(Received for publication, December 31, 1996, and in revised form, March 4, 1997)
From the Division of Allergy, La Jolla Institute for Allergy and
Immunology, San Diego, California 92121, the
Pharmaceutical Research Laboratory, Kirin Brewery Co.,
Ltd., Takasaki, Gunma 370-12, Japan, and the ¶ Laboratory of
Cellular Oncology, NCI, National Institutes of Health,
Bethesda, Maryland 20892
Pleckstrin homology (PH) domains comprised of
loosely conserved sequences of ~100 amino acid residues are a
functional protein motif found in many signal-transducing and
cytoskeletal proteins. We recently demonstrated that the PH domains of
Tec family protein-tyrosine kinases Btk and Emt (equal to Itk and Tsk)
interact with protein kinase C (PKC) and that PKC down-regulates Btk by
phosphorylation. In this study we have characterized the PKC-BtkPH
domain interaction in detail. Using pure PKC preparations, it was shown
that the Btk PH domain interacts with PKC with high affinity
(KD = 39 nM). Unlike other tested
phospholipids, phosphatidylinositol 4,5-bisphosphate, which binds to
several PH domains, competed with PKC for binding to the PH domain
apparently because their binding sites on the amino-terminal portion of
the PH domains overlap. The minimal PKC-binding sequence within the Btk
PH domain was found to correspond roughly to the second and third
-sheets of the PH domains of known tertiary structures. On the other
hand, the C1 regulatory region of PKC
containing the pseudosubstrate and zinc finger-like sequences was found to be sufficient for strong
binding to the Btk PH domain. Phorbol 12-myristate 13-acetate (PMA), a
potent activator of PKC that interacts with the C1 region of PKC,
inhibited the PKC-PH domain interaction, whereas the bioinactive PMA
(4-
-PMA) was ineffective. The
isoform of PKC, which has a single
zinc finger-like motif instead of the two tandem zinc finger-like
sequences present in conventional and novel PKC isoforms, does not bind
PMA. Thus, as expected, PH domain binding with PKC
was not
interfered with by PMA. Further, inhibitors that are known to attack
the catalytic domains of serine/threonine kinases did not affect this
PKC-PH domain interaction. In contrast, the presence of physiological
concentrations of Ca2+ induced less than a 2-fold increase
in PKC-PH domain binding. These results indicate that PKC binding to PH
domains involve the
2-
3 region of the Btk PH domain and the C1
region of PKC, and agents that interact with either of these regions
(i.e. phosphatidylinositol 4,5-bisphosphate binding to the
PH domain and PMA binding to the C1 region of PKC) might act to
regulate PKC-PH domain binding.
The importance of functional protein motifs in various signal transduction pathways is well documented. For example, Src homology (SH)1 2 and SH3 domains play essential roles in signal transduction for cell activation and growth by interacting with phosphotyrosine residues in the context of surrounding sequences (1-3) and short proline-rich stretches (2-4), respectively. Pleckstrin homology (PH) domains are comprised of loosely conserved sequences of approximately 100 amino acid residues (5, 6). Originally recognized as repeated sequences in the platelet protein pleckstrin (a prominent substrate for protein kinase C (PKC)), PH domains have been found in more than 60 proteins (7-9). Most PH domain-containing proteins have been implicated in signal transduction or in cytoskeletal functions and include guanosine triphosphatases (GTPases), GTPase-activating proteins, guanine nucleotide exchange factors, serine/threonine kinases, tyrosine kinases, and phospholipases C (PLCs).
