Interactions between Protein Kinase C and Pleckstrin Homology Domains
INHIBITION BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE AND PHORBOL 12-MYRISTATE 13-ACETATE*

(Received for publication, December 31, 1996, and in revised form, March 4, 1997)

Libo Yao , Hidefumi Suzuki Dagger , Koichiro Ozawa §, Jianbei Deng , Csaba Lehel , Hiromi Fukamachi Dagger , Wayne B. Anderson , Yuko Kawakami and Toshiaki Kawakami par

From the Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, the Dagger  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

ABSTRACT
INTRODUCTION
Experimental Procedures
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 beta -sheets of the PH domains of known tertiary structures. On the other hand, the C1 regulatory region of PKCepsilon 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-alpha -PMA) was ineffective. The zeta  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 PKCzeta 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 beta 2-beta 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.


INTRODUCTION

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, beta -spectrin, dynamin, and PLC-delta 1. The core of the compact domains is an antiparallel beta -sheet consisting of seven strands. The amino-terminal four strands form a pocket-like structure, suggestive of a ligand-binding site. The carboxyl-terminal alpha -helix follows the last beta -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 beta -adrenergic receptor kinase and several other proteins interacts with the beta gamma complex of heterotrimeric GTP-binding proteins (G-proteins). The invariant tryptophan residue in the carboxyl-terminal alpha -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 beta  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 beta -sheets (15, 21, 22). PIP2 interactions with the amino-terminal PH domain of pleckstrin (21) and the PLC-delta 1 PH domain (22) occur with low affinity (KD = ~30 and 1.7 µM, respectively) while the PLC-delta 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 beta gamma 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 beta 2-beta 3 region of the Btk PH domain and the C1 regulatory region of PKCepsilon . 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.


Experimental Procedures

Reagents

Purified rat brain PKC (>95% pure; a mixture of alpha , beta , and gamma  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.

Cells

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 PKCepsilon or c-Raf-1 fragments containing the carboxyl-terminal 12 residues of PKCepsilon as an epitope tag (36-38) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

DNA Constructs

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.

Immobilization of Fusion Proteins

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-beta -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.

Solid-phase Binding Assay with GST Fusion Protein Beads

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; alpha , beta , and gamma ) 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-PKCepsilon (Life Technologies Inc.) was used for detection of PKCepsilon holoenzyme and epsilon -epitope-tagged fragments of PKCepsilon or c-Raf-1.


Fig. 5. Effects of cPKC activation factors on the PKC-PH domain interaction. A, effects of Ca2+. GST-BtkPH (1 µg) beads were mixed with MCP-5 mast cell lysates in the absence or presence of various concentrations of CaCl2 or 1 mM EGTA, and bound cPKC was detected as described in Fig. 2B. As controls, GST was used in the presence or absence of 5 µM CaCl2. B, effects of PMA. GST or GST-BtkPH (1 µg each) beads were mixed with HMC-1 cell lysates in the absence or presence of various concentrations of PMA (upper panel) or 4-alpha -PMA (lower panel), and bound cPKC was detected as described in Fig. 2B. C, effects of PMA on the PKCzeta -PH domain interaction. GST or GST-BtkPH (1 µg) beads were incubated with detergent lysates of HMC-1 cells in the absence or presence of various concentrations of PMA. Bound PKCzeta was detected by immunoblotting. DMSO, dimethyl sulfoxide.
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Fig. 1. Kinetics of the interaction between rat brain PKC and the Btk PH domain. A, GST or GST-BtkPH (1 µg each) immobilized onto glutathione-agarose beads was incubated with the indicated amounts of rat brain PKC, and bound PKC was detected by immunoblotting with anti-cPKC (MC5) monoclonal antibody. The enhanced chemiluminescence method was used to visualize the immunoreactive bands (upper panel). Immunoblotting of known amounts of PKC was carried out to generate the standard curve (lower panel). B, amounts of bound PKC were plotted as a function of input PKC. Scatchard analysis based on this experiment gave a KD value of 39 nM. A representative result out of three similar experiments is presented.
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RESULTS

The PH Domain of Btk Interacts with Pure Preparations of PKC with High Affinity

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 alpha , beta , and gamma  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").

