From the Division of Allergy and Immunology, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
Received for publication, January 30, 2001
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ABSTRACT |
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Bruton's tyrosine kinase (Btk) binds to
phosphatidylinositol-3,4,5-trisphosphate
(PtdIns-3,4,5-P3) through the Btk pleckstrin homology
(PH) domain, an interaction thought to be required for Btk membrane
translocation during B cell receptor signaling. Here, we report that
interaction of PtdIns-3,4,5-P3 with the PH domain of Btk
directly induces Btk enzymatic activation in an in vitro kinase assay. A point mutation that reduces interaction of
PtdIns-3,4,5-P3 with the Btk PH domain blocks in
vitro PtdIns-3,4,5-P3-dependent Btk
activation, whereas the PH domain deletion enhances Btk basal activity
but eliminates the PtdIns-3,4,5-P3-dependent
stimulation. Btk kinase activity and the Btk activation loop
phosphorylation site are both required for the
PtdIns-3,4,5-P3-mediated stimulation of Btk kinase
activity. Together, these results suggest that the Btk PH domain is
positioned such that it normally suppresses both Btk kinase activity
and access to substrates; when interacting with
PtdIns-3,4,5-P3, this suppression is relieved, producing apparent Btk activation. In addition, using Src family kinase inhibitors and Btk catalytically inactive mutants, we demonstrate that
in vivo, the activation of Btk is due to both Lyn
phosphorylation and PtdIns-3,4,5-P3-mediated direct
activation. Thus, the Btk-PtdIns-3,4,5-P3 interaction
serves to translocate Btk to the membrane and directly regulate its
signaling function.
The B cell antigen receptor
(BCR)1 produces an
inositol-1,4,5-trisphosphate (IP3)-mediated calcium signal
that is required for antigen-driven B cell development (1, 2). One of
the most important components of this BCR calcium signaling pathway is
mediated by Bruton's tyrosine kinase (Btk), the predominant member of
the Tec kinase family that is expressed in B cells (3). Btk is required
for the majority of BCR-stimulated IP3 production, and its function in this process is tightly linked to the accumulation of phosphatidylinositol-3,4,5-trisphosphate
(PtdIns-3,4,5-P3) (4-6). The critical role of
Btk/PtdIns-3,4,5-P3-dependent IP3 production in the B cell receptor signal is demonstrated by the ability
of targeted deletions of Btk, the p85 subunit of phosphatidylinositol 3-kinase or phospholipase C The Btk recruitment into activated BCR signaling complexes is thought
to be a direct result of its interaction with
PtdIns-3,4,5-P3 in the plasma membrane following local
accumulation after BCR engagement (4, 5). This model of Btk activation
is based on evidence from direct binding of the Btk PH domain and
PtdIns-3,4,5-P3 in vitro (15, 16) and by
phosphatidylinositol 3-kinase-dependent membrane
translocation of the Btk PH domain fused to green fluorescent protein
(15-17). After binding to PtdIns-3,4,5-P3, Btk then
becomes activated via transphosphorylation through Src family
kinase-dependent mechanisms and autophosphorylation of its
kinase domain activation loop tyrosine (18). However, whether Btk
activation also might be directly regulated by the Btk PH
domain/PtdIns-3,4,5-P3 interaction has not been addressed.
Here, we have investigated this question using an in vitro
Btk kinase assay. Our data show that PtdIns-3,4,5-P3 is
able to directly induce Btk enzymatic activity in vitro and, therefore, support a dual role for PtdIns-3,4,5-P3 in the
activation of Btk signaling function, namely membrane translocation in
proximity to activated B cell receptor complexes and direct regulation
of Btk kinase domain signaling function.
Cell Culture, Recombinant Virus Production and
cDNAs--
A20 mouse B cells and Ramos human B cells were grown in
RPMI 1640 with 10% fetal bovine serum and
10
A20 infections were performed by adding 5 plaque-forming unit/cell of
recombinant virus to ~20% confluent A20 cells and allowing infections to proceed for 12-15 h. Where appropriate, control recombinant virus was added so that all samples were exposed to an
equal number of plaque-forming unit/cell. A recombinant virus containing a cDNA-encoding human G
The pSC-FLAG4 vector was constructed by ligating the FLAG
epitope coding sequence and the following multienzyme restriction site
sequence
(GTCGATTGGCCGCCACCATGGGCGACTACAAGGACGACGATGACAAGGCGGCCGCGGTACCACTAGTGCATGCGTCGACCCCGGG) into the pSC-66 vaccinia recombination plasmid using 5'SalI
and 3'XbaI restriction sites.