The tertiary structures of four PH domains to date have been determined
by nuclear magnetic resonance spectroscopic or x-ray crystallographic
techniques (10-16). Basic structural features are shared by the PH
domains of pleckstrin, -spectrin, dynamin, and PLC-
1. The core of
the compact domains is an antiparallel
-sheet consisting of seven
strands. The amino-terminal four strands form a pocket-like structure,
suggestive of a ligand-binding site. The carboxyl-terminal
-helix
follows the last
-sheet. Studies by Lefkowitz and co-workers (17,
18) established that the sequence encompassing the carboxyl-terminal
portion and downstream sequences of the PH domain of the
-adrenergic
receptor kinase and several other proteins interacts with the
complex of heterotrimeric GTP-binding proteins (G-proteins). The
invariant tryptophan residue in the carboxyl-terminal
-helical
region of the PH domain was later shown to be important for this
interaction (19). The interacting counterpart is the WD40 repeats of
the
subunit of G-protein (20). More recently, several studies
showed that various PH domains bind to phosphatidylinositol
4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate
(IP3) through their positively charged residues in the
amino-terminal four
-sheets (15, 21, 22). PIP2
interactions with the amino-terminal PH domain of pleckstrin (21) and
the PLC-
1 PH domain (22) occur with low affinity (KD = ~30 and 1.7 µM, respectively)
while the PLC-
1 PH domain binds IP3 with high affinity
(KD = 210 nM, Ref. 22). Membrane
localizing functions have been implicated for the binding of PH domains
to G-protein
complexes and PIP2. Indeed, many PH
domain-containing proteins are known to be localized at the plasma
membrane or other membrane structures.
We recently demonstrated that the PH domains of protein-tyrosine
kinases (PTKs) Btk and Emt (equal to Itk and Tsk) interact directly
with PKC (23). We also presented evidence that PKC phosphorylates Btk
and inhibits the kinase activity of the latter enzyme, suggesting that
the PH domain-PKC interaction plays a regulatory role for Btk.
Mutations in the gene encoding Btk lead to immunodeficiencies in humans
(X-linked agammaglobulinemia, see Refs. 24 and 25) and mice (X-linked
immunodeficient mice; xid, see Refs. 26 and 27). Btk and Emt
constitute a distinct subgroup (Tec family) of PTKs along with TecII
(28), Dsrc28 (29), Bmx (30), and Txk/Rlk (31, 32). In the present study we have characterized in detail the interaction between the Btk PH
domain and PKC using an in vitro binding assay. A high
affinity binding between PKC and the Btk PH domain was demonstrated by this assay. The interaction sites were mapped to the amino-terminal 2-
3 region of the Btk PH domain and the C1 regulatory region of
PKC
. In accordance with the mapping data, PIP2 competes
with PKC for binding to the PH domains of Btk and Emt while bioactive PMA that binds to the C1 region of PKC also inhibited the PKC-PH domain
interaction.
Purified rat brain PKC (>95% pure; a mixture of
,
, and
isoforms) was purchased from Calbiochem.
Anti-glutathione S-transferase (GST) antiserum and pGEX-3T
(33) were a kind gift from Dr. Wolfgang Northemann (ELIAS
Entwicklungslabor). Glutathione-agarose beads, PIP2,
and other chemicals were obtained from Sigma unless otherwise described. Another source of PIP2 was Calbiochem.
An immortalized murine mast cell line, MCP-5 (34),
was cultured in RPMI 1640 medium supplemented with 10% fetal bovine
serum, 50 µM 2-mercaptoethanol, and interleukin-3
(culture supernatants of mouse interleukin-3 gene-transfected cells).
HMC-1 human mast cells (35) were cultured in RPMI 1640 medium
containing 10% fetal bovine serum and 50 µM
2-mercaptoethanol. NIH/3T3 transfectants overexpressing PKC or
c-Raf-1 fragments containing the carboxyl-terminal 12 residues of
PKC
as an epitope tag (36-38) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum.
