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Department of Cell Signaling, DNAX Research Institute, Palo Alto, CA 94304
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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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Among the first steps in BCR signaling is the recruitment of the
protein tyrosine kinases Lyn and Syk to the receptor complex in a
phosphorylation-dependent manner (2, 3). B cells from
Syk-/- mice accumulate in
the late pro-B stage, suggesting an important function for Syk in B
cell development (4, 5). Phosphorylation targets
identified for Syk include the adapter molecule BLNK/SLP-65 (6, 7) and phospholipase C-
(PLC-) (8).
Phosphorylation of BLNK/SLP-65 is required for membrane localization of
PLC-, NCK, and Vav. BLNK/SLP-65 also binds the GRB2-SOS complex, and
thus contributes to the activation of the Ras/mitogen-activated
protein-kinase cascade (6, 7). In contrast to Syk, Lyn
serves both positive and negative functions during BCR signaling. Lyn
phosphorylates and activates the pleckstrin homology (PH)
domain-containing tyrosine kinase Btk. Btk and Syk cooperate to
activate PLC-, which is required for the BCR-stimulated
Ca2+ response (9, 10, 11). Lyn mediates
its negative regulatory function by phosphorylating the BCR coreceptor
CD22. Once phosphorylated, CD22 recruits and activates the protein
tyrosine phosphatase SHP-1, which leads to down-regulation of BCR
signaling (12, 13, 14, 15). Another important component of the BCR
signaling cascade is the coreceptor CD19. CD19 is tyrosine
phosphorylated in response to BCR ligation and recruits Vav and
phosphatidylinositol 3-kinase (PI 3-kinase) (13, 16, 17).
While effects of mitogen-activated protein kinase activation and calcium mobilization have been well studied in B cells (reviewed in Ref. 3), the consequences of PI 3-kinase activation have only recently been addressed. Mice that lack the regulatory p85 subunit of PI 3-kinase exhibit profound defects in B cell function, including decreased proliferative responses and impaired B cell survival (18, 19). PI 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3) (reviewed in Ref. 20). The PH domains of several signaling molecules have been demonstrated to specifically bind PIP3, resulting in the recruitment of the respective proteins to the plasma membrane. In B cells, the PH domain-containing kinases Btk and Akt (also termed protein kinase B) are activated in a PI 3-kinase-dependent manner (2, 21, 22). While Btk is found to play a role in the sustained calcium response, a function for the serine/threonine kinase Akt in B cells has not yet been established in BCR signaling.
Akt is the cellular homologue of the product of v-Akt, the
oncogene of the acutely transforming rodent retrovirus Akt-8 that
causes T cell leukemias and lymphomas in mice (23, 24). To
date, three closely related isoforms of Akt (Akt1, Akt2, and Akt3) have
been identified in mammals (4, 25, 26, 27, 28, 29). All three isoforms
are widely expressed and are regulated by the levels of PIP3 in the
plasma membrane. Upon activation in response to various growth factors,
such as platelet-derived growth factor, epidermal growth factor,
insulin, and insulin-like growth factor-1, Akt generates a survival
signal in nonhemopoietic cells (29, 30, 31). Consistent with
its survival function, overexpression of Akt can lead to cell
transformation (30, 32). Recently, the tumor suppressor
MMAC1 (also termed PTEN and TEP-1) has been identified as an important
negative regulator of Akt (33). MMAC1 is a phospholipid
phosphatase and catalyzes the dephosphorylation of the D3 position in
PIP3, thereby antagonizing PI 3-kinase. Interestingly, heterozygous
MMAC1 knockout mice develop spontaneous tumors with a high frequency of
lymphomas (19, 34, 35), suggesting an important role for
MMAC1 in lymphocyte biology. While it is clear that PH
domain-containing signaling molecules such as Btk and PLC- are
regulated by PIP3 levels and important for B cell function, a role for
Akt in lymphocyte activation has not been addressed.