The FLAG-tagged wild type and mutant forms of Btk were constructed by
subcloning the wild type (nucleotides 1-1977), Pharmacologic Reagents, Antibodies, Cell Lysis,
Immunoprecipitations, and Western Blotting--
Phosphatidylserine
(PS) was purchased from Avanti Polar. Lipids,
phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2),
and PtdIns-3,4,5-P3 were purchased from Biomol. PP2 was
purchased from Calbiochem. The enolase substrate was purchased from
Sigma. Anti-FLAG M2 antibody was obtained from Sigma.
Anti-phosphotyrosine antibody 4G10 was obtained from Upstate
Biotechnology. Polyclonal anti-Btk has been described previously (18).
Stimulation of A20 B cells was performed with F(ab')2
rabbit anti-mouse IgG (15 µg/ml) purchased from Jackson Immunoresearch.
Cells were lysed in Tris-HCl-buffered saline (pH 7.4) containing 1%
Brij 97 or 0.5% Triton X-100, 2 mM sodium vanadate,
5 mM EDTA, 5 mM sodium fluoride, and 5 µg/ml
of leupeptin and pepstatin. Immunoprecipitations, Western transfer, and
immunoblotting were described previously (20). The precipitates were
subjected to Western blot analysis using enhanced chemifluorescent
detection system (Amersham Pharmacia Biotech). The blots were analyzed
using a Molecular Dynamics STORM imager and ImageQuant software.
Stimulation by Lipids and in Vitro Kinase Assays--
In
vitro kinase assay was carried out utilizing previously described
standard protein kinase C vesicle assay (21). Briefly, mixed vesicles
containing PS, PtdIns-4,5-P2, or
PtdIns-3,4,5-P3 were sonicated on ice for 10 min in 10 mM Hepes, pH 7.5. Reaction mixtures (50 µM)
contained 20 mM Hepes, pH 7.5, 5 mM
MgCl2, 5 mM MnCl2, 10 µM ATP, 10 µCi of [ Quantification and Calculation of the Stimulation and
Phosphorylation Indexes--
The volume of the bands was measured
using ImageQuant software (Molecular Dynamics). The stimulation index
and tyrosine phosphorylation index were determined by comparing the
density rations of the bands to those of the base-line sample and using
the formula: stimulation index = (density of phosphorylation
band/density of Btk protein band)test sample:(density of
phosphorylation band/density of Btk protein band)base-line control
sample, tyrosine phosphorylation index = (density of
tyrosine phosphorylation band/density of Btk protein band)test
sample:(density of tyrosine phosphorylation band/density of Btk
protein band)base-line control sample.
PtdIns-3,4,5-P3 Induces Btk Phosphorylation--
To
analyze whether interactions between PtdIns-3,4,5-P3 and
the Btk PH domain might regulate Btk function, we first examined whether the presence of PtdIns-3,4,5-P3 might be able to
affect Btk in vitro kinase activity in recombinant Btk
immunoprecipitates or add to lysates from cells overexpressing Btk.
Fig. 1A shows that the
presence of PtdIns-3,4,5-P3 significantly increased Btk autophosphorylation under these assay conditions and was highly specific. The effect observed with PtdIns-3,4,5-P3 present
was not detected with the substitution of the related polar lipid, PtdIns-4,5-P2 (compare middle and far
right lanes of top panel). When added to whole cell lysates,
PtdIns-3,4,5-P3 also dramatically increased the
phosphorylation of a 75-80-kDa protein in the whole cell lysates; the
size of this protein corresponded precisely with the size of expressed
Btk by Western blot against the anti-FLAG tag antibody (bottom
panel).
Next, to eliminate the potential contribution of other B cell-specific
signaling pathways, such as through the endogenous Lyn as well as
through other hematopoietic phosphatases, we examined Btk produced in a
non-hematopoietic cell line (Fig. 1B). As little as 1 µM PtdIns-3,4,5-P3 significantly activated
Btk produced in NIH3T3 cells compared with PtdIns-3,4-P2 or
PS alone. We also observed the same activation of endogenous Btk in the
human B cell line (Fig. 1C).
