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INTRODUCTION |
Phosphoinositides are important precursor molecules that generate
multiple second messengers in stimulated cells. Phospholipase C
(PLC)1-mediated hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) yields
the water-soluble messenger molecule inositol 1,4,5-trisphosphate,
which mobilizes intracellular Ca2+, and the hydrophobic
moiety diacylglycerol, which activates protein kinase C isozymes (1,
2). In addition, PI(4,5)P2 can be phosphorylated by PI
3-kinase(s) to form phosphatidylinositol 3,4,5-trisphosphate
(PI(3,4,5)P3), a lipid product found only in stimulated
cells (3, 4). The multiplicity and divergent regulation of PI 3-kinases
by receptor and non-receptor tyrosine kinases as well as by GTP-binding
proteins (5) and the resistance of PI(3,4,5)P3 to
hydrolysis by any known phospholipase C have led to the proposal that
3-phosphorylated inositides, in particular PI(3,4,5)P3,
serve important regulatory functions (5).
The pleckstrin homology (PH) domains of several regulatory proteins
have been shown to bind PI(3,4,5)P3 in vitro
(6-10). One of these proteins is Bruton's tyrosine kinase (Btk), a
member of the Tec family of non-receptor tyrosine kinases (11),
mutations of which are associated with the human disease X-linked
agammaglobulinemia and its murine equivalent, X-linked immunodeficiency
(12, 13). Although Btk also contains a protein kinase as well as SH2
and SH3 domains, its PH domain alone has been shown to be sufficient to
bind PI(3,4,5)P3 selectively in vitro (6, 8).
Many of the Btk mutations that cause the B-cell defect that leads to
X-linked agammaglobulinemia in humans (14) are within the Btk PH domain of the protein, and one of these, the R28C substitution, is responsible for X-linked immunodeficiency in mice (13). The latter mutation has
also been shown to abolish the binding of Btk to inositol lipids
in vitro (6, 8). In addition, a transforming mutant of Btk
(Btk*, E41K) has been reported to show increased membrane association,
which further indicates that PH domain-mediated binding of Btk to cell
membrane(s) is critical for its activation (15).
This study was designed to investigate whether the isolated PH domain
of Btk is sufficient to interact with membrane phosphoinositides within
intact living cells with similar specificity to that described in
vitro and whether this interaction can localize the protein to the
membrane without additional binding motifs. Expression of the Btk PH
domain fused to the enhanced green fluorescent protein (BtkPH-GFP) has
demonstrated that PI 3-kinase activation recruits these molecules
to the plasma membrane, suggesting that they specifically recognize
3-phosphorylated inositol lipids without binding to PI(4,5)P2. This methodology also allows visualization
of dynamic changes in 3-phosphorylated inositides in single living cells.
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EXPERIMENTAL PROCEDURES |
Materials--
myo-[3H]Inositol (80 Ci/mmol) was purchased from Amersham Pharmacia Biotech. EGF
(recombinant human) and PDGF-AB (recombinant human) were obtained from
Life Technologies, Inc. Ionomycin, LY 294002, bisindolylmaleimide I,
and BAPTA were purchased from Calbiochem, and wortmannin was a gift
from Kyowa Hakko Laboratories (Tokyo, Japan). All other chemicals were
of high pressure liquid chromatography or analytical grade.
Plasmid Constructs--
The PH domains of PLC
1
(amino acids 1-170) and of Bruton's tyrosine kinase (amino acids
1-177) were amplified with the Advantage Klentaq polymerase mixture
(CLONTECH) from human cDNAs (Marathon cDNA
from brain and K562 leukemia cells, CLONTECH) with
the following primer pairs: PLC
, 5'-GGCATGGACTCGGGCCGGGACTTCCTG-3'
and 5'-AAGATCTTCCGGGCATAGCTGTCG-3'; Btk,
5'-CCAAGTCCTGGCATCTCAATGCATCTG-3' and 5'-TGGAGACTGGTGCTGCTGCTGGCTC-3'. The amplified products were subcloned into the pGEM-Easy T/A cloning vector (Promega) and sequenced with dideoxy sequencing
(Thermosequenase, Amersham Pharmacia Biotech). A second amplification
reaction was performed from these plasmids with nested primers that
contained restriction sites for appropriate cloning into the pEGFP-N1
plasmid (CLONTECH) to preserve the reading frame.
