PKN Regulates Phospholipase D1 through Direct Interaction*

Kumiko OishiDagger , Mikiko Takahashi§, Hideyuki MukaiDagger §, Yoshiko Banno, Shigeru Nakashima, Yasunori Kanaho||, Yoshinori Nozawa**, and Yoshitaka OnoDagger §DaggerDagger

From the Dagger  Graduate School of Science and Technology, and the § Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, the  Department of Biochemistry, Gifu University School of Medicine, Gifu 500-8076, Japan, the || Department of Pharmacology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan, and the ** Department of Environmental Cell Responses, Gifu International Institute of Biotechnology and Institute of Applied Biochemistry, Mitake, Gifu 505-0116, Japan

Received for publication, November 26, 2000, and in revised form, March 16, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The association of phospholipase (PLD)-1 with protein kinase C-related protein kinases, PKNalpha and PKNbeta , was analyzed. PLD1 interacted with PKNalpha and PKNbeta in COS-7 cells transiently transfected with PLD1 and PKNalpha or PKNbeta expression constructs. The interactions between endogenous PLD1 and PKNalpha or PKNbeta were confirmed by co-immunoprecipitation from mammalian cells. In vitro binding studies using the deletion mutants of PLD1 indicated that PKNalpha directly bound to residues 228-598 of PLD1 and that PKNbeta interacted with residues 1-228 and 228-598 of PLD1. PKNalpha stimulated the activity of PLD1 in the presence of phosphatidylinositol 4,5-bisphosphate in vitro, whereas PKNbeta had a modest effect on the stimulation of PLD1 activity. The stimulation of PLD1 activity by PKNalpha was slightly enhanced by the addition of arachidonic acid. These results suggest that the PKN family functions as a novel intracellular player of PLD1 signaling pathway.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PKNalpha is a serine/threonine protein kinase whose regulatory domain in the N-terminal region contains three repeats of leucine zipper-like sequences (CZ1-3) and a D region, and whose catalytic domain in the C-terminal region is highly homologous to those of PKC1 family members (1). The protein kinase activity is enhanced by fatty acids such as arachidonic acid (2) and the small GTP-binding protein Rho through the direct interaction with the CZ1 region containing the RhoA effector motif class I in a GTP-dependent manner (3-5). This protein kinase makes a family that comprises at least three gene products including PKNalpha /PRK1, PKNbeta , and PRK2 (6, 7). The CZ1 regions of PKNbeta and PRK2 have the RhoA effector motif class I sequences (8), and they can interact with RhoA (6, 9-11). We previously reported that the D region of PKNalpha contains the autoinhibitory domain (12). It blocks the access of the catalytic domain to the substrate. Arachidonic acid can recover the catalytic activity from this autoinhibition (12), suggesting that arachidonic acid causes conformational change of PKNalpha to release the autoinhibitory domain. PKNbeta and PRK2 conserve the domains corresponding to the autoinhibitory domain of PKNalpha (6, 12); however, they are only slightly activated by arachidonic acid (6, 13). Therefore, PKNbeta and PRK2 may need some other factors for conformational changes to release their autoinhibitory domains.

PKNalpha is ubiquitously expressed but is enriched in the brain (14). In neurons, PKNalpha is concentrated in a subset of endoplasmic reticulum as well as late endosomes, multivesicular bodies, Golgi bodies, secretary vesicles, and nuclei (15). Thus we have identified several binding partners of PKNalpha by employing a two-hybrid screening using the N-terminal regulatory region of PKNalpha as a bait (16-20). One of these partners is the centrosome- and Golgi-localized PKN-associated protein (CG-NAP), localized to centrosomes throughout the cell cycle and the Golgi apparatus at interphase (17). PKNbeta is localized to the Golgi apparatus and nuclei in cells (6). Thus, there is the possibility that PKNalpha and PKNbeta may play roles in the control of physiological events in the Golgi apparatus and endosomal compartment such as mitotic Golgi fragmentation and intracellular membrane traffic through the interaction with some other molecules.