In this study, we asked whether Akt plays a role in the regulation of B cell survival and how the activity of Akt is regulated. Using the B cell lymphoma line DT40 as a model system, we found that overexpression of Akt in these cells blocked receptor-induced apoptosis, indicating that Akt does provide a survival signal. Consistent with a requirement for PI 3-kinase in Akt activation, inhibition of PI 3-kinase abolished BCR-induced Akt activation and resulted in enhanced apoptosis. Similarly, exogenous expression of MMAC1 led to enhanced apoptosis and inhibition of Akt. Furthermore, our data suggest antagonistic roles for Syk and Lyn kinases in regulating Akt activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DT40 cell lines carrying targeted deletions in btk,
lyn, syk, or plc- have been
described previously (8, 9). All DT40 cell lines were
maintained in RPMI 1640 supplemented with 10% FCS, 1% chicken serum
(Sigma, St. Louis, MO), penicillin, streptomycin, glutamine, and 50
µM mercaptoethanol. The chicken cell lines SL29 and Gd1T were
optained from American Type Culture Collection (Manassas, VA) and
cultured as recommended. U373 cells and U373 cells ectopically
expressing MMAC (36) were grown in DMEM, supplemented with
10% FCS, penicillin, and streptomycin. Anti-chicken µ mAb M4 was
described previously (9). Anti-Akt and
anti-phosphoserine Akt were purchased from New England Biolabs
(Beverly, MA). Anti-phosphotyrosine Ab 4G10 was from Upstate
Biotechnology (Lake Placid, NY). Anti-MMAC1 Ab BL72 was described
previously (36). PI 3-kinase inhibitors wortmannin and
LY294002 were from Calbiochem (La Jolla, CA). Histone H2B and insulin
were from Sigma.
Cloning and transfections
Avian Akt was cloned from a DT40 cDNA library (prepared by T. McClanahan, DNAX, Palo Alto, CA) by PCR using primers based on the chicken Akt1 cDNA sequence (GenBank accession AF039943). The human MMAC1 cDNA and the catalytically inactive MMAC1 mutant MMAC1(C/S) were described previously (36, 37). Both avian Akt and human MMAC1 were subcloned into the pApuroII vector, and cells were stably transfected by electroporation (9).
Biochemical analysis
Cells were washed twice in room temperature PBS, resuspended in
serum-free RPMI medium, and then incubated at 37°C for 2 h. For
inhibition of PI 3-kinase, cells were pretreated for 15 min with
inhibitor before stimulation with M4 or insulin. A total of 2–4
x 107 cells was lysed in 1% Nonidet P-40, 50 mM
MOPS, pH 7, 150 mM NaCl, 2 mM EDTA, 5 mM sodium orthovanadate, 20
µg/ml aprotinin, 10 µg/ml leupeptin, 5 mM sodium fluoride, and 2 mM
PMSF (lysis buffer) for 15 min on ice. Cell lysates were clarified by
centrifugation at 12,000 x g for 10 min. Abs were
added to clarified lysates, incubated on ice for 45 min, followed by
addition of protein A-agarose beads. Lysates were mixed at 4°C for
1–2 h and washed in lysis buffer. Precipitated proteins were separated
by SDS-PAGE, followed by Western blot analysis. For in vitro Akt kinase
assays, the precipitates were washed three times in washing buffer (25
mM HEPES, pH 7, 1 M NaCl, 0.1% BSA, 10% glycerol, 1% Triton X-100)
and once in kinase buffer (20 mM HEPES (pH 7), 10 mM
MgCl2, 10 mM MnCl2, 0.2 mM
EGTA). After the final wash, beads were resuspended in kinase buffer
containing 1 mM DTT, 5 mM ATP, 10 mCi
[-32P]ATP (Amersham), and 500 ng histone H2B
(Roche Molecular Biochemicals, Burlington, Boehringer Mannheim,
Indianapolis, IN). Reactions were incubated for 30 min at 30°C and
terminated by the addition of SDS sample buffer.
Apoptosis assay
Cells (5 x 105/ml) were incubated with mAb M4 at 2 µg/ml. Flow cytometry analyses of apoptotic cells were conducted after 20 h using the TUNEL in situ cell death detection kit (Roche Molecular Biochemicals). Flow cytometry was performed using a FACScan (Becton Dickinson, San Diego, CA) and analyzed using CellQuest software (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of lymphoid cells to respond to extracellular stimuli with the activation of cell death or survival pathways is an important regulatory mechanism during an immune response. The identification of Akt as the cellular homologue of v-Akt, which induces lymphomas in rats (24), suggests that this kinase might play an important role in lymphoid cell survival. To investigate a possible function of Akt in B cell survival, we chose the chicken DT40 cell system, an established model for the analysis of BCR-mediated signal transduction.