Plasmids were transfected into COS-7 or NIH 3T3 cells and analyzed by
SDS-polyacrylamide gel electrophoresis followed by Western blotting for
the presence of the GFP fusion constructs using a polyclonal antibody
against GFP (CLONTECH).
Mutations were created with the QuickChangeTM mutagenesis
kit (Stratagene). All mutations were confirmed by dideoxy sequencing, and the expression of the protein was confirmed by Western blotting.
Transfection of Cells for Confocal Microscopy--
Cells were
plated onto poly-L-lysine-coated 30-mm diameter circular
glass coverslips at a density of 5 × 104 cells/dish
and cultured for 3 days before transfection with plasmid DNAs (1 µg/ml) using the LipofectAMINE reagent (10 µg/ml; Life Technologies, Inc.) and Opti-MEM (Life Technologies, Inc.). Forty-eight hours after transfection, cells were washed twice with a modified Krebs-Ringer buffer containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose, and 10 mM Na-Hepes, pH 7.4, and the coverslip was placed into a
chamber that was mounted on a heated stage with the medium temperature
kept at 33 °C. Cells were incubated in 1 ml of the Krebs-Ringer
buffer, and stimuli were added in 0.5 ml of prewarmed buffer
after removing 0.5 ml of medium from the cells. Cells were examined in
an inverted microscope under a 40× oil immersion objective (Nikon
Inc.) and a Bio-Rad laser confocal microscope system (MRC-1024) with
Lasersharp acquisition software (Bio-Rad).
Analysis of Inositol Phosphates in COS-7 Cells--
Inositol
phosphates were analyzed in COS-7 cells transfected with the
AT1a angiotensin II receptor together with selected PH
domain-GFP fusion constructs after labeling with
myo-[3H]inositol for 24 h as described
previously (16, 17). 3H-Labeled inositol phosphates
were analyzed with Dowex minicolumns.
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RESULTS |
Distribution of PH Domains Fused to the Enhanced Green Fluorescent
Protein--
To follow the localization of isolated PH domains within
intact cells, they were fused to GFP and expressed in NIH 3T3 cells. As
shown in Fig. 1A (left
panel), expressed GFP (without any attached sequence) was found to
be cytosolic and was also present in the nucleus. Addition of the Btk
PH domain (amino acids 1-177) to the N terminus of GFP had no effect
on its localization in quiescent cells (after serum deprivation) and
was indistinguishable from GFP alone (Fig. 1A, center
panel). In contrast, fusion of the PLC
PH domain to GFP caused
prominent membrane localization of the construct due to its interaction
with membrane PI(4,5)P2 (18) (Fig. 1A,
right panel). These results suggest that, unlike the PH
domain of PLC
, the PH domain of Btk does not possess high enough
affinity to interact with PI(4,5)P2 so that it would be sufficient for its membrane targeting.

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Fig. 1.
Localization of the PH domains of Btk and
PLC in transfected NIH 3T3 cells. A, the PH domain of Btk
(amino acids 1-177) or PLC (amino acids 1-170) was fused to the N
terminus of GFP, and the respective constructs were transiently
expressed in NIH 3T3 cells. Confocal images were taken from
serum-starved (>5 h) cells incubated at 33 °C. Alignment of the
protein sequences within the PH domains (covering the 2-
and 3-strands) of Btk and PLC is shown. The
arrows indicate the positions of the mutations analyzed in
this study. B, redistribution of fluorescence in NIH 3T3
cells after a 5-min stimulation with 50 ng/ml PDGF (left
panels). The fluorescent intensity changes across the white
lines were plotted as line intensity histograms in the right
panels. Calculation of
Ipm/Icyt was used to
quantitate the extent of membrane localization.