Mapping of the PKC-binding Site within the Btk PH Domain

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 beta -sheets 2 and 3 as deduced from tertiary structural studies on several PH domains (Fig. 2C).


Fig. 2. Mapping of the PKC-binding site within the Btk PH domain. A, a summary of the in vitro binding assay data with GST fusion proteins containing the PH domain subregions. The diagram at the top shows the deduced secondary structure of the PH domain of Btk based on the tertiary structural analyses of other PH domains. beta 1-beta 7 and alpha 1 indicate beta -sheets and an alpha -helix, respectively. Residue numbers (1 and 139) of the amino and carboxyl termini are also indicated. Subregion fusion proteins were designated by the residue numbers of the amino- and carboxyl-terminal ends of the Btk portions. B, in vitro binding assays with some subregion fusion proteins are shown. MCP-5 cell lysates (equivalent to 1 × 107 cells) were mixed with 2 µg each of GST or GST-PH domain fusion proteins immobilized onto glutathione-agarose beads. Bound proteins were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-P membranes. Bound PKC was detected by probing the blots with anti-cPKC (MC5). 1/45 and 28/77 bound PKC with efficiencies of approximately 30 and 10%, respectively, compared with the full-length construct, 1/139. C, the minimal PKC-binding sequence of Btk PH domain. Amino acid sequences of the amino-terminal portions (corresponding to the beta -sheets 1-4) of the PH domains of Btk (39), Emt (39), and pleckstrin (55) are depicted in the single-letter code. Positions of the first residues are indicated in parentheses. Residues forming the beta -sheets of the amino-terminal PH domain of pleckstrin (Ple N) are underlined and indicated by beta 1-beta 4 at the top (10). Residues in Ple N involved in binding to PIP2, as deduced from chemical shift (21), are shaded. Overlined is the minimal PKC-binding sequence of the Btk PH domain determined in this study. The asterisk indicates the position of the Btk residue (Arg-28) that is mutated in xid mice and in some X-linked agammaglobulinemia patients. # indicates position 41 (Glu-41) whose substitution with Lys resulted in a constitutively active mutant of Btk (47). The consensus sequences in the second and third beta -sheet regions are taken from Ref. 6. Plus signs, basic residues; small phi, hydrophobic residues; large phi, aromatic residues.
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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-delta 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).


Fig. 3. PIP2 competes with PKC for binding to the Btk PH domain. GST-BtkPH (2 µg) beads were mixed with MCP-5 cell lysates in the presence of 0, 4.6, 23, or 115 µg/ml sonicated lipid vesicles. Bound cPKC was detected as described in Fig. 2B. As a control, a result with GST together with PIP2 is also shown. PIP, phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; PC, phosphatidylcholine. PIP2 competition experiments were carried out four times with similar results, while experiments with other lipids were repeated twice.
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Mapping of the PH Domain-binding Site within the Regulatory Region of PKCepsilon

GST-BtkPH bound to both Ca2+-dependent (alpha , beta I, and beta II) and Ca2+-independent isoforms (epsilon  and zeta ) in mast cell lysates (data not shown). To determine the PH domain-binding region within PKCepsilon we utilized stably transfected NIH/3T3 cell lines that overexpress the epitope-tagged domain fragments of PKCepsilon 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 PKCepsilon fragments were detected by immunoblotting with anti-epsilon (epitope) antibody. The results showed that the relative band intensities of the GST-BtkPH-bound PKCepsilon fragments normalized to their expression level in cell lysates were high with the PKCepsilon holoenzyme and with fragments epsilon 1 and epsilon 2 (Fig. 4). However, fragments epsilon 3 and epsilon 7, which lack the pseudosubstrate region, exhibited lower but significant binding capacities to GST-BtkPH. GST control beads bound negligible amounts of these PKCepsilon fragments. These data demonstrated that the C1 region of PKCepsilon is sufficient for interaction with the Btk PH domain. Neither RI-epsilon nor RIII-epsilon fragments derived from the regulatory and catalytic domains, respectively, of c-Raf-1 (38) bound to the PH domain (data not shown). RI-epsilon 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-epsilon indicates a high specificity of PKC-PH domain interactions. An epitope-tagged rat PKCbeta I construct encompassing residues 312-671, which correspond to the catalytic domain, failed to bind to GST-BtkPH (data not shown).