Comparison of the primary structure of Akt kinases from various species
reveals a high degree of sequence conservation. Chicken Akt, for
example, shares 96% sequence identity with human Akt1, thus allowing
use of anti-human Akt reagents for detection. DT40 B cells express
easily detectable levels of Akt when either immunoprecipitated and
blotted with an anti-Akt Ab (Fig. 1
,
left panel) or by blotting whole cell lysate (not shown).
Using a variety of different Akt antisera, we could only detect
expression of one Akt isoform in DT40 cells (data not shown).
Stimulation of DT40 cells through the B cell Ag receptor using an
anti-chicken IgM Ab, M4, led to increased Akt kinase activity after
5 min of stimulation, as measured by phosphorylation of histone H2B in
an in vitro kinase assay (Fig. 1, right panel).
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A, the timing and magnitude
of Akt activation are nearly identical between the two stimuli given
slight differences in protein-loading levels, yet stimulation with M4
led to 63% apoptotic cells, while insulin receptor stimulation did not
induce apoptosis (Fig. 2B). In addition, costimulation of
the insulin receptor and BCR did not augment Akt activation nor enhance
survival (Fig. 2B). Similarly, a 30-min pretreatment with
insulin followed by M4 did not reduce the apoptotic response (not
shown). This suggests that other signaling pathways besides Akt
contribute to the outcome of B cell survival and that these pathways
exist in a balance, which together produce the appropriate B cell
response.
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To investigate the hypothesis that the balance of death and
survival pathways determines cell survival, we artificially altered
this balance to favor either Akt activation or inhibition. To shift the
balance of signals to favor Akt activation, stable clones
overexpressing chicken Akt were compared with parental cells for their
ability to rescue BCR-induced apoptosis. Cells were stimulated with M4
and cultured for 18 h. Apoptotic cells were enumerated using the
TUNEL assay method. As shown in Fig. 3A, high levels of Akt
expression (clones 7 and 9) led to increased cell survival (Fig. 3A). In the lower expressing clone (clone 3), while still
expressing more Akt than parental cells, no effect on apoptosis was
observed, suggesting that activation of Akt is tightly controlled by
factors other than expression levels, and only when levels of Akt are
above a certain threshold can this regulation be overcome. The
inhibition of apoptosis observed with clone 9 was not sensitive to the
PI 3-kinase inhibitor LY294002 (data not shown), suggesting that high
levels of Akt are active independent of PI 3-kinase.
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B). DT40 cells
were transfected with the Akt expression vector (clones 6', 8', and
13') or with empty vector as control (C), and stable lines were
selected as before. Akt clone 6' and the vector control (C) show Akt
expression levels comparable with parental DT40 cells (P). In contrast,
Akt clones 8' and 13' exhibit highly elevated Akt protein levels. In
this experiment, we also analyzed the constitutive phosphorylation of
Akt on S473 as a relative readout for activity. As shown in Fig. 3B (bottom panel), only in clone 13', which shows
the highest level of Akt expression, could we detect constitutive S473
phosphorylation. Consistent with the previous experiment (Fig. 3A), BCR-induced apoptosis is significantly reduced (24% of
the rate seen with parental DT40 cells) in clone 13', but not in clone
8', which shows high levels of Akt, but no constitutive activity (Fig. 3B, bar graph). Inhibition of Akt activation by LY294002 or exogenous expression of MMAC1 enhances apoptosis
Since artificially shifting the balance of BCR-induced signals to favor Akt activation-promoted cell survival, we explored the effect of inhibiting Akt signals. Our initial approach was to inactivate Akt by homologous recombination. While we were successful in inactivating one allele, we were not able to obtain viable clones in which both alleles are inactivated. Given our previous observation that DT40 cells express only one Akt isoform, it is likely that inactivation of both alleles is lethal in these cells. Therefore, we used inhibitors of PI 3-kinase to study the function of Akt in DT40 cells.