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Stimulation of Cells Causes Translocation of the Btk PH Domain to
Membranes--
Next we examined whether stimulation of cells with
growth factors that are known to activate PI 3-kinases and to increase formation of PI(3,4,5)P3 causes any change in the
distribution of the BtkPH-GFP protein. NIH 3T3 cells were transfected
with the BtkPH-GFP construct alone or together with the cDNA
encoding the human EGF receptor and were stimulated either with PDGF
(25-100 ng/ml) (to activate the endogenous PDGF receptor) or with EGF (100 ng/ml) after serum starvation. As shown in Figs. 1B and
2, stimulation of either receptor caused
a translocation of the cytosolic fluorescence to the plasma membrane.
The decrease in cytosolic fluorescence was most obvious when compared
with the bright nuclear signal that did not change in intensity
following stimulation in the NIH 3T3 cells. This was in contrast with
our previous finding in the same cell type, where the nuclear intensity
of the PLC
PH domain-GFP fusion protein slowly followed the
cytoplasmic changes, although with a slight delay (19).

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Fig. 2.
Agonist-induced changes in the localization
of BtkPH-GFP in NIH 3T3 cells stimulated with EGF or PDGF or in
hepatocytes stimulated with insulin. NIH 3T3 cells were
transiently cotransfected with plasmids encoding the human EGF receptor
and BtkPH-GFP. Serum-starved cells were stimulated either with EGF (100 ng/ml) (A) or with PDGF (50 ng/ml) (B) at
33 °C. Immortalized hepatocytes were transfected with the BtkPH-GFP
construct for 48 h and were stimulated with insulin (100 nM) after serum deprivation at 33 °C (C).
Confocal images were taken at every 30 s. The numbers
show the time in seconds at which images were recorded. Stimuli were
added at 15 s.
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The redistribution of fluorescence was clearly demonstrated by
comparing line intensity histograms calculated at selected "cross-sections" of the cell. Dividing the fluorescence intensity of the plasma membrane (Ipm) by that of the
cytosol (Icyt) yields a ratio that can be used
as an index of membrane localization (Fig. 1B, right
panels).
To determine whether a similar translocation of BtkPH-GFP takes place
in response to stimulation of other receptors that activate PI
3-kinases, NIH 3T3 cells were also stimulated with EGF (via expressed
EGF receptors). EGF stimulation also evoked a translocation response,
and we could not detect a notable difference in the localization of the
fluorescent signal whether PDGF or EGF was used as the agonist (Fig. 2,
A and B). To examine the effects of insulin,
another known activator of PI 3-kinases, immortalized hepatocytes (20)
were transfected with the BtkPH-GFP construct and subjected to a
6-12-h serum deprivation. Addition of insulin (100 nM) to
such cells also stimulated the translocation of the fluorescent signal
to the plasma membrane. Interestingly, in this case, localization of
the construct to intracellular vesicular structures that moved toward
the plasma membrane was also observed. It is also noteworthy that, in
the hepatocytes, the nuclear signal intensity decreased in parallel
with the insulin-induced membrane localization (Fig.
2C).
Membrane Localization of BtkPH-GFP Depends on Increased PI 3-Kinase
Activity and Membrane PI(4,5)P2 Levels--
To determine
whether the Btk PH domain binds to PI(3,4,5)P3, the lipid
product of PI 3-kinase, we used the PI 3-kinase inhibitors wortmannin
and LY 294002 to prevent formation of the lipid in growth
factor-stimulated cells. Addition of wortmannin or LY 294002 to cells
after EGF or PDGF stimulation caused dissociation of BtkPH-GFP from the
plasma membrane (Fig. 3, A and
C). This finding suggested that the Btk PH domain localizes
to the membrane by a mechanism that requires the sustained activity of
PI 3-kinase(s) and supports the notion that PI(3,4,5)P3 is
the membrane component to which the PH domain of Btk binds in living
cells. These results also show that PI(3,4,5)P3 is actively
dephosphorylated during stimulation since, after blocking its increased
synthesis, its level rapidly decreases as indicated by the dissociation
of BtkPH-GFP from the plasma membrane.