Furthermore, we observed the enhanced kinase activity of Btk by
PtdIns-3,4,5-P3 toward acid-denatured enolase used here as a substrate (Fig. 1D).
We then examined the dose/response relationship of
PtdIns-3,4,5-P3-dependent activation of Btk
in vitro by measuring the phosphorylation of Btk and
acid-denatured enolase (Fig. 2). The
activation of Btk by various amounts of PtdIns-3,4,5-P3 was
studied in the presence of PS to maintain a constant total lipid
concentration (200 µM). The activation of Btk was
observed at concentrations of 10 nM to 10 µM,
thus yielding a 50% maximal activation (EC50) of ~100 nM. This result closely corresponds to the value reported
by others for the affinity of the Btk PH domain for
PtdIns-3,4,5-P3 (100-700 nM) and is consistent
with a direct interaction between PtdIns-3,4,5-P3 and the
Btk PH domain being involved in the kinase activation effect (16,
22).
PtdIns-3,4,5-P3-Dependent Phosphorylation of Btk
Is Due to Autophosphorylation--
To discriminate between
autophosphorylation of Btk versus the presence of
contaminating kinases such as endogenous Lyn or other Src family
kinases, we first compared the effect of the Src family-specific
inhibitor, PP2, to the enhanced phosphorylation of Btk by PtdIns-3,4,5-P3.
Even at a concentration of 10 µM, PP2 did not affect the
enhanced phosphorylation (Fig. 3A). (Note that we will show
in Fig. 4, A and B
that 10 µM PP2 inhibits Lyn activity efficiently but not
Btk).
We then analyzed two different inactive versions of Btk (a kinase
domain deletion mutant and the K430R ATP binding site mutant) (see Fig.
3, B and C). Neither of these mutants
demonstrated any apparent kinase activity (either basal or
PtdIns-3,4,5-P3-dependent) in the in
vitro assay conditions. These data demonstrate unequivocally that
the kinase activity observed in these assays is entirely due to the
autophosphorylation kinase activity of Btk.
The Btk PH Domain Allosterically Regulates Btk Activation in
Vitro--
To analyze further the mechanism through which
PtdIns-3,4,5-P3 produced Btk activation in our assay, we
examined whether PtdIns-3,4,5-P3 would be able to activate
various other mutant versions of Btk in a series of separate
experiments (Fig. 3, B-D). We analyzed a PH
deletion mutant of Btk, the R28C PH domain mutant with known reduced
affinity for PtdIns-3,4,5-P3, and the Y551F activation loop
mutant (Y551F). All three mutants revealed a lack of
PtdIns-3,4,5-P3-stimulated activity. However, the R28C and
Y551F mutants showed normal levels of basal activity whereas the PH
domain deletion mutant had a highly elevated basal activity, which was
the same as or greater than the PtdIns-3,4,5-P3-stimulated
activity of wild type Btk (compare lanes 2, 3,
and 4 of the top panel). Together,
these results suggest that the PH domain is normally positioned so that it suppresses Btk kinase activity in this assay and that
PtdIns-3,4,5-P3 relieves this suppression when it is
available to bind to the PH domain of Btk.
An interesting additional question elucidated by our findings is
whether a PH domain deletion mutant would be able to signal normally
given that it is fully active in vitro and its SH2 domain, which is thought to mediate interaction with specific target
substrates, would be expected to retain normal binding activity (25).