DNA fragments encoding the PH domains of Btk
and Emt were amplified with polymerase chain reaction using the cloned
murine btk and emt cDNAs (39), respectively,
as templates. 5-Polymerase chain reaction primers contain a
BamHI recognition sequence attached to the downstream
sequence of interest. The polymerase chain reaction products were
cloned into pCRII vector (Invitrogen, San Diego). Limited DNA sequence
analysis was performed to verify the pCRII clones. The insert sequences
released from the vector sequence by digestion with BamHI
and EcoRI were ligated to pGEX-3T. GST proteins used in this
study were GST-BtkPH (coding for residues 1-139 of Btk), GST-EmtPH
(residues 1-109), and truncated Btk PH domain fragments. GST fusion
proteins containing the subregions of the Btk PH domain were designated
according to the Btk residue numbers at the amino and carboxyl termini,
e.g. 28/77, which codes for the region from residue 28 to
77.
pGEX-3T constructs were
transformed into an Escherichia coli strain XL1 Blue
(Stratagene). Fusion proteins were expressed by inducing 1 liter of
log-phase bacteria with 0.4 mM
isopropyl--D-thiogalactopyranoside overnight at
26 °C. Cells were collected and sonicated in phosphate-buffered saline, containing 1 mg/ml lysozyme, 1 mM
phenylmethylsulfonyl fluoride, and 1% Triton X-100. Lysates were
cleared by centrifugation at 12,000 × g for 10 min at
4 °C and filtrated through 0.22-µm filter Millex-GS (Millipore,
Bedford, MA). Five hundred µl of glutathione-agarose beads were
incubated with lysates overnight at 4 °C and then washed thoroughly
with phosphate-buffered saline, containing 1% Triton X-100 and 0.02%
sodium azide. Purities and amounts of fusion proteins bound to beads
were assessed after elution with SDS-sample buffer by SDS-polyacryamide
gel electrophoresis and Coomassie Brilliant Blue staining.
MCP-5 or HMC-1 cells were used to examine PKC binding
activities of GST fusion proteins in vitro. Cells were lysed
in 1% Nonidet P-40 buffer (Nonidet P-40 lysis buffer) containing 20 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1 mM CaCl2, 0.1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 16.5 µg/ml aprotinin, 10 µg/ml leupeptin, 25 µM
p-nitrophenyl p-guanidinobenzoate, and 0.1% sodium azide. Experiments shown in Fig. 5A were done using
Nonidet P-40 lysis buffer with various concentrations of
Ca2+ in place of 0.1 mM CaCl2.
Lysates (equivalent to 1 × 107 cells) were clarified
by centrifugation at 16,000 × g at 4 °C for 10 min
and then mixed with 1-2 µg of GST fusion proteins immobilized onto
glutathione-agarose beads for 4 h at 4 °C. Experiments shown in
Fig. 1 were done using rat brain PKC in place of cell lysates. In some
experiments sonicated lipid vesicles or other chemicals were included
in the mixture of GST fusion protein beads and mast cell lysates. Beads
were washed six times with Nonidet P-40 lysis buffer, and bound
proteins were separated by SDS-polyacrylamide gel electrophoresis and
blotted to Immobilon-P membranes (Millipore Corp.). PKC binding ability
was evaluated by incubation with anti-PKC monoclonal antibody (MC5,
Santa Cruz Biotechnology, Santa Cruz, CA) that detects conventional
isoforms of PKC (cPKC;
,
, and
) or PKC isoform-specific
antibodies (Santa Cruz Biotechnology and Transduction Laboratories,
Lexington, KY), followed by incubation with a horseradish
peroxidase-conjugated secondary antibody and visualization with the
enhanced chemiluminescence kit (Amersham Corp.). Anti-PKC
(Life
Technologies Inc.) was used for detection of PKC
holoenzyme and
-epitope-tagged fragments of PKC
or c-Raf-1.
A far Western blotting experiment showed that
GST-BtkPH immobilized onto a polyvinylidene difluoride membrane binds
to purified rat brain PKC (23), indicating the direct interaction
between the PH domain and PKC. The PKC preparations used in the above and following experiments are a pure (>95%) population of the Ca2+-dependent isoforms of ,
, and
collectively termed cPKC. To analyze the kinetics of the interaction,
rat brain PKC was incubated with GST-BtkPH immobilized onto
glutathione-agarose beads. Bound PKC was detected by immunoblotting
with a monoclonal anti-cPKC antibody (MC5) in conjunction with the
enhanced chemiluminescence method. The amounts of bound PKC were
quantified using the standard curve obtained by immunoblotting of known
amounts of rat brain PKC. The results shown in Fig. 1
demonstrated that a pure population of rat brain PKC binds to GST-BtkPH
in a concentration-dependent manner. The dissociation
constant KD of this interaction was 39 nM as revealed by Scatchard analysis (data not shown). This
affinity is much higher than those of the PIP2-PH domain interactions (see "Discussion").