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Fig. 3.
EGF-induced changes in the localization of
the wild-type and R28C mutant BtkPH-GFP constructs in transfected NIH
3T3 cells. NIH 3T3 cells expressing the human EGF receptor
together with wild-type BtkPH-GFP (A) or the R28C mutant of
BtkPH-GFP (B) were stimulated with 100 ng/ml EGF after serum
starvation. Wortmannin (WT; 300 nM) was added 4 min after EGF (A). Experiments were carried out at 33 °C,
and confocal images were taken at every 30 s. The
numbers indicate the elapsed time in seconds. Fluorescence
ratios calculated from line intensity plots (see legend to Fig.
1) obtained from each of the frames were plotted against time
(C). , wild-type BtkPH-GFP (means ± S.E.,
n = five cells quantitated); , R28C mutant (based on
a large number of cells observed in several independent
experiments).
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The finding that the maintenance of PI(3,4,5)P3 levels
requires the sustained phosphorylation of PI(4,5)P2 by PI
3-kinases prompted us to investigate whether decreasing the level of
PI(4,5)P2 has an indirect effect on the localization of the
Btk PH domain. Addition of ionomycin (which elevates intracellular
[Ca2+] and thereby induces hydrolysis of
PI(4,5)P2) to EGF-stimulated cells caused the rapid release
of the fluorescence from the membranes back to the cytosol (data not
shown). Since PI(3,4,5)P3 is not hydrolyzed directly by any
known PLC, these findings are consistent with the sensitivity of
PI(3,4,5)P3 levels both to PI 3-kinase activity and to
changes in the level of its substrate, PI(4,5)P2.
Membrane-targeted PI 3-Kinase Leads to Membrane Localization of the
Btk PH Domain--
Next we tested whether cellular production of
3-phosphorylated inositides without receptor stimulation is sufficient
to cause the translocation of the Btk PH domain construct to the plasma membrane. For this, we cotransfected NIH 3T3 cells with a
membrane-targeted form of PI 3-kinase
(PI3K
-CAAX)
(21) and BtkPH-GFP. Expression of this PI 3-kinase construct has been
shown to cause production of PI(3,4,5)P3 and a high level
of activation of the Akt protein kinase in COS-7 cells (21). As shown
in Fig. 4A, this manipulation yielded cells in which the fluorescent construct was localized to the
membranes, but unlike after hormonal stimulation, this localization was
not confined to the plasma membrane. In many cells, there were
intracellular bright spots that, in some cases, formed "aggregates"
with high fluorescent intensities (data not shown). Moreover, very
little nuclear localization of fluorescence was observed in these
cells. Addition of wortmannin (300 nM) caused the release
of fluorescence from the plasma membrane to the cytosol, but it
required a significantly longer time than in cases of acute stimulation
by agonists. Also, the spotty intracellular signal appeared to be more
resistant to wortmannin treatment.

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Fig. 4.
Effects of membrane-targeted PI 3-kinase
(PI3K -CAAX) and PKC
activation on the localization of BtkPH-GFP in NIH 3T3 cells.
A, NIH 3T3 cells were cotransfected with BtkPH-GFP and a
membrane-targeted version of PI 3-kinase (21). Addition of 300 nM wortmannin (WT) slowly reversed plasma
membrane localization. B, NIH 3T3 cells coexpressing
BtkPH-GFP and the human EGF receptor were stimulated with
12-O-tetradecanoylphorbol-13-acetate (TPA; 200 nM) and subsequently with 50 ng/ml PDGF (upper
panels) or with 100 ng/ml EGF after pretreatment with 500 nM bisindolylmaleimide I (lower panels). Shown
are representative cells from several independent observations.
inh., inhibitor.
