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© The Rockefeller University Press,
0021-9525/2003/3/929 $5.00
The Journal of Cell Biology, Volume 160, Number 6, 929-937
Article |
Association of diacylglycerol kinase 
with protein kinase C 
2 Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112
3 Department of Internal Medicine, University of Utah, Salt Lake City, UT 84112
Address correspondence to Matthew K. Topham, The Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112. Tel.: (801) 585-0304. Fax: (801) 585-6345. E-mail: matt.topham{at}hci.utah.edu
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Abstract |
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Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGK
inhibits PKC
activity and that DGK activity is required for this inhibition. We also show that DGK directly interacts with PKC in a signaling complex and that the binding site in DGK is located within the catalytic domain. Because PKC can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGK, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGK and PKC and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGK mutant that was unable to bind PKC did not inhibit PKC activity. Together, our results suggest that DGK spatially regulates PKC activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKC-mediated DGK phosphorylation.
Key Words: diacylglycerol; diacylglycerol kinase; protein kinase C; spatial regulation; phosphorylation
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Introduction |
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DAG kinases (DGKs)* are critical regulators of DAG signaling. DGKs metabolize DAG by phosphorylating it to generate phosphatidic acid (PA). To date, nine mammalian DGK isoforms have been cloned and divided into five classes based on common structural motifs (Topham and Prescott, 1999; van Blitterswijk and Houssa, 1999; Kanoh et al., 2002). Their structural diversity, together with their different subcellular localization (Topham and Prescott, 1999), suggests that each DGK isoform may regulate distinct DAG signaling events. DGK, a type IV DGK, contains a unique region homologous to the phosphorylation site domain (PSD) of the myristoylated alanine-rich C-kinase substrate (MARCKS) protein, a prominent substrate for PKC in cells (Blackshear, 1993; Bunting et al., 1996). This MARCKS motif is the predominant nuclear localization signal of DGK, and its phosphorylation by PKC isoforms reduces nuclear localization of DGK, which alters nuclear DAG accumulation (Topham et al., 1998). Thus, cells regulate the concentration of PKC-activating nuclear DAG by controlling nuclear localization of DGK. Interestingly, overexpression of wild-type DGK leads to decreased levels of nuclear DAG (Topham et al., 1998). This, combined with the fact that several DAG pools have been found in distinct, spatially separated compartments within the cell (Wakelam, 1998; D'Santos et al., 1999), suggests that the regulation of DAG signaling is achieved locally.
PKC is strongly activated by DAG, and its regulation is likely spatially controlled (Wagner et al., 2000; Newton, 2001). We considered the possibility that DGK, by metabolizing signaling DAG, spatially regulates PKC activity. We demonstrate here that DGK associates with PKC and inhibits its activity in a signaling complex. This association was abolished when the MARCKS motif of DGK was phosphorylated by PKC. Dissociation of the complex, in turn, attenuated the inhibition of PKC activity; a phosphorylation-mimicking DGK mutant that could not bind to PKC did not inhibit PKC activity. Together, these data suggest that PKC facilitates its own activation by phosphorylating DGK. This sequence may allow transient or even prolonged activation of PKC in stimulated cells while inhibiting its activity in the basal state.
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regulates PKC activity regulates the activity of Ras guanyl nucleotide–releasing protein (RasGRP) (Topham and Prescott, 2001), an enzyme that is allosterically activated by DAG. Because DAG is a crucial component of PKC activation, we considered the possibility that DGK could also regulate the enzymatic activity of some PKC isoforms. PKC was an ideal candidate because the pattern of tissue and cellular expression closely parallels that of DGK (unpublished data). To test this possibility, we cotransfected PKC along with wild-type DGK or a mutant, catalytically inactive DGK (
ATP) into HEK293 cells and then measured PKC activity in the cell lysates. Lysates from PKC-transfected cells demonstrated 
10-fold higher PKC activity than the endogenous PKC activity, demonstrating that the majority of PKC activity that we measured was from transfected PKC (Fig. 1). We found that simultaneous expression of DGK reduced PKC activity by 50% (P < 0.001), whereas expression of a similar amount of inactive DGK had no significant effect on PKC activity. Thus, DGK activity was required for PKC inhibition. PA, the product of the DGK reaction, did not affect PKC activity (unpublished data), indicating that its accumulation was not responsible for the inhibition. Supporting our contention that DGK regulates PKC activity by converting DAG to PA, we found that DGK did not inhibit PKC activity in the presence of PMA, a DAG analogue that cannot be metabolized by DGK (unpublished data). Demonstrating that the inhibition was selective to specific PKC isoforms, expression of DGK did not affect PKC
activity under the same experimental conditions (unpublished data).