Others have shown that chemical inhibition of PI 3-kinase activity
effectively inhibits activation of Akt in DT40 cells (38).
To investigate the effect of Akt inhibition on cell survival, DT40
cells were pretreated with LY294002 compound or vehicle, DMSO,
stimulated with M4, and assayed for Akt activation (Fig. 4A) and cell survival (Fig. 4B). In agreement with previous reports, activation of Akt
is efficiently blocked by LY294002 in DT40 cells and, consistent with
Akt playing a survival role, LY294002 significantly enhanced
BCR-induced apoptosis from 37.4% (M4 alone) to 74.8% (M4 plus
LY294002) (Fig. 4B). Similar results were observed with
wortmannin (data not shown).
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). To analyze MMAC expression in DT40
cells, we performed immunoblots on whole cell lysates using a
polyclonal anti-MMAC antiserum, which was raised against human MMAC
(36). To see whether our antiserum would cross-react with
the chicken protein, we performed database searches and identified an
EST sequence (AI981745) that encodes for chicken MMAC. Conceptual
translation of this sequence gives a peptide with 100% sequence
identity to aa residues 18–148 of human MMAC. To demonstrate
cross-reactivity of our anti-MMAC antiserum with chicken MMAC in
immunoblot, we also analyzed MMAC protein levels in two additional
chicken cell lines (SL29 and Gd1T). MMAC protein was readily detectable
in these two lines as well as in the mammalian control cell lines (see
legend to Fig. 5 for details), but not in DT40 cells and MMAC-deficient
U373 cells.
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B). Comparison of Akt
activation following Ag receptor ligation revealed an almost complete
block in Akt activation in MMAC1 (wt) cells (Fig. 6A),
whereas MMAC1(C/S) appeared to have a slightly enhanced activation.
Consistent with the lack of Akt activation, cells expressing MMAC1 (wt)
were much more sensitive to Ag receptor-induced apoptosis (Fig. 6B) than parental cells, whereas MMAC1(C/S) cells responded
similar to parental cells. Addition of LY294002, which also inhibits
levels of PIP3 in the plasma membrane, enhanced apoptosis in parental
and MMAC1(C/S) cells to levels of MMAC1 (wt) cells (Fig. 6B), suggesting that inhibition of PIP3-mediated Akt
activation by both LY294002 and MMAC1 blocks cell survival.
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Our data clearly implicate Akt as a potent survival factor
activated by BCR ligation. We next investigated the signaling
mechanisms required for activation of Akt. Early signaling events in
BCR activation have been well characterized both biochemically and
genetically through the use of cell lines and genetically altered mice.
Specific requirements for the activation of PI 3-kinase and Akt in B
cells, however, have only recently been addressed. Two recent studies
that analyzed the requirements for Akt activation in DT40 cells
reported contradicting results (21, 39). The study by Li
et al. (21) provided evidence that Syk kinase is a
requirement for Akt activation and, further, that Lyn kinase acts as an
endogenous inhibitor of Akt activation. In contrast, Gold et al.
reported that activation of Akt was completely dependent on Lyn kinase
and that Syk kinase was required for sustained phosphorylation of Akt.
Thus, to definitively identify upstream molecules required for Akt
activation in B cells, we utilized mutant DT40 cell lines previously
established by Kurosaki and coworkers (reviewed in Ref.