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Protein Kinase C Activation Is Not a Major Determinant of the Btk
PH Domain Translocation Response--
The C1 region of various protein
kinase C (PKC) isozymes has been shown to interact with the Btk PH
domain based on in vitro binding assay (22). This
interaction was found to be inhibited by agents that bind either to the
C1 domain of PKC (such as phorbol esters) or to the Btk PH domain (such
as PI(4,5)P2). Moreover, recently, the PI
3-kinase-dependent kinase PDK-1 has been shown to
phosphorylate and activate PKC
(23). Therefore, we examined the
effect of phorbol esters and PKC inhibitors on the membrane translocation response of the Btk PH domain. Stimulation of NIH 3T3
cells with phorbol esters
(12-O-tetradecanoylphorbol-13-acetate, 100 nM to
1 µM) had no effect on the distribution of BtkPH-GFP (Fig. 4B). Moreover, stimulation with EGF (or PDGF) induced
translocation of BtkPH-GFP in cells that either were pretreated with
the PKC inhibitor bisindolylmaleimide I (Gö 6850, up to 500 nM, 10 min) (Fig. 4B) or were treated with 100 nM phorbol 12-myristate 13-acetate for 12 h before
PDGF stimulation (data not shown). Similarly, addition of the PKC
inhibitor to PDGF-stimulated cells (after Btk PH domain translocation
to the membrane had taken place) had no significant effect on the
distribution of fluorescence, whereas subsequent addition of wortmannin
rapidly released the fluorescence from the membranes (data not shown).
(The PKC inhibitor applied at this concentration reversed the effects
of phorbol 12-myristate 13-acetate on a variety of cell responses in
our laboratory.) Although these data cannot rule out PKC being a
regulator of the holoprotein Btk, they did not suggest an important
role of PKC in the PI 3-kinase-mediated membrane targeting of the Btk
PH domain.
Mutations within the Btk PH Domain Affect Its Ability to Localize
to the Membrane--
Several mutations of Btk that are responsible for
X-linked agammaglobulinemia are located within the PH domain of the
protein and impair its membrane association (11). Therefore, we
examined the ability of the best characterized mutant, R28C (which is
unable to bind PI(3,4,5)P3 in vitro (6, 8)), to
localize to the membrane in living cells in response to stimulation. As
shown in Fig. 3 (B and C), no membrane
localization of the R28C mutant of BtkPH-GFP was observed after PDGF or
EGF stimulation in NIH 3T3 cells (or in COS-7 cells; data not shown).
The E41K mutation, which causes transformation in NIH 3T3 cells and
shows enhanced membrane association (15), was also introduced into the
BtkPH-GFP construct, and its localization within intact cells was
examined. As shown in Fig. 5A,
the E41K mutant displayed significant membrane localization even in
quiescent NIH 3T3 cells and still displayed translocation in response
to EGF (100 ng/ml). Application of wortmannin (300 nM)
reversed the EGF-induced increase in membrane association, but did not
reduce it below its initial level. Wortmannin also failed to affect the
basal localization of the construct when added to cells that were not
stimulated with EGF (data not shown). In view of the ability of the
E41K mutant to associate with membranes of unstimulated cells, which contain only small amounts (if any) of PI(3,4,5)P3, we
investigated the possibility that this mutant BtkPH-GFP has a
diminished binding specificity for PI(3,4,5)P3 and hence is
also able to bind to PI(4,5)P2 that is present in membranes
of unstimulated cells. As shown in Fig. 5A (panels
d-h), PI(4,5)P2 breakdown evoked by the addition of
the Ca2+ ionophore ionomycin caused a complete release of
the membrane-bound fluorescence into the cytosol, whereas subsequent
chelation of Ca2+ by BAPTA caused a reappearance of the
signal at the plasma membrane. The effect of ionomycin was inhibited by
preincubation with 10 mM neomycin (to inhibit PLC; data not
shown). The relocalization of fluorescence after Ca2+
chelation was not prevented by 300 nM wortmannin, but was
abolished by the addition of 100 µM quercetin, which
inhibits PI(4,5)P2 resynthesis (19) (Fig. 5B).