In the present study, we have focused attention on phospholipase D (PLD)-1 showing overlapping subcellular localization with PKNalpha and PKNbeta : endoplasmic reticulum, Golgi apparatus, late endosomes, and plasma membrane (21, 22). PLD hydrolyzes phosphatidylcholine to generate the signaling lipid phosphatidic acid. Phosphatidic acid has been hypothesized to act as a fusogenic lipid in vesicular transports such as exocytosis and endocytosis. Many cell types express the known PLD1 and/or PLD2 isozymes, which become activated in response to a wide variety of agonists through heterotrimeric G protein-coupled or tyrosine kinase receptors. It has been proposed that the link between receptor stimulation and PLD activation is mediated by PKC, ADP-ribosylation factor, and Rho family members, which have been shown to stimulate PLD1 directly using in vitro reconstitution assays (23-29). They can act alone to stimulate PLD1 and in combination to elicit a synergistic activation (26-28, 30).

In this report, we show that PLD1 associates with PKC-related protein kinases, PKNalpha and PKNbeta , in vitro and in vivo. Furthermore, we demonstrate that PKNalpha stimulates the PLD1 activity through the direct interaction in vitro. These results suggest that the PKN family participates in the PLD1 signaling pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies-- Mouse monoclonal anti-FLAG (M2), anti-(His)6, anti-PKN (PRK1), rat monoclonal anti-HA (3F10), and rabbit polyclonal anti-PLD were purchased from Sigma, Covance Research Products, Transduction Laboratories, Roche Molecular Biochemicals, and BIOSOURCE, respectively.

Plasmid Construction-- The pTB701/HA/hPKNalpha plasmid used to express HA-tagged human PKNalpha in COS-7 cells was constructed by subcloning the EcoRI fragment of pMhPKN-H4 (1) containing the full length of hPKNalpha to the EcoRI site of pTB701/HA (35). The pTB701/HA/hPKNalpha N and pTB701/HA/hPKNalpha CA plasmids were constructed as follows: the cDNA fragment encoding hPKNalpha (residues 1-540) was obtained by digesting phPKNH4 with EcoRI and BamHI; the cDNA fragment encoding hPKNalpha (residues 561-942) was obtained by polymerase chain reaction; and each cDNA fragment was inserted into pTB701/HA. The pTB701/HA/hPKNbeta plasmid was made by inserting the EcoRI fragment of pRc/CMV/hPKNbeta into pTB/701/HA. The pTB701/HA/hPKNbeta N and pTB701/HA/hPKNbeta CA plasmids for the expression in mammalian cells were made by digesting pRc/CMV/hPKNbeta with EcoNI, filling the ends with T4 polymerase, ligating with EcoRI linker, digesting with EcoRI, and inserting the resulting fragments encoding hPKNbeta (residues 1-520 and 520-889) into the EcoRI site of pTB701/HA, respectively. The pTB701/FLAG/hPLD1 used to express FLAG-tagged human PLD1 in mammalian cells was constructed as described previously (28). The pTB701/FLAG/hPLD1N plasmid was constructed by deleting the BglII fragment from pTB701/FLAG/hPLD1. The pTB701/FLAG/hPLD1I plasmid was made by digesting pTB701/FLAG/hPLD1 with NcoI, filling the ends with T4 polymerase, ligating with BglII linker, digesting with BglII, and inserting the resulting fragment encoding hPLD1 (residues 228-598) to the BglII site of pTB701/FLAG (35). The pTB701/FLAG/hPLD1C was constructed by digesting pTB701/FLAG/hPLD1 with NcoI, filling the ends with T4 polymerase, ligating with BglII linker, digesting with BglII, and ligating the resulting fragment encoding hPLD1 (residues 598-1070) to the BglII site of pTB701/FLAG. The pBlueBacHis/GST/hPKNalpha was made as described (12). The pAcGHLTC/hPKNbeta plasmid was made by digesting pTB701/HA/hPKNbeta with EcoRI and inserting the resulting fragment into the EcoRI site of pAcGHLTC. The pRSETB/hPLD1N for (His)6-tagged hPLD1N (residues 1-228) was constructed as follows: the cDNA fragment encoding the amino acid residues 1-228 of hPLD1 generated by polymerase chain reaction was ligated into the BamHI site of pRSETB. The pRSETB/hPLD1I plasmid used to express (His)6-tagged hPLD1I (residues 228-598) was made by digesting pTB701/FLAG/hPLD1I with BglII, and the resulting fragment was inserted into the BglII site of pRSETB.