To
define the determinant for PKC binding within the PH domain of Btk, the
solid-phase binding assay (23) was used. We prepared several
carboxyl-terminal deletion mutants derived from the parental
pGEX-3T-BtkPH plasmid and other truncated mutants and determined their
ability to bind PKC in MCP-5 murine mast cell lysates (Fig.
2A). As shown in Fig. 2B, the
carboxyl-terminal truncated PH domain fusions that retain the
amino-terminal sequence up to residue 45, as well as the amino- and
carboxyl-terminal truncated PH domain fusion 28/77, bound to cPKC.
Therefore, the minimal PKC-binding sequence corresponds to the region
encompassing -sheets 2 and 3 as deduced from tertiary structural
studies on several PH domains (Fig. 2C).
Effects of Phospholipids on PKC Binding to the Btk PH Domain
The above mapping results indicated that the minimal
PKC-binding sequence overlaps the binding site for PIP2 and
IP3 shown for several PH domains including that of Emt
(Fig. 2C). Therefore, we examined possible competition for
binding to the Btk PH domain between PKC and various phospholipids.
GST-BtkPH beads were incubated with MCP-5 mast cell lysates in the
presence of various concentrations of phospholipids. No effects on the
interaction between cPKC and GST-BtkPH were found with such lipids as
phosphatidylserine (up to 32 µg/ml, data not shown),
phosphatidylcholine (up to 115 µg/ml), phosphatidylethanolamine (up
to 115 µg/ml, data not shown), phosphatidylinositol (up to 115 µg/ml), and phosphatidylinositol 4-phosphate (up to 115 µg/ml)
(Fig. 3). In contrast, PIP2 inhibited cPKC
binding to the Btk PH domain in a dose-dependent manner
(Fig. 3). The IC50 (~1 µM) estimated by
densitometric measurements of the bound PKC bands was similar to the
reported dissociation constant between PIP2 and the
PLC-1 PH domain (1.7 µM). Similar data were obtained using PIP2 from two different suppliers. The
PIP2 competition for binding with cPKC also was observed
with GST-EmtPH (data not shown).
Mapping of the PH Domain-binding Site within the Regulatory Region of PKC
GST-BtkPH bound to both
Ca2+-dependent (,
I, and
II) and
Ca2+-independent isoforms (
and
) in mast cell
lysates (data not shown). To determine the PH domain-binding region
within PKC
we utilized stably transfected NIH/3T3 cell lines that
overexpress the epitope-tagged domain fragments of PKC
depicted in
Fig. 4 (37, 38). For comparison, we also used NIH/3T3
cells expressing c-Raf-1 fragments tagged with the same epitope (data
not shown). These cells were lysed, and the extracts were used in the
solid-phase binding assay with GST-BtkPH beads. Bound PKC
fragments
were detected by immunoblotting with anti-
(epitope) antibody. The results showed that the relative band intensities of the
GST-BtkPH-bound PKC
fragments normalized to their expression level
in cell lysates were high with the PKC
holoenzyme and with fragments
1 and
2 (Fig. 4). However, fragments
3 and
7, which lack
the pseudosubstrate region, exhibited lower but significant binding
capacities to GST-BtkPH. GST control beads bound negligible amounts of
these PKC
fragments. These data demonstrated that the C1 region of PKC
is sufficient for interaction with the Btk PH domain. Neither RI-
nor RIII-
fragments derived from the regulatory and catalytic domains, respectively, of c-Raf-1 (38) bound to the PH domain (data not
shown). RI-
contains the zinc finger-like sequence of c-Raf-1 that
has a low level homology (~30% identity) with that of the C1 region
of PKC. Therefore, the lack of PH domain binding to RI-
indicates a
high specificity of PKC-PH domain interactions. An epitope-tagged rat
PKC
I construct encompassing residues 312-671, which correspond to
the catalytic domain, failed to bind to GST-BtkPH (data not shown).