These changes were very similar to those observed with the PLC
PH
domain-GFP construct (which binds to PI(4,5)P2 (18, 19,
24)) and are consistent with the assumption that the E41K mutation
impairs the ability of the Btk PH domain to discriminate between
PI(3,4,5)P3 and PI(4,5)P2 effectively.

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Fig. 5.
Effects of EGF and ionomycin on the
localization of the E41K mutant of the BtkPH-GFP construct in
transfected NIH 3T3 cells. NIH 3T3 cells expressing the human EGF
receptor and the E41K mutant of BtkPH-GFP were stimulated with EGF (100 ng/ml) and/or ionomycin (10 µM). Shown in A
are confocal images taken at selected times during the experiment from
a representative cell taken through a full protocol. Shown in
B (left panel) are fluorescence ratios
(calculated and plotted as described in the legend to Fig. 1) from
cells stimulated with EGF followed by the addition of wortmannin
(WT; 300 nM) (n = 3). Cells
stimulated with ionomycin (Iono) after a 10-min treatment
with 300 nM wortmannin followed by the addition of BAPTA (2 mM) (n = 6) are shown in B
(right panel). Dashed lines represent cells that
were pretreated with quercetin (querc; 100 µM,
10 min) before addition of ionomycin to inhibit PI(4,5)P2
resynthesis (n = 3). The letters on the
images correspond to the letters on the fluorescence ratio-time plot,
to indicate the time of the recording.
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The E41K Mutant, but Not the Wild-type or R28C Mutant Btk PH
Domain, Interferes with Agonist-induced Hydrolysis of
PI(4,5)P2--
To further investigate whether the E41K
mutant Btk PH domain is able to bind to membrane PI(4,5)P2
within the intact cell, we examined the ability of this construct to
interfere with agonist-induced PI(4,5)P2 hydrolysis. As
shown earlier for the PLC
PH domain (25) and for the pleckstrin PH
domain (26) (both of which bind to PI(4,5)P2), expression
of these molecules inhibited receptor-mediated inositol phosphate
production, presumably by interfering with PLC-mediated hydrolysis of
PI(4,5)P2. As shown in Fig.
6, neither the wild-type Btk PH domain
nor the R28C mutant had a significant effect on angiotensin
II-stimulated inositol phosphate production in COS-7 cells expressing
the AT1a receptor together with the GFP construct of
interest. In contrast, E41K mutant BtkPH-GFP showed a significant
inhibitory effect, although this inhibition was not as strong as that
caused by expression of PLC
PH domain-GFP (Fig. 6).

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Fig. 6.
Effects of expressed PH domains on
angiotensin II-induced formation of inositol phosphates in COS-7
cells. COS-7 cells coexpressing the AT1a angiotensin
II (Ang II) receptor and the respective PH domain-GFP
constructs were labeled with myo-[3H]inositol
(20 µCi/ml, 24 h) and stimulated with 1 µM
angiotensin II in the presence of 10 mM LiCl for 30 min.
Inositol phosphates were extracted and separated by Dowex AG 1-X8
minicolumns. Means ± S.E. from four separate experiments, each
performed in duplicate, are shown. *, significant difference from the
control response (p < 0.001). Ins,
inositol; GFPN, pEGFP-N1 plasmid.