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and DGK physically associate in the cell may interact with PKC in cells. To test this possibility, we cotransfected HEK293 cells with PKC and DGK–FLAG and then immunoprecipitated DGK with anti-FLAG antibody. We assayed for coprecipitation of PKC by immunoblotting (Fig. 2 A). In these experiments, we found that PKC clearly coimmunoprecipitated with DGK, whereas no PKC was detected when we used a nonimmune antibody for the immunoprecipitation. Catalytically inactive DGK (ATP) still efficiently coimmunoprecipitated with PKC. This result, combined with the fact that ATP did not inhibit PKC activity (Fig. 1), confirms that association with PKC is not sufficient for DGK to inhibit its activity. Demonstrating the specificity of this interaction, we could not detect coimmunoprecipitation of DGK and PKC under the same experimental conditions (unpublished data). Coimmunoprecipitation cannot distinguish direct from indirect protein–protein interactions. To test whether DGK and PKC can directly bind to each other, we examined their interaction in vitro by incubating purified DGK–FLAG immobilized on anti-FLAG beads with recombinant PKC. After precipitating the complexes, we observed by immunoblotting that PKC specifically bound to DGK but did not bind to control beads (Fig. 2 B). Together, these data demonstrate that DGK and PKC interact in vivo and that they likely bind to each other directly.
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and PKC could interact in rat brain extracts. Consistent with our initial results, endogenous PKC coimmunoprecipitated with DGK from rat brain extracts (Fig. 2 C). We could not detect PKC in immunoprecipitates using nonimmune IgG, demonstrating the specificity of their association. And, consistent with our results from transfected cells, we did not observe coprecipitation of endogenous DGK and PKC in rat brain extracts (unpublished data). We also tested for binding of endogenous DGK and PKC in A172 cells, a glioblastoma cell line known to express both proteins (Xiao et al., 1994; Topham et al., 1998). Again, we observed a specific interaction (Fig. 2 D). Together, these data demonstrate that endogenous PKC and DGK associate with the same signaling complex in vivo, and that they appear to bind directly to each other.
A portion of the catalytic domain of DGK is sufficient to bind PKC
To map the domain in DGK that binds PKC, we examined a series of DGK deletion mutants to determine which ones could associate with PKC. We cotransfected each construct along with PKC and then immunoprecipitated the FLAG-tagged DGK mutant with anti-FLAG antibodies and assessed coprecipitation of PKC by immunoblotting. We found that a region (BD) near the COOH terminus of the catalytic domain was necessary for PKC to coprecipitate with DGK (Fig. 3 A, lane 5). This region (BD), other than being part of the catalytic domain, lacks any identifiable protein motifs. Importantly, we also noted in these experiments that two different mutants (L and M) lacking the MARCKS motif could still bind PKC (Fig. 3 A, lanes 6 and 7). This binding in the absence of the MARCKS motif (the site of PKC phosphorylation; Topham et al., 1998) demonstrates that their association was not simply a result of DGK being a substrate for PKC.
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was sufficient to interact with PKC, we incubated recombinant PKC with GST fusion proteins containing full-length DGK (GST–DGK), the putative binding domain (GST–BD), or control GST protein. We assessed coprecipitation of PKC by immunoblotting and found that PKC coprecipitated with both the GST–DGK and GST–BD fusion proteins, but not with the GST control protein (Fig. 3 B). Taken together, our results demonstrate that a region in the COOH terminus of the catalytic domain of DGK is both necessary and sufficient to bind to PKC.
Activation of PKC impairs its association with DGK
Because activated PKC regulates the subcellular localization of DGK (Topham et al., 1998), we wondered if the association of DGK and PKC was similarly dependent on the activation state of PKC. To assess this possibility, we examined whether phorbol esters, potent activators of PKC, affected coprecipitation of DGK and PKC. Initially, we monitored the binding between DGK and PKC when both proteins were overexpressed in HEK293 cells and observed that treating the cells with PMA significantly reduced coprecipitation of PKC and DGK (Fig. 4 A). A PKC inhibitor abolished this reduction, indicating that PKC activity was required to attenuate their interaction. To examine this more directly, we tested whether PMA inhibited in vitro binding of recombinant PKC and purified DGK. As demonstrated in Fig. 4 B, the recombinant PKC could specifically bind to purified DGK (lane 2), but PMA dramatically attenuated their association (lane 1). To test this in vivo, we used confocal microscopy to examine whether endogenous DGK and PKC colocalized in NIH 3T3 cells and whether PMA had any effect. We found that the proteins colocalized in the basal state but not after addition of PMA (Fig. 5). Thus, activation of PKC inhibits its association with DGK.