We therefore compared the effect of Btk At Least Half of Btk Is Autophosphorylated by the Interaction of
PtdIns-3,4,5-P3 in Vivo--
The above results showed
direct interaction of PtdIns-3,4,5-P3 with the Btk PH
domain with an enhancement of the kinase activity of Btk. On the other
hand, we and others have described that Btk is also activated by the
transphosphorylation of Src family kinase, Lyn, in B cells. Indeed, Btk
is activated by both interaction of PtdIns-3,4,5-P3 and by
transphosphorylation with Lyn. How significant is the
autophosphorylation of Btk by the interaction of
PtdIns-3,4,5-P3 while transphosphorylated by Lyn? To answer
this question, we examined the tyrosine phosphorylation of Btk in A20 B
cells with the overexpression of the constitutively active form of
phosphatidylinositol 3-kinase, P110 We have presented biochemical evidence that the interaction
between PtdIns-3,4,5-P3 and the Btk PH domain directly
regulates the catalytic activity of Btk. This conclusion is based on
the following results: the stimulation of the in vitro
activity of wild type Btk by PtdIns-3,4,5-P3, the
disruption of this effect either by a PH domain mutation (R28C) known
to eliminate its PtdIns-3,4,5-P3 binding activity or by
deletion of the Btk PH domain, and the observation of enhanced basal
activity in our Btk PH domain deletion mutant. Together, these findings
suggest that the Btk PH domain normally acts to suppress Btk kinase
activity and that this domain may in addition hinder access of the
kinase domain to substrates. In either case, interaction with
PtdIns-3,4,5-P3 or PH domain deletion relieves the
inhibition, producing a form of Btk that is now able to more
effectively autophosphorylate and transphosphorylate substrates.
We and others have previously noted significant similarity between the
role of PtdIns-3,4,5-P3 in Btk activation and that proposed
for D-3-phosphoinositides in regulation of the
serine-threonine kinase, Akt/protein kinase B (Fig.
5) (5, 23). Full activation of wild type
Akt requires phosphorylation on its activation loop (24). This
phosphorylation occurs only in the presence of
D-3-phosphoinositides. D-3-Phosphoinositides
fail to promote the 3-phosphoinositide-dependent kinase 1 (PDK1)-mediated phosphorylation of a phosphoinositide-binding PH-deficient Akt mutant (R25C) and deletion of the Akt PH domain allows the Akt
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2, to produce highly similar B
cell immunodeficiencies in the mouse (7-11) and by the association of
Btk mutations that abrogates phospholipase C
2 activation with X-linked agammaglobulinemia in humans (12, 13). Furthermore, Btk/PtdIns-3,4,5-P3-dependent IP3
signaling is blocked by the Fc
RIIb1 inhibitory receptor, whose
coengagement with the BCR has been shown to inhibit BCR-mediated
IP3 production, BCR-mediated calcium signaling,
BCR-dependent antibody production, and B cell proliferation
(14).
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5 M 2-mercaptoethanol. NIH3T3
cells were grown in RPMI 1640 with 10% calf serum.
l inserted in an antisense orientation was used as the control virus because the transcript generated was similar in length to that of Btk.
PH (nucleotides 400-1977),
kinase (nucleotides 1-1182) cDNA into the pSC-FLAG4 plasmid using 5'NotI and 3'KpnI restriction
sites. Recombinant viruses were then selected with
5-bromo-2'-deoxyuridine/
-galactosidase selection, amplified, and
titered using standard techniques (19). Other recombinant Btk and
vaccinia viruses have been described previously (4, 6, 18).
-32P]ATP, and mixed
phospholipid/phosphoinositide vesicles. For transphosphorylation experiments, 5 µg of acid-denatured enolase were added to the reaction mixture. Reactions were started by addition of Btk and Btk
mutant-precipitated beads and incubated at room temperature for 3 min.
Reactions were stopped by addition of 2× SDS-polyacrylamide gel
electrophoresis sample buffer and heating to 100 °C for 5 min.
Proteins were separated by 8% SDS-polyacrylamide gel electrophoresis. After electrophoresis the gels were stained with Coomassie Blue. Autoradiograms of the gels were obtained with an overnight exposure to
a storage phosphor screen and measurement of incorporated radioactivity using a Molecular Dynamics STORM imager and ImageQuant software. For
some experiments, the bands of Btk and enolase were excised, and
the incorporated radioactivity was measured by liquid scintillation counter.
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Fig. 1.