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DISCUSSION |
The important role of the Btk PH domain was originally recognized
after identifying and analyzing mutations that cause X-linked agammaglobulinemia in humans (14). Several (although not all) of these
mutations are within the PH domain of Btk. Some mutations cause a
folding defect, and others affect its function, presumably interfering
with the binding characteristics of this module (27). The ability of
Btk to interact with membranes appears to be the key regulatory element
in determining the function(s) of the kinase, and several lines of
evidence suggest that the PH domain is a critical region of the
molecule for membrane association (27). Although 
-subunits of
heterotrimeric G-proteins (28) and various PKC isozymes (29) have been
shown to interact with the PH domain of Btk, more recent studies
indicate that the 3-phosphorylated inositides,
PI(3,4,5)P3 in particular, are its binding partners in the
membrane. The isolated recombinant PH domain of Btk in the form of a
GST fusion protein has been demonstrated to bind PI(3,4,5)P3 in a BIAcore assay system (6) or by utilizing
lipid micelles in vitro (8). Binding of the lipid to the PH
domain of Btk was found to depend on the ionic composition (6), and the
fatty acid side chains of PI(3,4,5)P3 were also shown to be important for the interaction (8). In contrast, soluble inositol 1,3,4,5-tetrakisphosphate was found to bind to the Btk PH domain with
high affinity in one report (30). These in vitro studies also showed that the binding of the Btk PH domain to
PI(4,5)P2 is much weaker, providing the specificity that
would be required for its general regulation by 3-phosphorylated
inositides. In all of these studies, the R28C mutant PH domain,
which causes X-linked immunodeficiency in mice, was found to be unable
to bind PI(3,4,5)P3.
In this report, we provide evidence that the isolated PH domain of Btk
fused to the fluorescent reporter molecule GFP exhibits agonist-dependent membrane association when expressed in a
variety of cells. This method allowed analysis of the binding
specificity as well as imaging of spatiotemporal changes in the
membrane association of this protein module inside single living cells.
Our results show that the Btk PH domain does not localize to membranes
inside quiescent cells (suggesting that its binding to
PI(4,5)P2 is probably too weak for such membrane targeting)
and that the agonist-induced translocation of BtkPH-GFP to membranes is
dependent on PI 3-kinase activation as well as on membrane
PI(4,5)P2 levels. Moreover, the production of
PI(3,4,5)P3 by membrane-targeted PI 3-kinase was sufficient
to target the Btk PH domain to membranes. These results, together with
in vitro binding data (6, 8), further support the idea that
the Btk PH domain binds to PI(3,4,5)P3 of the plasma
membrane and that this mechanism is sufficient to regulate membrane
localization of the protein.
These results do not rule out the possibility that additional factors
(proteins) participate in the membrane anchoring of the Btk PH domain,
similarly to the protein anchors (RACK proteins) (31) that stabilize
the various motifs of PKC. Similarly, even though the Akt kinase has a
PH domain that appears to be sufficient to localize this protein to
3-phosphorylated inositides (32), its activation requires PDK-1, a
kinase that also possesses a PH domain and that also is regulated by
3-phosphorylated lipids (33). Although in vitro studies
suggested that the C1 domain of various PKC isozymes can interact with
the Btk PH domain (22), our studies in intact cell did not provide
evidence for a role of PKC in the membrane targeting of the Btk PH
domain. Based on the studies of Yao et al. (22), the
association of the Btk PH domain with PKCs would be the strongest in
quiescent cells, and either the binding of diacylglycerol (or phorbol
12-myristate 13-acetate) to PKC or the inositol lipid head group to the
Btk PH domain (i.e. activation of the respective enzymes)
would eliminate this association. Certainly, stimulation with phorbol
12-myristate 13-acetate, which has been widely demonstrated to induce
membrane translocation of various PKC isozymes (34, 35), failed to induce membrane translocation of the Btk PH domain in the present study, and a PKC inhibitor did not affect translocation induced by
PDGF. The existence of additional factors contributing to the membrane
localization of PH domains has been suggested by recent studies in
which the cytohesin-1 PH domain was shown to require a small basic
flanking sequence to effectively localize it to membranes and to exert
a dominant-negative effect on cell adhesion (36). In this context, it
is important to note that our Btk PH domain construct also contained
the small adjacent Btk motif that had been found important for proper
folding in bacteria (27). Clearly, more studies are needed to fully
explore the complexity of inositol lipid-PH domain interactions that
regulate Btk and other PI 3-kinase-regulated effectors.