Expression and Purification of Recombinant PKNalpha and PKNbeta -- The recombinant baculovirus was generated as described (12). The Sf9 cells expressing recombinant protein were lysed for 15 min in buffer A (20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 5 mM MgCl2, 150 mM NaCl, and 10 µg/ml leupeptin) containing 1% Nonidet P-40 at 4 °C. The lysate was then centrifuged at 30,000 × g for 20 min at 4 °C, and the resulting supernatant was collected. An appropriate volume of glutathione-Sepharose 4B was added to the supernatant, and the mixture was rotated for 1 h at 4 °C. After washing with buffer A, the bound proteins were eluted with elution buffer (50 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml leupeptin, and 10 mM reduced glutathione).

Cell Culture-- COS-7 and HeLa cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. U937 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. Transfection to COS-7 cells by electroporation was performed as described (6).

Immunoprecipitation-- Cells were lysed with buffer A containing 1% Nonidet P-40 at 4 °C, and the resulting lysate was centrifuged at 10,000 × g for 10 min. The supernatants were incubated with the indicated antibodies at 4 °C for 2 h and with protein A-Sepharose for 1 h. The resulting immunoprecipitates were subjected to PLD assay, SDS-polyacrylamide gel electrophoresis, and immunoblot analysis with the indicated antibodies.

In Vitro Binding Assay-- Two µg of the indicated protein was incubated at 4 °C for 2 h with 2 µg of either (His)6-tagged hPLD1N (residues 1-228) or hPLD1I (residues 228-598) and then precipitated with glutathione-Sepharose 4B beads. The (His)6-tagged hPLD1 fragment that was co-precipitated with the indicated GST-fused proteins was detected by immunoblotting with an anti-(His)6 antibody.

In Vitro PLD Assay-- The immunoprecipitate with anti-FLAG from COS-7 cells expressing FLAG-tagged hPLD1 was reconstituted with the purified proteins in the presence of phosphatidylethanolamine/phosphatidylinositol 4,5-bisphosphate/[choline-methyl-[3H]dipalmitoylphosphatidylcholine vesicles in a molar ratio of 16:1.4:1 (36). The mixture was incubated in 20 mM Hepes, pH 7.5, 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2, 2 mM CaCl2, and 1 mM dithiothreitol with or without 40 µM arachidonic acid, and then the production of [3H]choline was determined (37). The incubation time was 30 min at 37 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Association of PKNalpha and PKNbeta with PLD1-- To analyze the association of PKN family members with PLD1, we performed the immunoprecipitation with the anti-FLAG antibody using the lysate of COS-7 cells expressing FLAG-tagged PLD1 and either HA-tagged PKNalpha or PKNbeta . The interaction was analyzed by Western blotting with an anti-HA antibody. As shown in Fig. 1, both PKNalpha and PKNbeta were clearly co-immunoprecipitated with FLAG-tagged PLD1.


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Fig. 1.   PKNalpha and PKNbeta interact with PLD1 in COS-7 cells. Either HA-tagged PKNalpha or PKNbeta and FLAG-tagged PLD1 were co-expressed in COS-7 cells. The cell lysate was immunoprecipitated with the anti-FLAG antibody as described under "Experimental Procedures." The cell lysate and the immunoprecipitate were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting. The anti-FLAG antibody was used to detect the expression (lanes l and 2) and immunoprecipitation level (lanes 3 and 4) of FLAG-tagged PLD1 (lower panel). The amounts of HA-tagged PKNalpha (lane 1) and PKNbeta (lane 2) expressed and co-immunoprecipitated (lanes 3 and 4) with PLD1 were visualized using the anti-HA antibody (upper panel). The immunoprecipitation with normal mouse IgG (lanes 5 and 6) was done as the control experiment.

To define the association between endogenous PLD1 and PKNalpha or PKNbeta in mammalian cells, we next carried out the immunoprecipitation experiments using the specific antibodies. As shown in Fig. 2B, endogenous PKNbeta was clearly co-immunoprecipitated with endogenous PLD1 from HeLa cells. Fig. 2A shows that PLD1 also interacted with PKNalpha in U937 cells, although the interaction with PKNalpha was weaker than that with PKNbeta . These data suggest that the association of PKNalpha and PKNbeta with PLD1 is physiological.