3). DT40 lines, which lack expression of Lyn, Syk,
PLC-, and Btk, were analyzed for their ability to activate Akt upon
BCR ligation. Activation of Akt in
Btk-/- and
PLC--/- cells was not
significantly altered compared with parental cells, indicating their
functions are not critical for Akt activation (Fig. 7, panels 1, 2, and
3). Surprisingly, DT40 cells, which lack Lyn tyrosine
kinase, consistently gave an earlier and more robust response compared
with parental cells (Fig. 7, panel 4), suggesting a negative
regulatory role for Lyn in Akt activation. By contrast, cells lacking
Syk tyrosine kinase showed no detectable activation of Akt, arguing
that Syk is an essential molecule in this activation pathway (Fig. 7, lower panels). In the double knockout cell line,
Lyn-/-Syk-/-,
a slight yet reproducible increase in Akt activation was observed,
possibly due to basal PI 3-kinase activity from other receptors. Our
findings are in agreement with Li et al. (21), and support
that Akt activation in response to BCR cross-linking occurs via
activation of PI 3-kinase in a pathway initiated by Syk activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB become integrated inside the cell, leading to a
response, which is largely determined by antigenic history, stage of
development, and microenvironment of the B cell. While multiple
pathways, which become activated following BCR ligation, have been
identified, the contribution of each path to cell fate is unclear. This
study addresses two important questions relating to the role of Akt in
B cell function. First, does activation of Akt following BCR ligation
influence B cell fate? And second, how is activation of Akt regulated
in B cells? As reported by others, Akt is activated following BCR ligation in DT40 cells, an immature B cell line, in A20 cells, a memory B cell line, and in primary B cells (21, 22, 38, 39). The functional consequence of Akt activation in B cells, however, has not been explored. We addressed this question in the DT40 cells. It is quite intriguing that Akt, a well-known survival factor, is activated following BCR ligation in DT40 cells, yet the cell’s response to this stimulus is programmed cell death. Thus, we suggest that both death and survival pathways are activated by BCR cross-linking, and that these pathways are in a fine balance with each other, and, depending on internal and external factors, the balance may be shifted. To test this, we artificially shifted the balance of pathways away from Akt activation either chemically with PI 3-kinase inhibitors, LY294002, or wortmannin (not shown), or by exogenous expression of MMAC1, a lipid phosphatase. BCR-induced apoptosis was significantly enhanced, suggesting that the PI 3-kinase/Akt pathway provides a survival signal to the cell. Furthermore, shifting the balance of pathways to favor Akt activation by increasing the expression levels of Akt led to an almost complete block of receptor-induced apoptosis, further supporting the idea that Akt activation sends a survival signal to B cells following Ag receptor ligation. Interestingly, at this high Akt expression level, the activity of Akt was PI 3-kinase independent, suggesting that localization to the plasma membrane is not necessary for activation in this case. High levels of Akt protein were also necessary to observe constitutive Akt phosphorylation/activation in DT40 transfectants, and it is possible that Akt levels need to exceed a certain threshold level for this constitutive phosphorylation to occur. Only in DT40 cells that showed constitutive Akt phosphorylation was BCR-mediated apoptosis significantly inhibited, suggesting that Akt is directly mediating the antiapoptotic effect in these cells. Consistent with this interpretation are findings of Li et al. (40), who studied the apoptotic effect of MMAC in human tumor cell lines. Interestingly, a constitutively active form of Akt, but not active PI 3-kinase, was able to suppress MMAC-mediated apoptosis in their experiments. Although the mechanism of Akt activation in our overexpressing DT40 clones is at present unclear, it is consistent with the transforming potential of Akt and the fact that it is overexpressed in a number of human tumors.
Interestingly, stimulation of DT40 cells with insulin or M4 resulted in Akt activation with very similar kinetics. Further costimulation with insulin plus M4 or pretreatment of cells with insulin had no significant effect on the apoptotic response or the overall activation of Akt kinase. This could suggest that in DT40 cells the majority of available Akt protein is activated in response to BCR stimulation. Alternatively, activation of an upstream component in the Akt signaling pathway, such as PI 3-kinase, could be a limiting factor in these cells. The data further underscore the notion that BCR ligation results in the activation of a proapoptotic pathway, which overrides the antiapoptotic signal generated by Akt.
Our findings that quantitative differences in Akt activation influence B cell fate may advance our understanding of why stimulation through the B cell Ag receptor can lead to multiple outcomes: anergy, apoptosis, proliferation, or differentiation. The integration of numerous signaling pathways influenced by both extrinsic factors such as microenvironment and context of Ag, and intrinsic factors such as levels of accessory molecules and coreceptors, leads to the appropriate response from the cell. We would predict that activation of the PI 3-kinase/Akt pathway would play a major role in determining the outcome of BCR ligation in B cells. Immature B cells generally respond to Ag receptor ligation with apoptosis, while mature B cells typically respond with proliferation and differentiation. A number of factors could potentially contribute to the signal balance. For instance, while immature B cells express only IgM isotype Ag receptors and lower levels of CD19, mature B cells express both IgM and IgD Ag receptors and elevated levels of CD19 (41, 42, 43, 44). While differences in signal transduction through IgM and IgD Ag receptors remain to be identified, it is conceivable that components of the respective receptor complexes may differ, leading to qualitatively distinct outcomes. In addition, memory B cell lines typically express IgG, IgE, or IgA Ag receptors. A possible shift in balance of signals may occur via the YXXM motif found in the cytoplasmic tail of IgG and IgE Ag receptors. This motif is characterized as a PI 3-kinase-binding region and may function in memory cells to enhance Akt activation either through bypassing the requirement for CD19 phosphorylation and its negative regulation by CD22 or by additive enhancement of PI 3-kinase activation. These questions remain to be addressed.