PIP2 binds to PH domains (21, 22). Since PIP2
also binds to the C2 region of PKC (40), there was a possibility that the observed PIP2 competition with cPKC holoenzyme for
binding to PH domains (Fig. 3) could be due to the allosteric effect of PIP2 bound to the C2 region. Therefore, effects of
PIP2 on the interactions between GST-BtkPH and the PKC
fragments
2 and
3, which have no PIP2-binding region,
were examined. The addition of 60 µg/ml PIP2 to the
binding mixtures inhibited these interactions by more than 70% (Fig.
4), suggesting that PIP2 binds to the PH domain to prevent
2 and
3 from interacting with GST-BtkPH. In contrast, the
presence of 60 µg/ml phosphatidylserine did not inhibit the
GST-BtkPH-PKC
fragment interactions (data not shown).
Activation of cPKCs requires
Ca2+ and diacylglycerol/PMA in addition to
phosphatidylserine (41). Effects of these factors on the PKC-PH domain
interaction were examined using GST-BtkPH and mast cell lysates. The
addition of 0.1-5 µM Ca2+ resulted in less
than a 2-fold increase in cPKC binding compared with the binding in the
absence of Ca2+ or in the presence of EGTA (Fig.
5A), although non-physiologically high
Ca2+ concentrations (10-1000 µM) induced up
to a 5-fold increase in cPKC binding (data not shown). 1 µM Ca2+ did not affect significantly the PH
domain binding to recombinant PKCI compared with that in the absence
of Ca2+ (data not shown). These data are consistent with
the above mapping data that the primary PH domain-binding site is the
C1 region.
PMA inhibited cPKC binding to GST-BtkPH in a dose-dependent
manner with an IC50 of ~1 µM, while the
bioinactive PMA (4--PMA) did not alter the level of bound cPKC (Fig.
5B). These results are consistent with the mapping results.
Thus, PMA (but not 4-
-PMA) binds to the C1 region of conventional
and novel PKC isoforms, which are composed of two tandem zinc
finger-like motifs. In contrast, the PKC
and PKC
/
isoforms
have only one zinc finger-like motif and thus lack the ability to bind
PMA (42, 43). The above mapping data strongly suggest that the C1
region of PKC binds to the amino-terminal region of the PH domain
containing the
2-
3 sheets. If this is the case, the PH domain
interaction with PKC
should not be susceptible to PMA-mediated
inhibition due to the lack of interaction between PKC
and PMA.
Indeed, the PKC
-GST-BtkPH interaction was not affected by increasing
concentrations of PMA up to 100 µM (Fig.
5C).
We also examined the effects of inhibitors of PKC and of other serine/threonine kinases on PKC-PH domain interaction. Staurosporine (a potent PKC inhibitor), KT-5720 (a selective protein kinase A inhibitor), and KT-5926 (a specific inhibitor of myosin light chain kinase), all showed little, if any, effect on the cPKC-PH domain interaction in HMC-1 human mast cell lysates (data not shown).
In this report we have described a biochemical
characterization on PKC-PH domain interactions. Pure PKC preparations
interact with the Btk PH domain with high affinity. The
interaction sites were mapped to the 2-
3 region of the
Btk PH domain and the C1 region of PKC
. This assignment of
the interaction sites was supported by several pieces of
experimental evidence. First, PIP2, whose binding
site within PH domains overlaps that of PKC, competed for PH
domain binding with PKC. Second, PMA that binds to the C1
region of PKC also interferes with PKC-PH domain interactions. Further, PMA-mediated inhibition was not observed with
PKC
-PH domain interactions because of the lack of binding capacity
of PMA to the C1 region of PKC
.