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Fig. 2.   Endogenous PKNalpha and PKNbeta interact with endogenous PLD1 in cells. A, the association of PKNalpha with PLD1 in U937 cells. B, the association of PKNbeta with PLD1 in HeLa cells. The cell lysates of HeLa and U937 cells were immunoprecipitated with the anti-PLD1 antibody (alpha PLD1) followed by immunoblots with alpha PRK1 (A) and alpha C131beta (B) to detect PKNalpha and PKNbeta , respectively. The immunoprecipitation with normal rabbit IgG was done as the control experiment (normal). sup, supernatant of cell lysate; IP, immunoprecipitate.

To identify the interaction site(s) of PKNalpha or PKNbeta with PLD1, we made the deletion mutants of PKNalpha and PKNbeta as shown in Fig. 3A. FLAG-tagged PLD1 was co-expressed with HA-tagged PKNalpha N (residues 1-540), HA-tagged PKNalpha CA (residues 561-942), HA-tagged PKNbeta N (residues 1-520), or HA-tagged PKNbeta CA (residues 520-889) in COS-7 cells. The cell lysate was subjected to immunoprecipitation with the anti-FLAG antibody, and the interaction was analyzed by Western blotting with the anti-HA antibody. As shown in Fig. 3B, right, PKNbeta clearly bound to PLD1 at the N- and C-terminal regions. PKNalpha also associated with PLD1 at both N- and C-terminal regions (Fig. 3B, left).


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Fig. 3.   The N terminus and C terminus of PKNalpha and PKNbeta interact with PLD1 in COS-7 cells. A, the constructs of PKNalpha and PKNbeta . Schematic representations of the structures of PKNalpha and PKNbeta are shown on the top. CZ, charged amino acid-rich region with leucine zipper-like sequences; D, ~130 amino acid stretch between the CZ region and catalytic domain, which has weak homology to the E4/V0/C2 domain of PKC, and the C-terminal part of it functions as a fatty acid-sensitive autoinhibitory domain; P1 and P2, two distinct proline-rich regions. B, HA-tagged N-terminal or C-terminal fragments of PKNalpha (left) and PKNbeta (right) were co-expressed with FLAG-tagged PLD1 in COS-7 cells. After immunoprecipitation of FLAG-tagged PLD1 with the anti-FLAG antibody, the amounts of co-immunoprecipitated HA-tagged N-terminal (lane 3) and C-terminal (lane 4) fragments were detected by immunoblotting using the anti-HA antibody (upper panel). The expression (lanes 1 and 2) and immunoprecipitation (lanes 3 and 4) levels of PLD1 were visualized by immunoblotting with the anti-FLAG antibody (lower panel). As a control, immunoprecipitation with normal mouse IgG was done (lanes 5 and 6).

Next, we analyzed the interaction site(s) of PLD1 with PKNalpha or PKNbeta . The lysate from COS-7 cells co-expressing either HA-tagged PKNalpha or PKNbeta and three different deletion mutants of FLAG-tagged PLD1 (Fig. 4A) was immunoprecipitated with the anti-FLAG antibody and analyzed by Western blotting with the anti-HA antibody. Fig. 4B shows that both PKNalpha and PKNbeta associated with the PLD1 (residues 1-228) containing phox domain and PLD1 (residues 228-598) containing pleckstrin homology-like domain, conserved regions I and II, and a part of the loop domain of PLD1 (31-34).


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Fig. 4.   Dissection of the PLD1 interaction with PKNalpha and PKNbeta . A, schematic representation of the structure of PLD1 is shown on the top. The possible functions of each box have been proposed or demonstrated in Ref. 31. PX, phox domain; PH, pleckstrin homology-like domain; CRI-IV, highly conserved regions in the PLD family (31); LOOP, loop region; CT, C terminus. B, the N-terminal (lane 1), internal (lane 2), and C-terminal (lane 3) fragments of PLD1 and either PKNalpha (left) or PKNbeta (right) were co-transfected into COS-7 cells. After immunoprecipitation with the anti-FLAG antibody, the amounts of co-immunoprecipitated HA-tagged PKNalpha and PKNbeta were detected by immunoblotting with the anti-HA antibody (upper panel). The immunoprecipitate of FLAG-tagged PLD1 with the anti-FLAG antibody is indicated in the middle panel. The expressions of PKNalpha and PKNbeta were visualized using immunoblotting with the anti-HA antibody (lower panel).