Introduction of the R28C mutation into the BtkPH-GFP construct
prevented its membrane localization in response to stimulation, in
agreement with the data regarding the diminished affinity of this
mutant for PI(3,4,5)P3 (6, 8, 30). Arg-28 is located within
the predicted inositide-binding pocket of the Btk PH domain and, based
on structural alignments, corresponds to Arg-40 of the PLC
PH
domain. Mutation of this residue in the latter molecule (which makes
contact with the 5-phosphate of PI(4,5)P2 (24)) prevents
its binding to PI(4,5)P2 and its membrane association (18,
19). Based on this analogy, it is expected that replacing the
positively charged Arg-28 with a non-charged residue would result in
the loss of binding affinity for PI(3,4,5)P3.
Another mutation within the Btk PH domain, E41K (15), was found to show
increased membrane localization in quiescent cells and further
translocation in response to EGF stimulation. Although there are a
number of reasons why this mutant protein could bind to the membrane
(including a higher affinity for PI(3,4,5)P3), its similar
behavior compared with the PLC
PH domain raised the possibility that
it also binds to membrane PI(4,5)P2. Membrane association
of the E41K mutant of BtkPH-GFP was not abolished by PI 3-kinase
inhibitors, but showed correlation with PI(4,5)P2 levels
after manipulations of the latter by Ca2+ ionophores,
Ca2+ chelators, and inhibitors of PI(4,5)P2
resynthesis. Also, it showed a significant inhibitory effect on
agonist-induced PI(4,5)P2 hydrolysis, a feature of PH
domains that bind PI(4,5)P2. Comparison of the crystal
structure of PLC
and Btk reveals that Glu-41 is located in a
position that corresponds to a region of the PLC
PH domain (Ser-55
and Arg-56) that makes important contacts with the phosphates at the 4- and 5-positions of the inositol ring in PI(4,5)P2 (24). An
acidic amino acid (Glu-41) in this position could provide a significant
repulsive force to prevent association of the mutant protein with
PI(4,5)P2. Mutation of Glu-41 to Lys would therefore be
expected to increase PI(4,5)P2 binding. Indeed, an
analogous mutation within the PH domain of PLC
(E54K) has been
reported to enhance the catalytic activity of the enzyme, presumably by
increasing its affinity to PI(4,5)P2 (37). Such an affinity
increase in the E41K mutant of Btk toward both PI(4,5)P2 and PI(3,4,5)P3 could explain why the E41K substitution did
not significantly affect the ability of Ins(1,4,5)P3 to
displace Ins(1,3,4,5)P4 in the binding studies performed on
the isolated BtkPH-GST fusion protein (30).
Members of the Tec tyrosine kinase family have been recognized recently
as important modulators of Ca2+ influx pathways in B- and
T-lymphocytes (38) via a mechanism that amplifies inositol
1,4,5-trisphosphate formation after PLC
activation (39, 40). This
function of the kinases also relies upon the interaction of their PH
domains with membrane PI(3,4,5)P3 (39). However, it is also
important to note that the present results only explore the aspect of
Btk function from the standpoint of its PH domain and that additional
interactions mediated by other domains of the molecule may greatly
affect the overall localization of the holoprotein. Nevertheless, the
ability of the isolated Btk PH domain to confer
PI(3,4,5)P3-dependent membrane localization may
also be utilized as a probe that can detect changes in the level of
this lipid in single living cells with fine spatial resolution. Such a
feature of other PH domain-GFP fusion constructs that can interact with
PI(3,4,5)P3 has been recently demonstrated in
insulin-stimulated adipocytes (41) and EGF-stimulated PC-12 cells
(42).
In summary, we have shown that the isolated PH domain of Btk interacts
with plasma membranes with characteristics that are consistent with its
binding to membrane PI(3,4,5)P3. This interaction appears
to be a fundamental aspect of the Btk protein that is severely
compromised in a human disease and can now be monitored in single
living cells. This methodology will also help to better understand the
role of inositide phospholipids in membrane-protein interactions.