The second objective of this study was to identify mechanisms for regulation of Akt activity in B cells. While regulation of PI 3-kinase activity in B cells has not been well characterized, two conflicting reports indicate that the tyrosine kinases Syk and Lyn play significant roles in Akt activation in B cells (21, 39). Our findings are in agreement with Li et al., who demonstrate that Syk kinase is absolutely required for BCR-induced Akt activation and that Lyn kinase plays an antagonistic role in Akt activation. Similar findings have recently been reported by Craxton et al. (45). Craxton et al. (45) also reported that Btk was required for full Akt activation. In our experiments, however, we could not detect a significant effect on Akt activation in Btk-/- cells.
CD22, which is tyrosine phosphorylated by Lyn upon BCR ligation (13), and CD19 have been shown to have counterregulatory effects on BCR-mediated signaling pathways, including activation of extracellular signal-related kinase 2, c-Jun N-terminal kinase, and p38 (46). Negative regulation by CD22 occurs via recruitment of SHP-1 to the phosphorylated tyrosine in the immunoreceptor tyrosine-based inhibitory motif. As one substrate for SHP-1 is CD19 (47), and phosphorylation of CD19 is the major pathway for PI 3-kinase activation in B cells (16), we postulate that activation of Akt following BCR ligation is regulated by Syk phosphorylation of CD19 and that phosphorylation of CD22 mediated by Lyn kinase may inhibit the activation of Akt by inhibiting activation of PI 3-kinase. Experiments are in progress to further analyze the regulation of Akt in B cells.
At many stages during B cell development, the decision to live or die is mediated through Ag receptor signals. This study points to the PI 3-kinase/Akt pathway as a major factor in determining cell fate. While Akt has been shown to play an important role in maintenance of cell survival in fibroblasts and epithelial cells (48, 49, 50), its effects on cell survival have not been investigated in lymphocytes. Our data clearly demonstrate for the first time that the PI 3-kinase/Akt is critical in determining B cell fate by preventing apoptosis. While a number of potential substrates for Akt have been identified in many cell types, further studies will be necessary to identify the specific targets Akt utilizes to block apoptosis in lymphocytes and especially in tumor cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Molecular Genetics, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi 570, Japan.
3 Current address: Oncology Disease Group, Millennium Pharmaceuticals, 75 Sidney Street, Cambridge, MA 02139.
4 Address correspondence and reprint requests to Dr. Ronald Herbst, Department of Cell Signaling, DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304.
5 Abbreviations used in this paper: BCR, B cell receptor; BLNK, B cell linker protein; PH, pleckstrin homology; PI 3-kinase, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PLC, phospholipase C; SHP, Src homology 2 domain-containing phosphatase; SLP, Src homology 2 domain leukocyte protein; wt, wild type.
Received for publication September 24, 1999. Accepted for publication May 16, 2000.
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2 activation in surface immunoglobulin M-induced B cell apoptosis. J. Exp. Med. 184:907.