Fig. 4. The C1 regulatory region of PKCepsilon interacts with the Btk PH domain. NIH/3T3 cells overexpressing the holoenzyme or epsilon -epitope-tagged fragments of PKCepsilon were used for in vitro binding assays. The domain organization and features of the expressed PKCepsilon truncated derivatives are shown in the schematic diagram (upper panel). PS, pseudosubstrate region. The residues retained in the indicated fragments are epsilon 1, 1-297; epsilon 2, 133-297; epsilon 3, 166-297; and epsilon 7, 166-400 (36, 37). Lysates prepared from these cells were incubated with GST or GST-BtkPH (2 µg each) beads in the absence or presence of 60 µg/ml of PIP2. Bound PKCepsilon sequences were detected by immunoblotting with anti-epsilon (lower left panel). Expression levels of the PKCepsilon sequences in the cells were measured by immunoblotting of total cell lysates with anti-epsilon (lower right panel).
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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 PKCepsilon fragments epsilon 2 and epsilon 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 epsilon 2 and epsilon 3 from interacting with GST-BtkPH. In contrast, the presence of 60 µg/ml phosphatidylserine did not inhibit the GST-BtkPH-PKCepsilon fragment interactions (data not shown).

Effects of PKC Activators or Inhibitors on the Interaction between PKC and the Btk PH Domain

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 PKCbeta I 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-alpha -PMA) did not alter the level of bound cPKC (Fig. 5B). These results are consistent with the mapping results. Thus, PMA (but not 4-alpha -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 PKCzeta and PKClambda /iota 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 beta 2-beta 3 sheets. If this is the case, the PH domain interaction with PKCzeta should not be susceptible to PMA-mediated inhibition due to the lack of interaction between PKCzeta and PMA. Indeed, the PKCzeta -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).


DISCUSSION

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 beta 2-beta 3 region of the Btk PH domain and the C1 region of PKCepsilon . 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 PKCzeta -PH domain interactions because of the lack of binding capacity of PMA to the C1 region of PKCzeta .

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-delta 1 PH domain) and 5-fold lower than for IP3 binding to the PLC-delta 1 PH domain (KD = 210 nM). The concentrations of PKCalpha and PKCbeta , two major PKC isoforms in a mast cell line, MC-9, were shown to be 26.0 and 63.9 nM, respectively (44). Accordingly, PKCbeta 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-delta 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 PKCbeta 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 beta -sheets 2 and 3, which form a major part of the beta -barrel. It is noteworthy that the hydrophobic residues of the beta 2-beta 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 beta gamma 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. Fcepsilon 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 beta gamma 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; alpha , beta I, and beta II) and Ca2+-independent (novel (epsilon ) and atypical (zeta )) 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 PKCepsilon demonstrated that the C1 region of PKCepsilon 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 PKCepsilon ) 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 PKCepsilon ) in the C1 region. The involvement of these hydrophobic residues in interactions with the hydophobic beta 2-beta 3 region of PH domains is implied since PH domain interactions with fragments epsilon 2 and epsilon 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 PKCbeta 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 PKCbeta I or c-Raf-1 (RIII-epsilon ).

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 PKCbeta II. 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 beta -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 PKCbeta gene knock-out mice (54). The phenotype of PKCbeta null mice was shown to be quite similar to those of xid and btk null mice.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants AI33617 and AI38348.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.
§   On a leave of absence from the Laboratory of Analytical Chemistry, Inst. of Pharmaceutical Sciences, Hiroshima University School of Medicine.
par    To whom correspondence should be addressed. Tel.: 619-558-3500; Fax: 619-558-3526; E-mail: toshi_kawakami{at}liai.org.
1   The abbreviations used are: SH, Src homology; cPKC, conventional isoforms of protein kinase C; G-proteins, GTP-binding proteins; GST, glutathione S-transferase; IP3, inositol 1,4,5-trisphosphate; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; PTKs, protein-tyrosine kinases.
2   Y. Kawakami and T. Kawakami, unpublished data.

ACKNOWLEDGEMENTS

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.


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