PtdIns-3,4,5-P3 induces Btk
phosphorylation. A, PtdIns-3,4,5-P3 is able
to induce Btk in vitro kinase activity and Btk tyrosine
phosphorylation in A20 B cells overexpressing Btk. A20 B cells were
plated to 20% confluency and subsequently infected for 15 h with
recombinant vaccinia viruses expressing FLAG-tagged Btk. Upper
panel, cells were harvested, washed once with calcium buffer (135 mM NaCl, 10 mM KCl, 10 mM Hepes (pH
7.5), 5.6 mM glucose, 0.1% bovine serum albumin, 1 mM MgCl2, 1 mM CaCl2),
lysed, and then postnuclear supernatants were immunoprecipitated with
anti-FLAG antibody. Precipitates were divided into three aliquots and
treated with mixed lipids containing phosphatidylserine 200 µM alone (PS), PS 100 µM and
PtdIns-4,5-P2 100 µM (PS/PIP2), or
PS 100 µM and PtdIns-3,4,5-P3 100 µM (PS/PIP3). The precipitates were then
subjected to an in vitro kinase assay. Bottom
panel, cells were harvested, washed once with calcium buffer,
resuspended at 107 cells/ml in calcium buffer, and
stimulated or not stimulated for 2 min with 30 µg/ml
F(ab')2 rabbit anti-mouse IgG. Cells were quickly
centrifuged and lysed, and postnuclear supernatants were incubated with
and without PtdIns-3,4,5-P3 100 µM for 5 min
at room temperature. Lysates were then subjected to SDS-polyacrylamide
gel electrophoresis and analyzed by Western blotting with the indicated
antibody. B, PtdIns-3,4,5-P3 activates in
vitro kinase activity of Btk expressed in a non-hematopoietic cell
context. NIH3T3 fibroblasts were infected for 3 h with recombinant
vaccinia viruses expressing FLAG-tagged Btk. Precipitates of Btk were
prepared, treated with the indicated concentration of mixed lipids, and
assayed in the same fashion as in the top panel of A. C, PtdIns-3,4,5-P3 enhanced in vitro
kinase activity of endogenous Btk in B cells. Ramos cells were lysed
and then postsupernatants were immunoprecipitated with anti-Btk
antibody. Precipitates were divided and assayed as in the top
panel of A. D, PtdIns-3,4,5-P3 enhance the
kinase activity of Btk toward acid-denatured enolase. Precipitates of
Btk were prepared in the same fashion as in the top panel of
A. Precipitates were then assayed with the presence of 5 µg of
acid-denatured enolase in the same fashion as in the top panel of
A. The in vitro kinase data shown here are
representatives of three separate experiments. Stimulation indexes
(SI) were calculated as described under "Experimental
Procedures." From the calculation of 32P incorporation,
the in vitro kinase activity of Btk produced in A20 cells
was increased 2.0-4.0-fold by PtdIns-3,4,5-P3 when
compared with PS alone. PtdIns-4,5-P2 increased
1.1-1.5-fold compared with PS alone. The in vitro kinase
activity of Btk in NIH3T3 cells was increased 2.0-3.0-fold by 1 µM PtdIns-3,4,5-P3 and 2.4-4.3-fold by 100 µM PtdIns-3,4,5-P3 compared with PS alone,
whereas 100 µM PtdIns-4,5-P2 increased less
than 1.5-fold. The in vitro kinase activity of endogenous
Btk in Ramos cells was increased 2.0-2.9-fold by
PtdIns-3,4,5-P3 and 1.1-1.6-fold by
PtdIns-4,5-P2. IP, immunoprecipitated;
IB, immunoblotted; IVK, in vitro
kinase.
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Fig. 2.
Activation of Btk as a function of
PtdIns-3,4,5-P3 concentration. NIH3T3 cells were
infected for 3 h with recombinant vaccinia viruses expressing
FLAG-tagged Btk. Precipitates of Btk were prepared and processed in the
same fashion as in Fig. 1B. The bands of Btk and
enolase were excised and the incorporated radioactivity was measured by
liquid scintillation counter. A dose response curve for activation of
Btk by PtdIns-3,4,5-P3 was obtained by varying the
concentration of PtdIns-3,4,5-P3 and maintaining a fixed
concentration of total phospholipids (200 µM) by
adjusting the concentration of PS. The dose response curve of Btk was
representative of three separate experiments.
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Fig. 3.