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and PKC. Control A172 cells or those treated with PDGF were lysed, and then endogenous PKC was immunoprecipitated. We then measured DGK activity in PKC immunoprecipitates (Fig. 4 C). Under the experimental conditions, PDGF, which stimulates PKC through PLC
(Valius and Kazlauskas, 1993), activated endogenous PKC (unpublished data). We found that PKC immunoprecipitates had 2.1 times more DGK activity than control (nonimmune IgG) immunoprecipitates. Stimulation with PDGF almost completely removed DGK activity from PKC immunoprecipitates, but not in the presence of a PKC inhibitor. Thus, activation of PKC by PDGF impaired its association with DGK in A172 cells. Supporting our contention that the associated DGK was DGK, we found by immunoblotting that PDGF significantly attenuated coprecipitation of DGK and PKC, and this was blocked by a PKC inhibitor (Fig. 4 D). These data demonstrate that the association between endogenous DGK and PKC depends on the activation state of PKC.
Phosphorylation of the MARCKS motif causes DGK and PKC to dissociate
Because PKC can phosphorylate the MARCKS motif of DGK (Topham et al., 1998), we considered the possibility that this phosphorylation regulated the association of DGK and PKC. Initially, we investigated whether the MARCKS motif was essential for regulation of their association. To test this, we cotransfected HEK293 cells with PKC and the MARCKS motif deletion mutant (M) and then examined the effect of PMA on the coprecipitation of PKC and M. As shown in Fig. 6 A, treating cells with PMA did not significantly affect coprecipitation of PKC and M (lane 4) but markedly reduced the binding between PKC and wild-type DGK (lane 2), indicating that the MARCKS motif was necessary for the binding regulation. Under these experimental conditions, PKC phosphorylated wild-type DGK on the MARCKS motif upon PMA stimulation (unpublished data). Thus, these data suggested that phosphorylation of the MARCKS motif inhibited association of DGK and PKC. To further test this possibility, we cotransfected HEK293 cells with PKC and either wild-type DGK or a mutant of DGK in which serine residues in the MARCKS motif were changed to aspartates (DGK S/D) to mimic phosphorylation of these residues (Swierczynski and Blackshear, 1995; Topham et al., 1998). We compared the ability of these DGK proteins to coimmunoprecipitate PKC. As shown in Fig. 6 B, DGK S/D did not interact with PKC (lane 3), whereas wild-type DGK (lane 2) and a second mutant of DGK in which the same serines in the MARCKS motif were changed to asparagines (DGK S/N) (lane 4) efficiently coimmunoprecipitated with PKC. These results demonstrated that phosphorylation of the MARCKS motif by PKC inhibited the interaction between DGK and PKC. To verify this observation, we examined the effect of PMA on association of PKC and DGK S/N. We expected that activation of PKC by PMA would not cause their dissociation because the S/N mutation prevents phosphorylation in the MARCKS motif (unpublished data). Indeed, we found that PMA did not significantly affect coprecipitation of PKC and DGK S/N (Fig. 6 C).
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does not inhibit PKC activity binds to and locally inhibits PKC activity. To extend our observation that phosphorylation of DGK caused PKC and DGK to dissociate, consequently relieving PKC inhibition, we tested whether a mutant DGK that could not bind to PKC could still affect inhibition of PKC. We transfected HEK293 cells with wild-type DGK, a mutant DGK (S/D) that could not bind efficiently to PKC, or another mutant DGK (S/N) that could still bind to PKC. After immunoprecipitation of endogenous PKC, we compared its activity in the immunoprecipitates. We found in these experiments that expression of wild-type DGK significantly reduced endogenous PKC activity, consistent with our previous results (Fig. 1), whereas expression of DGK S/D did not inhibit endogenous PKC activity (Fig. 7, top). Protein expression levels of these DGK were similar in these experiments (Fig. 7, bottom). The lack of inhibition by DGK S/D was not caused by loss of its DGK activity, because it demonstrated a high DGK activity level in the cell lysates (Fig. 7, middle). A control mutant DGK S/N also efficiently inhibited endogenous PKC in cells, demonstrating that negative charge (i.e., phosphorylation) within the MARCKS motif, which causes dissociation of DGK and PKC, is necessary to relieve inhibition of PKC. Consistent with this, we found that wild-type DGK did not inhibit PKC activity when PMA was included in the PKC activity reaction (unpublished data). Taken together, our data demonstrate that DGK associates with PKC to negatively regulate its protein kinase activity. But, PKC can relieve this inhibition by phosphorylating DGK, thus causing dissociation of the two proteins.