Direct Interaction PKNalpha and PKNbeta with PLD1-- To investigate whether PKNalpha and PKNbeta directly bind to PLD1, we performed an in vitro binding analysis using the purified proteins. (His)6-PLD1N (residues 1-228) and (His)6-PLD1I (residues 228-598) were bacterially expressed and purified as described (17). The PKNalpha or PKNbeta fused to GST was expressed and affinity-purified from Sf9 cells. The proteins were mixed, and each GST-fusion protein was pulled down by glutathione-Sepharose beads. The bound proteins were detected by Western blotting with the anti-(His)6 antibody. Fig. 5 shows that GST-PKNbeta pulled down both (His)6-PLD1N and (His)6-PLD1I. On the other hand, GST-PKNalpha associated with (His)6-PLD1I, but not with (His)6-PLD1N. GST itself did not interact with (His)6-PLD1N or (His)6-PLD1I. In the co-immunoprecipitation study, PKNalpha associated with both PLD1N and PLD1I in COS-7 cells, suggesting that the interaction of PKNalpha with PLD1 (residues 1-228) in COS-7 cells is mediated by some other molecules.


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Fig. 5.   In vitro interactions of the N-terminal and internal fragments of PLD1 with PKNalpha and PKNbeta . GST-fused PKNalpha , PKNbeta , and GST were expressed in Sf9 cells and affinity-purified with glutathione-Sepharose beads. (His)6-PLD1N and PLD1I expressed and purified from Escherichia coli were incubated with GST-PKNalpha (alpha ) or GST-PKNbeta (beta ). Each GST-fused protein was precipitated as described under "Experimental Procedures." As a control, GST was used instead of these protein kinases (GST). The amounts of (His)6-PLD1N (left) or PLD1I (right) co-precipitated with GST-fused proteins were detected by immunoblotting with the anti-(His)6 antibody.

Stimulation of PLD1 by PKNalpha in Vitro-- Because the activators for PLD1 such as PKC and RhoA stimulate the activity of PLD1 through direct interaction, we investigated whether PKNalpha and PKNbeta can stimulate PLD1. FLAG-tagged PLD1 expressed in COS7 cells was immunoprecipitated with the anti-FLAG antibody and was used for this assay. Fig. 6 shows that PKNalpha stimulated the activity of PLD1 in the presence of phosphatidylinositol 4,5-bisphosphate, whereas PKNbeta had a modest effect on the stimulation of PLD1 activity. The PLD1 stimulation by PKNalpha was slightly enhanced by the addition of arachidonic acid, which is a potential activator of PKNalpha . This stimulation was independent of ATP (data not shown).


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Fig. 6.   In vitro activation of PLD1 by PKNalpha and PKNbeta . Immobilized FLAG-tagged PLD1 was incubated at 37 °C for 30 min with the indicated concentrations of GST-PKNalpha (alpha ) or PKNbeta (beta ) in 20 mM Hepes, pH 7.5, 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2, 2 mM CaCl2, and 1 mM dithiothreitol with (right) or without (left) 40 µM arachidonic acid (AA). As a control, the indicated concentrations of GST were incubated instead of these protein kinases (mock). The PLD1 activity was determined as described under "Experimental Procedures." The error bars represent the differences of triplicate determinations. The results shown are representative of three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrated that the members of the PKN family, PKNalpha and PKNbeta , associated with PLD1 and that PKNalpha stimulated PLD1 activity in vitro. This stimulation was ATP-independent, suggesting that it is mediated by direct binding rather than phosphorylation of PLD1. The interaction of PKNalpha with PLD1 was weaker than PKNbeta (Figs. 2 and 3). Nevertheless, PKNalpha stimulated PLD1 activity to a greater extent than PKNbeta (Fig. 6). There may be at least two possible explanations for the lower stimulatory effect of PKNbeta . First, the binding nature of PKNbeta with PLD1 was somewhat different from that of PKNalpha . PKNbeta directly interacted with the N-terminal (residues 1-228) as well as the internal (residues 228-598) region of PLD1 (Fig. 5), whereas PKNalpha interacted directly with the internal region (Fig. 5) and indirectly with the N-terminal region (Fig. 4). Although PKNalpha and PKNbeta are closely related, this difference in the binding may reflect the distinct effects of these proteins on PLD1 activity. Second, insect cell-expressed PKNbeta used in the PLD1 assay (Fig. 6) was relatively unstable and susceptible to proteolysis (data not shown). Because endogenous PKNbeta in mammalian cells is distributed in membrane and nuclear fractions (6), PKNbeta may need some lipids or binding proteins to form a stable structure. This instability of the enzyme may lower the effect on the PLD1 activity measured in vitro. Further, in the case of RhoA, lipid modification such as geranylgeranylation greatly enhance the stimulatory effect on PLD1 but does not affect the binding (38), suggesting that RhoA-lipid interaction may help to induce catalytically active conformation of PLD1. Similarly, some conformational change of PKNbeta by association with lipids (or other proteins) might be required to achieve effective stimulation of PLD1. We observed that endogenous PLD activity in COS-7 cells was significantly enhanced by the expression of PKNbeta (data not shown), suggesting that PKNbeta may work as an effective stimulator in vivo.