PtdIns-3,4,5-P3-induced Btk
phosphorylation is due to autophosphorylation: evidence for allosteric
regulation of Btk activity. A, the Src family
kinase-specific inhibitor does not affect the
PtdIns-3,4,5-P3-enhanced in vitro kinase
activity of Btk. Precipitates of Btk were prepared in the same fashion
as in the top panel of Fig. 1A. Precipitates were
treated with the indicated concentration of PP2 at 37 °C for 1 h and then assayed in the same fashion as in the top panel
of Fig. 1A. B-D, analysis of various
Btk mutants. NIH3T3 fibroblasts were infected for 3 h with the
indicated vaccinia viruses, harvested, and lysed, and postnuclear
supernatants were immunoprecipitated with the indicated antibody or
analyzed directly. Top panels, precipitates were divided in
two aliquots, treated or untreated with PtdIns-3,4,5-P3,
and analyzed for in vitro kinase activity as in Fig. 1.
Bottom panels, whole cell lysates were analyzed by Western
blotting using the indicated antibody. In vitro kinase
activities of Btk mutants were representative from three sets of
different experiments. Stimulation indexes (SI) were
calculated as described under "Experimental Procedures." From the
calculation of 32P incorporation, PH mutant showed
4.0-4.3-fold comparison to the basal level of in vitro
kinase activity of wild type Btk. The basal level of in
vitro kinase activity of R28C mutant was reduced by 50-90% of
the wild type. No 32P incorporation was detected in the
precipitates of the
kinase or the K430R mutant. IP,
immunoprecipitated; IB, immunoblotted; IVK,
in vitro kinase.
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Fig. 4.
Effect of the Src family kinase-specific
inhibitor on the tyrosine phosphorylation of Btk. A and
B, incubation of A20 B cells with PP2 specifically affects
the kinase activity of Lyn but does not affect the kinase activity of
Btk. A20 B cells were infected with recombinant vaccinia viruses
expressing FLAG-tagged Btk as shown in Fig. 1A. Cells were
then incubated with the indicated concentration of PP2 at 37 °C for
30 min. Precipitates of Btk were prepared, treated with PS/PIP3, and
tested with in vitro kinase assay in the same fashion as in
the top panel of Fig. 1A. C, at least
half of Btk can be autophosphorylated by PtdIns-3,4,5-P3
without transphosphorylation by Lyn. A20 B cells were infected with
indicated viruses as in Fig. 1A. Cells were incubated or not
with 10 µM PP2 at 37 °C for 30 min, harvested, washed,
and lysed, and postnuclear supernatants were immunoprecipitated with
anti-Btk antibody. Precipitates were then subjected to
SDS-polyacrylamide gel electrophoresis and analyzed by Western blot
using anti-phosphotyrosine antibody (4G10) and anti-Btk antibody. The
volume of the tyrosine phosphorylation and the content of protein bands
were measured. Tyrosine phosphorylation indexes were calculated as
described under "Experimental Procedures." The results are
represented from two sets of different experiments. IP,
immunoprecipitated; IVK, in vitro kinase.
PH overexpression to that of
wild type Btk through BCR calcium signaling and IP3 assays in the A20 B
cell line. The Btk
PH mutant produced no enhancement in either the
BCR calcium signal or IP3 production in A20 B cell in
conditions when wild type Btk does produce enhancements in both (data
not shown). These data suggest that the PH domain membrane targeting is
required also for proper integration of Btk into the BCR signaling
complex, although we cannot exclude that PH domain deletion alters the
folding/conformation of the remaining Btk domains in an idiosyncratic
manner that prohibits normal Btk signaling function.
, using PP2 and the K430R Btk to
eliminate each one of the phosphorylation pathways. As seen in Fig. 4,
incubation of A20 cells with 10 µM PP2 did not affect the
kinase activity of Btk (Fig. 4A), whereas it significantly
inhibited the kinase activity of Lyn (Fig. 4B). The tyrosine
phosphorylation of wild type Btk in PP2-treated cells was half that
found in PP2-untreated cells; whereas in the K430R mutants, tyrosine
phosphorylation of Btk was equivalent to that of wild type Btk in
PP2-treated cells. These data suggest that in vivo, at least
half of the Btk is autophosphorylated by PtdIns-3,4,5-P3
(Fig. 4C).