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acts to regulate the activity of PKC. This regulation is accomplished by DGK binding to PKC and metabolizing local DAG that would otherwise activate the PKC enzyme, thus inhibiting PKC activity. Indeed, we observed that endogenous DGK coimmunoprecipitated with endogenous PKC in A172 cells and rat brain extracts and that the proteins colocalized in NIH 3T3 cells. In addition, we found that DGK directly interacted with PKC. Another finding confirmed that the regulation of PKC requires an association between DGK and PKC; the phosphorylation-mimicking DGK (DGK S/D) could not bind to PKC and inhibit its activity. Lack of PKC inhibition by DGK S/D likely resulted from its inability to access, and consequently remove, the local signaling molecule, DAG. Demonstrating that this inhibition was specific for the PKC isoform, we observed that DGK did not inhibit the activity of PKC, another DAG-dependent PKC isoform that did not associate with DGK. Thus, changes mediated by DGK in the concentration of signaling DAG occur locally and may not affect the function of PKCs found elsewhere within the cell. Taken together, these data strongly indicate that both the target (PKC) and the attenuator (DGK) of DAG signaling are spatially organized in a signaling complex that is able to fine tune DAG signaling.
From immunoprecipitation experiments of endogenous DGK and PKC, we estimated, using densitometry, that 10–20% of cellular DGK coimmunoprecipitated with 10–20% of cellular PKC (Fig. 4 D; unpublished data). Although rough estimates, they correlate well with the amount of overlap that we observed using confocal microscopy and indicate that this regulation affects a subset of cellular PKC. This is not surprising, given the numerous biologic functions of PKC and the importance of its spatial regulation, and it suggests that DGK regulates PKC in some, but not all, of its intracellular compartments. Because of the diverse functions of PKC, the lack of specific PKC or DGK inhibitors, and the absence of direct in vivo PKC activity assays, we were unable to determine a distinct physiologic consequence of this regulation. However, we previously demonstrated a functional association between DGK and PKC in the nucleus, indicating that DGK may regulate signaling mediated by nuclear PKC (Topham et al., 1998). In NIH 3T3 cells, though, we found no evidence that these proteins colocalized in the nucleus (Fig. 5). However, another DGK splice variant that is not present in NIH 3T3 cells predominantly localizes in the nucleus (unpublished data), suggesting that it might regulate nuclear PKC activity in cells where it is expressed.
PKC is not the only protein allosterically activated by DAG; several other proteins, including RasGRP, the chimaerins, Unc-13, and protein kinase D (Hurley et al., 1997; Kazanietz, 2002), have C1 domains and can bind and are activated by DAG. We have previously demonstrated (Topham and Prescott, 2001) that RasGRP associated with DGK and that DGK regulated the activation status of RasGRP by metabolizing local DAG. Additionally, Miller et al. (1999) and Nurrish et al. (1999) found that a DGK in Caenorhabditis elegans, an ortholog of mammalian DGK
, negatively regulated synaptic transmission by metabolizing DAG that would otherwise activate Unc-13, a protein that is involved in neurotransmitter secretion. Thus, it appears that regulation of DAG signaling is frequently spatially restricted and is achieved through association of DAG target proteins and DGKs. This may be a common mechanism to regulate the amplitude or duration of signaling events. Indeed, Tasken et al. (2001) and Dodge et al. (2001) demonstrated that phosphodiesterase, which metabolizes cAMP, associated with PKA, a cAMP-dependent protein kinase, in cells. In this signaling complex, phosphodiesterase could tightly control cAMP levels to regulate the activity of PKA and consequently the phosphorylation state of proteins regulated by PKA. Also, Divecha et al. (2000) observed that phosphatidylinositol 4-phosphate 5 kinase interacted with phospholipase D in a signaling complex. It has been shown that the products of the two enzymes each stimulate the opposite enzyme (Jenkins et al., 1994; Exton, 2000). Thus, they proposed a mutual positive regulation, leading to a rapid high local increase in both products, PA and phosphatidylinositol 4,5-bisphosphate (PIP2), which may be important in a series of cellular functions. These observations, along with ours, support a paradigm where regulation of second messengers, such as DAG, cAMP, and PIP2, is spatially controlled within an assembled signaling complex.