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 B. R. Herrin, A. L. Groeger, and L. B. Justement The Adaptor Protein HSH2 Attenuates Apoptosis in Response to Ligation of the B Cell Antigen Receptor Complex on the B Lymphoma Cell Line, WEHI-231 J. Biol. Chem., February 4, 2005; 280(5): 3507 - 3515. [Abstract] [Full Text] [PDF]
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K. D. Moon, C. B. Post, D. L. Durden, Q. Zhou, P. De, M. L. Harrison, and R. L. Geahlen Molecular Basis for a Direct Interaction between the Syk Protein-tyrosine Kinase and Phosphoinositide 3-Kinase J. Biol. Chem., January 14, 2005; 280(2): 1543 - 1551. [Abstract] [Full Text] [PDF]
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 M. Moroni, V. Soldatenkov, L. Zhang, Y. Zhang, G. Stoica, E. Gehan, B. Rashidi, B. Singh, M. Ozdemirli, and S. C. Mueller Progressive Loss of Syk and Abnormal Proliferation in Breast Cancer Cells Cancer Res., October 15, 2004; 64(20): 7346 - 7354. [Abstract] [Full Text] [PDF]
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A. Allam, H. Niiro, E. A. Clark, and A. J. Marshall The Adaptor Protein Bam32 Regulates Rac1 Activation and Actin Remodeling through a Phosphorylation-dependent Mechanism J. Biol. Chem., September 17, 2004; 279(38): 39775 - 39782. [Abstract] [Full Text] [PDF]
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 E. Rosati, R. Sabatini, E. Ayroldi, A. Tabilio, A. Bartoli, S. Bruscoli, C. Simoncelli, R. Rossi, and P. Marconi Apoptosis of human primary B lymphocytes is inhibited by N-acetyl-L-cysteine J. Leukoc. Biol., July 1, 2004; 76(1): 152 - 161. [Abstract] [Full Text] [PDF]
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M. J. Piatelli, C. Wardle, J. Blois, C. Doughty, B. R. Schram, T. L. Rothstein, and T. C. Chiles Phosphatidylinositol 3-Kinase-Dependent Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1/2 and NF-{kappa}B Signaling Pathways Are Required for B Cell Antigen Receptor-Mediated Cyclin D2 Induction in Mature B Cells J. Immunol., March 1, 2004; 172(5): 2753 - 2762. [Abstract] [Full Text] [PDF]
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A. P. Mone, P. Huang, H. Pelicano, C. M. Cheney, J. M. Green, J. Y. Tso, A. J. Johnson, S. Jefferson, T. S. Lin, and J. C. Byrd Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells Blood, March 1, 2004; 103(5): 1846 - 1854. [Abstract] [Full Text] [PDF]
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 C. C. Tomlinson and B. Damania The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Activates the Akt Signaling Pathway J. Virol., February 15, 2004; 78(4): 1918 - 1927. [Abstract] [Full Text] [PDF]
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M. Spaargaren, E. A. Beuling, M. L. Rurup, H. P. Meijer, M. D. Klok, S. Middendorp, R. W. Hendriks, and S. T. Pals The B Cell Antigen Receptor Controls Integrin Activity through Btk and PLC{gamma}2 J. Exp. Med., November 17, 2003; 198(10): 1539 - 1550. [Abstract] [Full Text] [PDF]
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R. Wen, Y. Chen, L. Xue, J. Schuman, S. Yang, S. W. Morris, and D. Wang Phospholipase C{gamma}2 Provides Survival Signals via Bcl2 and A1 in Different Subpopulations of B Cells J. Biol. Chem., October 31, 2003; 278(44): 43654 - 43662. [Abstract] [Full Text] [PDF]
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S. L. Christian, R. L. Lee, S. J. McLeod, A. E. Burgess, A. H. Y. Li, M. Dang-Lawson, K. B. L. Lin, and M. R. Gold Activation of the Rap GTPases in B Lymphocytes Modulates B Cell Antigen Receptor-induced Activation of Akt but Has No Effect on MAPK Activation J. Biol. Chem., October 24, 2003; 278(43): 41756 - 41767. [Abstract] [Full Text] [PDF]
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L. R. Whyburn, K. E. Halcomb, C. M. Contreras, C. A. Lowell, O. N. Witte, and A. B. Satterthwaite Reduced Dosage of Bruton's Tyrosine Kinase Uncouples B Cell Hyperresponsiveness from Autoimmunity in lyn-/- Mice J. Immunol., August 15, 2003; 171(4): 1850 - 1858. [Abstract] [Full Text] [PDF]