The KD value (39 nM) obtained for the
interaction between rat brain PKC and the Btk PH domain is at least
40-fold lower than the reported KD values for
PIP2 binding to PH domains (~30 µM for the
amino-terminal PH domain of pleckstrin and 1.7 µM for the
PLC-1 PH domain) and 5-fold lower than for IP3 binding
to the PLC-
1 PH domain (KD = 210 nM).
The concentrations of PKC
and PKC
, two major PKC isoforms in a
mast cell line, MC-9, were shown to be 26.0 and 63.9 nM,
respectively (44). Accordingly, PKC
is a major Btk-associated
isoform of PKC in mast cells (23). The PKC-binding site within PH
domains deduced from in vitro binding assays using truncated
PH domain fragments is overlapped by the PIP2- and
IP3-binding sites determined by nuclear magnetic resonance
and x-ray crystallographic methods. In accordance with this,
PIP2 competed with PKC for binding to the PH domain of Btk
with an IC50 of ~1 µM. This is similar to the KD (1.7 µM) for the
PIP2 interaction with the PLC-
1 PH domain. However,
cross-linking of the high affinity IgE receptor, which results in
increased levels of PIP2, did not induce significant changes in the levels of PKC
co-immunoprecipitated with Btk in the
cytosolic and membrane compartments for at least 10 min following activation (23).2 This may reflect the high
affinity nature of the PKC-PH domain interaction. The experiments shown
in Fig. 1 indicate that only 1 out of every 15 molecules of GST-BtkPH
protein in the binding mixture could bind PKC. This low efficient
binding could be due to low frequencies of properly folded PH domain,
denaturation of PKC preparation, or both.
The minimal PKC-binding sequence was localized to the amino-terminal
portion (residues 28-45) of the PH domain of Btk, which overlaps the
reported PIP2-binding site on the amino-terminal PH domain
of pleckstrin (Fig. 2C). This region corresponds roughly to
-sheets 2 and 3, which form a major part of the
-barrel. It is
noteworthy that the hydrophobic residues of the
2-
3 region following the basic residue corresponding to Arg-28 in Btk are well
conserved among most of PH domain-containing proteins. Therefore, these
conserved residues seem to be major determinants for PKC binding, and
most PH domains presumably have PKC-interacting capacity. Mutations of
Arg-28 in Btk are reported to be pathogenic for xid mice
(substitution of Cys for Arg-28 (26, 27) and for some X-linked
agammaglobulinemia patients (substitution of His for Arg-28 in two
unrelated cases (45, 46)). Therefore, defects in binding to either PKC
or PIP2 might be the mechanism for these immunodeficiencies. A gain-of-function mutation also was obtained by
the substitution of residue Glu-41 with Lys in this region of Btk. This
mutated form of Btk caused transformation of NIH/3T3 cells and resulted
in enhanced membrane localization of Btk (47). Therefore, it will be
interesting to determine if this mutant Btk has an altered PKC- or
PIP2-binding capacity. The competition for the subregion of
the PH domain between PKC and PIP2 raises an interesting
question as to a possible regulatory role for this competitive binding.
The capacity of PH domains to bind to PIP2 and the
G-protein
complex is implicated in localizing the PH domain-containing proteins to the membrane compartment. Since PKC can
be found localized both in the cytosol and membrane compartments, the
PKC binding property of PH domains gives more versatility to the
subcellular distribution of PH domain-containing signaling molecules.
It can be envisioned that a PH domain-containing signaling protein,
e.g. Btk, might translocate to the membrane in response to
cell stimulation (48) where the PH domain-containing signaling protein
could be sorted to either PIP2- or PKC-bound forms and be
subjected to distinct regulatory parameters to fulfill specific functions. Cell stimulation, e.g. Fc
RI cross-linking,
which can induce the activation of both G-proteins and tyrosine kinases such as Btk and Emt (48, 49), may complicate the regulation of the PH
domain-containing signaling proteins by possible interactions with the
subunits of G-protein, PKC, and PIP2 (and other
phospholipids). Further experiments are warranted to test these
interesting possibilities.
