compartments. As shown in recent gene disruption studies, signaling
components of B cell receptors, such as tyrosine kinases and
coreceptors, survival factors like B cell activating factor from the
TNF family/B cell activating factor from the TNF family receptor,
components controlling cell migration including protein tyrosin kinase
2 and Lsc (Lbc’s second cousin, the murine homolog of human p115 Rho
GEF), and transcription factors like, those of the NF-
B
family, Aiolos, and recombination signal binding protein-J are
all critical for MZ B cell development (3, 39). Cells that
could expand carrying high amounts of Lnk might preferentially
differentiate in B1 and MZ B cell compartments as a result of altered
expression of molecules critical for MZ B cell development.
How does Lnk control lymphocyte proliferation?
Using a c-Kit+ mast cell line, we previously demonstrated that Lnk is tyrosine-phosphorylated by c-Kit and interacts with phosphorylated c-Kit (20). Lnk specifically inhibited c-Kit-mediated signaling for cell growth by attenuating Gab2 (a family member of Gab1, Grb2-associated binder 1) phosphorylation and the subsequent activation of the mitogen-activated protein kinase pathway (20). However, previous studies demonstrated that a c-Kit signal is indispensable for T precursor expansion, whereas B cell generation occurs in the absence of c-Kit (40). Although the impaired expansion of CD4-CD8- double-negative T precursors in lnk-transgenic mice could result from an inhibitory effect of overexpressed Lnk on c-Kit signaling, the observed impairment of B cell production strongly indicates that c-Kit is not the sole target for Lnk. Consistent with this idea, the enhanced hemopoietic ability by lnk-deficient hemopoietic precursors was not significantly normalized by attenuating c-Kit signals with the introduction of the heterozygous W mutation in the c-Kit locus (20). Flt3/Flk-2 may be involved in such a pathway because it has been shown to support proliferation and differentiation of hemopoietic progenitor cells, while disruption of Flt3/Flk-2 perturbed the production of various blood cell lineages (41). W/Wvflk2-/- mice show impaired hemopoiesis with severe lymphocytopenia. However, injection of anti-Flt3/Flk-2 Abs into adult mice together with anti-c-Kit Abs does not inhibit B lymphopoiesis, while the treatment severely inhibits erythropoiesis and myelopoiesis (42). Injection of anti-Flt3/Flk-2 Abs into lnk-/- mice failed to normalize the B cell overproduction (S. Takaki, unpublished observation). These observations suggest that Flt3/Flk-2 signaling is not likely to be a target affected by Lnk overexpression. Further studies will be required to identify the molecular targets of Lnk and to understand how Lnk regulates the expansion of hemopoietic and lymphoid precursor cells in vivo.
It has been shown that Lnk associates with an actin binding protein, ABP-280 (43). It has also been shown that SH2-B, a member of the Lnk family adaptor proteins, is required for growth hormone induced actin reorganization and regulates cell motility (44, 45). These data suggest that Lnk could associate with an actin-containing complex and may control the actin cytoskeleton during cell division or migration. The relatively maintained splenic architecture in lnk-transgenic mice suggests that B cell migration during maturation was unperturbed. However, enlargement of T1 and T2 B cells in transgenic mice might reflect Lnk function in controlling the actin cytoskeleton. In line with the reduction in the fraction of cycling B cells, Lnk might regulate expansion of precursor and mature B cells by preventing both entry into the cell cycle and cell division, in part via effects on cytoskeletal remodeling involving actin.
A mutant form of Lnk lacking the C-terminal tyrosine residue conserved among Lnk family proteins still efficiently inhibited lymphopoiesis in transgenic mice. This is consistent with observations obtained from transfection experiments using the c-Kit+ mast cell line MC9 (20). Although Y536 of Lnk was the main c-Kit target phosphorylation site, SCF-dependent growth of MC9 cells was inhibited by Y536F as well as by wild-type Lnk. In contrast, APS inhibits Janus kinase 2- or platelet-derived growth factor receptor-mediated signaling in combination with c-Cbl, and the phosphorylation of the C-terminal tyrosine is essential for c-Cbl-binding and subsequent APS inhibitory effects (46, 47). This suggests that the Lnk inhibitory function on c-Kit and as yet unidentified signaling cascade(s) is accomplished by Lnk subdomains other than the C-terminal tyrosine and may involve a mechanism unique to Lnk. In contrast to the negative regulatory role of Lnk in lymphopoiesis, positive regulatory roles for SH2-B and APS in signaling via receptors for various cytokines and growth factors have been reported (26, 27, 28, 29, 30, 31, 32). Thus, despite the significant structural similarities between Lnk, APS, and SH2-B, their functions appear to be quite different from each other.
In summary, we used transgenic mice overproducing Lnk to demonstrate that this adaptor protein is a critical regulator of lymphocyte production. Expansion of lymphoid precursors was severely impaired in lnk-transgenic mice. The skewed peripheral B cell subpopulations, enlarged size of transitional T1 B cells, reduced cycling splenic B cells, and altered humoral immune responses in lnk-transgenic mice suggest a potential function for Lnk in peripheral lymphocytes. Thus our results precisely complement studies of lnk-deficient mice. In addition, they suggest that Lnk may perform previously undescribed functions in peripheral B cells and during T cell development. Taken together, these genetic studies in mice illuminate the novel negative regulatory mechanism by the Lnk adaptor protein in controlling lymphocyte production and function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Satoshi Takaki, Division of Immunology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address: takakis{at}ims.u-tokyo.ac.jp
3 Current address: Genomics Institute, Novartis Research Foundation, San Diego, CA 92121.
4 Abbreviations used in this paper: BM, bone marrow; MZ, marginal zone; FO, follicular; TD, thymus-dependent; SCF, stem cell factor; SH, Src homology; PH, pleckstrin homology; KLH, keyhole limpet hemocyanin; TNP, trinitrophenyl; APS, adaptor molecule containing PH and SH2 domains; hGH, human growth hormone; HSA, heat stable Ag.
Received for publication October 1, 2002. Accepted for publication November 7, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1, Grb2, and phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA 92:11618.-dependent pathway plays a critical role in the pathogenesis of murine immunodeficiency syndrome induced by LP-BM5 murine leukemia virus. Int. Immunol. 6:1937.
as a potent cytoplasmic activator of the tyrosine kinase Janus kinase 2. Proc. Natl. Acad. Sci. USA 96:7172. is a Rac-binding protein that regulates cell motility. J. Biol. Chem. 277:10669.This article has been cited by other articles:
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  C. Simon, E. Dondi, A. Chaix, P. de Sepulveda, T. J. Kubiseski, N. Varin-Blank, and L. Velazquez Lnk adaptor protein down-regulates specific Kit-induced signaling pathways in primary mast cells Blood, November 15, 2008; 112(10): 4039 - 4047. [Abstract] [Full Text] [PDF]
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S. Gery, S. Gueller, K. Chumakova, N. Kawamata, L. Liu, and H. P. Koeffler Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders Blood, November 1, 2007; 110(9): 3360 - 3364. [Abstract] [Full Text] [PDF]
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W. Tong, J. Zhang, and H. F. Lodish Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways Blood, June 15, 2005; 105(12): 4604 - 4612. [Abstract] [Full Text] [PDF]
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