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X. Liu, A. Mayeda, M. Tao, and Z.-M. Zheng Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3' Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF/SF2 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway J. Virol., February 1, 2003; 77(3): 2105 - 2115. [Abstract] [Full Text] [PDF]
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D. E. Muscarella and S. E. Bloom Cross-linking of Surface IgM in the Burkitt's Lymphoma Cell Line ST486 Provides Protection against Arsenite- and Stress-induced Apoptosis That Is Mediated by ERK and Phosphoinositide 3-Kinase Signaling Pathways J. Biol. Chem., January 31, 2003; 278(6): 4358 - 4367. [Abstract] [Full Text] [PDF]
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K. Jiang, B. Zhong, C. Ritchey, D. L. Gilvary, E. Hong-Geller, S. Wei, and J. Y. Djeu Regulation of Akt-dependent cell survival by Syk and Rac Blood, January 1, 2003; 101(1): 236 - 244. [Abstract] [Full Text] [PDF]
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S.-C. Wong, W.-K. Chew, J. E.-L. Tan, A. J. Melendez, F. Francis, and K.-P. Lam Peritoneal CD5+ B-1 Cells Have Signaling Properties Similar to Tolerant B Cells J. Biol. Chem., August 16, 2002; 277(34): 30707 - 30715. [Abstract] [Full Text] [PDF]
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S. L. Christian, P. V. Sims, and M. R. Gold The B Cell Antigen Receptor Regulates the Transcriptional Activator {beta}-Catenin Via Protein Kinase C-Mediated Inhibition of Glycogen Synthase Kinase-3 J. Immunol., July 15, 2002; 169(2): 758 - 769. [Abstract] [Full Text] [PDF]
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H. Niiro, A. Maeda, T. Kurosaki, and E. A. Clark The B Lymphocyte Adaptor Molecule of 32 kD (Bam32) Regulates B Cell Antigen Receptor Signaling and Cell Survival J. Exp. Med., January 7, 2002; 195(1): 143 - 149. [Abstract] [Full Text] [PDF]
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H. Gary-Gouy, J. Harriague, A. Dalloul, E. Donnadieu, and G. Bismuth CD5-Negative Regulation of B Cell Receptor Signaling Pathways Originates from Tyrosine Residue Y429 Outside an Immunoreceptor Tyrosine-Based Inhibitory Motif J. Immunol., January 1, 2002; 168(1): 232 - 239. [Abstract] [Full Text] [PDF]
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A. Bernal, R. D. Pastore, Z. Asgary, S. A. Keller, E. Cesarman, H.-C. Liou, and E. J. Schattner Survival of leukemic B cells promoted by engagement of the antigen receptor Blood, November 15, 2001; 98(10): 3050 - 3057. [Abstract] [Full Text] [PDF]
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M. L. Woods and Y. Shimizu Signaling networks regulating {beta}1 integrin-mediated adhesion of T lymphocytes to extracellular matrix J. Leukoc. Biol., June 1, 2001; 69(6): 874 - 880. [Abstract] [Full Text] [PDF]
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 H. Bone and N. A. Williams Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase-dependent pathways to NF-{{kappa}}B activation in primary B cells Int. Immunol., June 1, 2001; 13(6): 807 - 816. [Abstract] [Full Text] [PDF]
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S.-j. Seo, J. Buckler, and J. Erikson Novel Roles for Lyn in B Cell Migration and Lipopolysaccharide Responsiveness Revealed Using Anti-Double-Stranded DNA Ig Transgenic Mice J. Immunol., March 15, 2001; 166(6): 3710 - 3716. [Abstract] [Full Text] [PDF]
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R. J. Ingham, L. Santos, M. Dang-Lawson, M. Holgado-Madruga, P. Dudek, C. R. Maroun, A. J. Wong, L. Matsuuchi, and M. R. Gold The Gab1 Docking Protein Links the B Cell Antigen Receptor to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway and to the SHP2 Tyrosine Phosphatase J. Biol. Chem., April 6, 2001; 276(15): 12257 - 12265. [Abstract] [Full Text] [PDF]
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M. Fujimoto, J. C. Poe, M. Hasegawa, and T. F. Tedder CD19 Amplification of B Lymphocyte Ca2+ Responses. A ROLE FOR Lyn SEQUESTRATION IN EXTINGUISHING NEGATIVE REGULATION J. Biol. Chem., November 21, 2001; 276(48): 44820 - 44827. [Abstract] [Full Text] [PDF]
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