PKCalpha is well known to stimulate PLD1 activity by direct interaction in a phosphorylation-independent manner. The interaction and the stimulation are enhanced by the presence of phorbol ester (25, 27-29), indicating the importance of the conformational change of PKCalpha induced by phorbol ester binding as well as intracellular translocation of PKCalpha to the membrane. Proteolysis studies indicate that the N-terminal regulatory region of PKCalpha mediates this stimulation (25). The mechanism of PLD1 stimulation by the PKN family presented here seems to be similar to that by PKCalpha . However, we observed stimulatory effects with the C-terminal catalytic region of PKNalpha but not with the N-terminal regulatory region (data not shown). Further analyses will be needed to clarify the difference in the mechanism of PLD stimulation between the PKN and PKC families.

PKNalpha and PKNbeta associated with PLD1 through the N- and C-terminal regions (Fig. 3). We have reported that the N-terminal region of PKNalpha directly interacts with the C-terminal catalytic region and that this interaction inhibits its catalytic activity (39). Thus, the PLD1 stimulation by the C-terminal region of PKNalpha may be affected by the interaction between its N-terminal region and PLD1. PLD1 stimulation by PKNalpha was enhanced by the presence of arachidonic acid, which is a potential activator of PKNalpha (Fig. 6). Arachidonic acid activates PKNalpha probably by inducing conformational change to release the N-terminal region and the autoinhibitory region from the catalytic region (12). This conformational change may help the associated PLD1 to form active conformation. Although PKNbeta has the conserved domain corresponding to the autoinhibitory domain of PKNalpha , arachidonic acid has little effect on the protein kinase activity (6) and PLD1 stimulation by this enzyme (Fig. 6, right), suggesting that conformation of PKNbeta is not sufficiently changed by arachidonic acid.

PKN family proteins interact with the Rho family small GTPase (3-5). PKNalpha is activated by the binding of RhoA to its leucine zipper-like domain in a GTP-dependent manner, suggesting that the interaction with GTP-bound RhoA also causes the conformational change of PKNalpha to expose the catalytic domain to substrate. Thus, RhoA might stimulate PLD1 activity by activating the PKN family in addition to the direct interaction with PLD1.

In summary, the PKN family protein kinases modulate the activity of PLD1 by direct protein-protein interaction. Further analysis including the relationship between other cofactors activating PLD1 and the PKN family will provide important insights into the physiological role of PLD1 and PKN family.

    ACKNOWLEDGEMENT

We thank Dr. Y. Nishizuka for encouragement.

    FOOTNOTES

* This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Research for Future program of the Japan Society for the Promotion of Science.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.

Dagger Dagger To whom correspondence should be addressed. Tel.: 81-78-803-5792; Fax: 81-78-803-5782; E-mail: yonodayo@kobe-u.ac.jp.

Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M010646200

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; PRK, protein kinase C-related protein kinase; PLD, phospholipase D; HA, hemagglutinin; h, human; GST, glutathione S-transferase.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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