DISCUSSION
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ABSTRACT
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DISCUSSION
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PH molecule to be phosphorylated by PDK1 in the absence of phosphoinositides in vitro. These findings have
led to the proposal that the Akt PH domain acts as an inhibitor of PDK1-dependent phosphorylation of Akt activation loop
residues required for full activation and that
D-3-phosphoinositide binding to the PH domain relieves
these inhibitory effects (Fig. 5A). In contrast, the Btk
activation mechanism has been proposed to consist of membrane targeting
by the binding of PtdIns-3,4,5-P3 to the Btk PH domain.
Then, tyrosine phosphorylation of its activation loop occurs through
Src family kinase, Lyn-dependent transphosphorylation and
Btk autophosphorylation (Fig. 5B). Our data demonstrate that enhanced PtdIns-3,4,5-P3-dependent Btk
autophosphorylation is blocked in the Btk-Y551F activation loop mutant.
This latter result demonstrates that this
PtdIns-3,4,5-P3-dependent Btk
autophosphorylation primarily occurs at the activation loop tyrosine.
This result is consistent with previous data (18) and, in conjunction
with the Btk PH domain mutant data, suggests that in this "opened" form of Btk the activation tyrosine Y551 is more accessible to both Lyn
and Btk. (Fig. 5B). Modification of our previous Btk activation model, in light of these data, suggests that
PtdIns-3,4,5-P3-mediated regulation of Btk resembles the
model proposed for D-3-phosphoinositide-mediated regulation
of Akt.
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Fig. 5.
Proposed mechanism for Btk activation
by comparison with Akt activation. The PH domains of PDK1, Akt,
and Btk are depicted in red, and their kinase domains are
depicted in dark blue. The yellow circles
represent phosphorylation sites whereas the orange hexagons
show D-3-phosphoinoitides. A,) the PH domains of
PDK1 and Akt bind to membrane-bound D-3-phosphoinositides.
This binding opens the enzymes and relieves the inhibitory
effect of PH domains, allowing PDK1 to phosphorylate the activation
loop threonine 308 (T308) of Akt. The R25C Akt, a
phosphoinositide-binding, PH-deficient mutant, fails to phosphorylate
on T308; deletion of the Akt PH domain results in the
phosphorylation of T308 by PDK1 in vitro, in the
absence of phosphoinositides. B, the PH domains of Btk binds
to membrane-bound D-3-phosphoinositides. This binding
relieves the inhibitory effect of the PH domain and provides access to
membrane-attached Src family kinases such as Lyn. This allows Btk to
autophosphorylate and permits Lyn-dependent
transphosphorylation of Btk on tyrosine 551 (Y551). The R28C
Btk, phosphoinositide-binding, PH-deficient mutant fails to
phosphorylate on Y551; deletion of the Btk PH domain results
in autophosphorylation of Y551 in vitro.
In conclusion, together with previous data, our results suggest that
PtdIns-3,4,5-P3 plays dual and probably functionally inseparable roles in the activation of PH-containing Tec family kinases; it provides proximity to target effector molecules (by targeting the kinase to the plasma membrane) as well as an activated and more accessible kinase domain (by opening up the kinase as suggested in this paper). These data suggest a modified model of Btk
activation, which is similar to one previously proposed for the
D-3-phosphoinositide-regulated serine-threonine kinase, Akt, in which D-3-phosphoinositides also played a dual
localization and allosteric regulatory role. Such a mechanism is
theoretically attractive for the control of signaling molecules like
Tec kinases and Akt whose functions are closely linked to cell fate
decisions as it provides a high level of assurance that their signaling function is directed toward the proper targets in the proper context.
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ACKNOWLEDGEMENTS |
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We thank Dr. Maria J. Denslow for proofreading the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Pediatrics, Univ. of Washington,
Seattle, WA 98195-6320.
§ To whom correspondence should be addressed. Tel.: 617-667-1324; Fax: 617-667-1323; E-mail: jkinet@caregroup.harvard.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M100873200
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ABBREVIATIONS |
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The abbreviations used are: BCR, B cell antigen receptor; IP3, inositol-1,4,5-trisphosphate; PtdIns-3, 4,5-P3, phosphatidylinositol-3,4,5-trisphosphate; PH, pleckstrin homology; PS, phosphatidylserine; PtdIns-4, 5-P2, phosphatidylinositol-4,5-bisphosphate; PDK1, 3-phosphoinositidedependent kinase 1.
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