PKC is a large family of serine/threonine kinases implicated in a
variety of cellular functions including proliferation, differentiation, and membrane receptor functions (50). Molecular characterization has
defined at least three classes of PKCs: conventional PKC isoforms that
contain both the C1 and C2 conserved regulatory regions and are
dependent for their activation on Ca2+; novel PKCs that
lack the C2 region and, therefore, are Ca2+-independent;
and atypical PKCs that contain an atypical C1 region composed of a
single zinc finger-like motif instead of the tandem duplicated zinc
finger-like motifs found in the other PKC classes. The PH domain
binding observed with both Ca2+-dependent
(conventional; ,
I, and
II) and Ca2+-independent
(novel (
) and atypical (
)) isoforms of PKC suggests that the PH
domain-binding site of PKC may be localized to the conserved sequences
found in various isoforms of PKC. In vitro binding assays
using NIH/3T3 cells overexpressing epitope-tagged fragments of PKC
demonstrated that the C1 region of PKC
is sufficient for binding to
the Btk PH domain. The sequence around the pseudosubstrate region
upstream of the zinc finger-like motifs contributed significantly to
strong binding to the PH domain. However, the tandem zinc finger-like region alone was sufficient for weak binding to the PH domain. There is
only limited homology around the pseudosubstrate sequence (contained in
residues 133-165 of PKC
) among the various PKC isoforms. However,
there is a cluster of several conserved residues with hydrophobic side
chains following the invariant Ala residue (Ala-157 in PKC
) in the
C1 region. The involvement of these hydrophobic residues in
interactions with the hydophobic
2-
3 region of PH domains is
implied since PH domain interactions with fragments
2 and
3 were
found to be resistant to 1 M NaCl washes (data not shown).
In addition to the C1 region, other regions also might play some role
in binding to the PH domain. One such candidate is the C2 region.
Indeed, non-physiologically high Ca2+ concentrations
(10-1000 µM) increased PKC-PH domain binding up to
5-fold over that in the absence of Ca2+. This effect might
be accounted for by allosteric effects on the PH domain-binding C1
region invoked by Ca2+ bound to the C2 region. However, the
normal physiological fluctuation range of intracellular
Ca2+ concentrations (less than 1 µM) would
lead to only a less than a 2-fold change in the PKC-PH domain binding.
Accordingly, we could not detect a significant change in the PKC
levels co-immunoprecipitated with Btk before and after mast cell
activation by cross-linking of the high affinity IgE receptor (23). The
possible involvement of the catalytic domain of PKC in PKC-PH domain
interactions was ruled out by the present study, since the Btk PH
domain did not bind to either kinase domains of either PKC
I or
c-Raf-1 (RIII-
).
Evidence for in vivo PKC-PH domain interactions so far has
been reported only for the PH domains of Btk, Emt, and Rac (23, 49,
51). However, this interaction seems to be more generally present.
There are various lines of circumstantial evidence that support this
possibility. In unstimulated T cells a PH domain-containing protein,
spectrin, is colocalized with PKCII. Upon stimulation with either
PMA or anti-T cell antigen receptor antibody, these two proteins are
coincidentally translocated to the same focal aggregates in the
cytoplasm (52). The PH domain of spectrin might be involved in this
colocalization event. Indeed, the PH domain of
-spectrin has been
shown to be capable of associating with brain membranes (53). Both PH
domain-containing proteins and PKC are implicated in various
signal-transducing pathways. Therefore, it is likely that some of the
signaling functions of PKC may be mediated through interactions with PH
domain-containing proteins. Physiological relevance of the PH
domain-mediated Btk-PKC interaction was supported by a recent study
with PKC
gene knock-out mice (54). The phenotype of PKC
null mice
was shown to be quite similar to those of xid and
btk null mice.
We thank Dr. Kimishige Ishizaka for encouragement and discussion during the course of this study, Dr. Alexandra C. Newton for kind advice, and Drs. Tomas Mustelin and Katsuji Sugie for critical reading of the manuscript.