Increasing evidence suggests that signaling proteins are organized into localized compartments where regulation of signaling events can be precisely controlled (Hunter, 2000). Targeting of protein kinases to the close proximity of their substrates ensures that they phosphorylate only the proper targets and prevents inappropriate phosphorylation events (Pawson and Nash, 2000; Smith and Scott, 2002). PKCs can directly bind to many substrates, such as adducin, GAP43, STICK72, MARCKS, and MARCKS-related proteins (Dekker and Parker, 1997; Jaken and Parker, 2000). The association of DGK and PKC, combined with the fact that PKC can phosphorylate DGK, suggests that DGK is a physiologic substrate for PKC. Interestingly, we showed that the interaction between DGK and PKC was abolished when PKC phosphorylated DGK. Jaken's group (Chapline et al., 1993; Dong et al., 1995) similarly demonstrated that adducin bound to PKC and that phosphorylation of adducin reduced their interaction. Because protein phosphorylation often leads to dramatic conformational changes, phosphorylation of DGK likely changes its structure, resulting in its dissociation from PKC. Supporting this idea, Bubb et al. (1999) observed a dramatic conformational change when a peptide corresponding to the PSD of the MARCKS protein was phosphorylated. This structural change may have resulted from the new negatively charged phosphates transiently interacting with positively charged amino acids in the PSD. The new structure was more compact and caused obliteration of an actin binding site in the MARCKS protein. The MARCKS motif of DGK is homologous to the PSD of the MARCKS protein (Bunting et al., 1996), so it would not be surprising if phosphorylation of the MARCKS motif in DGK caused similar conformational changes, resulting in dissociation of DGK and PKC. A functional consequence of this phosphorylation may be to prevent phosphorylated DGK from accessing and removing local DAG that activates PKC.
Our data support a model (Fig. 8) where in the basal state, when DAG levels in the cell are low, DGK associates with and regulates PKC. This allows DGK to metabolize local DAG and prevent PKC activation. Upon stimulation, when PKC activity is required, local DAG levels increase transiently and overcome the ability of DGK to remove DAG. Consequently, PKC becomes activated by DAG and then phosphorylates DGK, which causes dissociation of PKC and DGK. This sequence results in a transient increase in PKC activity, allowing it to phosphorylate other substrates. Presumably, PKC activity is eventually attenuated by a variety of mechanisms, including inactivation of PLCs, dephosphorylation of PKC or its proteolytic degradation, and reassociation with DGK. The duration of PKC activation likely depends on a variety of circumstances, and its regulation is probably complex. Some cellular responses, such as proliferation and differentiation, require sustained activation of PKC, whereas other responses require it to be activated only transiently (Nishizuka, 1995; Black, 2000). For example, Balciunaite et al. (2000) demonstrated in HepG2 cells that PDGF stimulated PKC activity at two distinct times, within 10 min after PDGF treatment and then for a longer duration, between 5 and 19 h. The late phase of PKC activity was required for the PDGF-dependent transition from G0 into S phase. Aihara et al. (1991) found that sustained activation of PKC is essential for differentiation of HL-60 cells to macrophages. Prolonged activation of PKC may occur by regulation of DGK protein levels, as we have observed that expression of DGK significantly decreases during differentiation of HL-60 cells (unpublished data). We have previously described a functional correlation of DGK and PKCs in the nucleus (Topham et al., 1998), and Shirai et al. (2000) observed that DGK and PKC showed spatially similar, but temporally different, translocation after purinergic receptor activation in living cells. They suggested that the time lag between the translocation of DGK and PKC may regulate the duration of PKC activation. Thus, regulation of conventional PKC isoforms by DGKs may be a common theme.
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, by metabolizing local DAG, negatively regulates PKC activity. In turn, by phosphorylating DGK, PKC removes this inhibition, allowing its own activation. This mechanism provides low basal PKC activity but allows for transient, or even prolonged, PKC activation, depending on the cellular context. |
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antibody, Gö 6983, and human recombinant PKC (prepared from Sf9 insect cells) were purchased from Calbiochem. [-32P]ATP (6,000 Ci/mmol) was purchased from Amersham Biosciences. Normal rabbit IgG, normal mouse IgG–coupled agarose, and protein A/G agarose were from Santa Cruz Biotechnology, Inc. Glutathione–sepharose 4B was obtained from Amersham Biosciences. PKC assay kit was purchased from Upstate Biotechnology. LipofectAMINE and all cell culture reagents were obtained from Invitrogen.
Expression plasmids
Wild-type DGK was cloned into pcDNA1/Amp, and a FLAG epitope tag was placed at the COOH terminus, as described previously (Topham and Prescott, 2001). Generation of the progressive COOH-terminal deletions of DGK constructs (B, H, and X) has been published previously (Topham and Prescott, 2001). The NH2-terminal deletion DGK L was generated by BamH1 digestion of the DGK–FLAG plasmid followed by religation. For the DGK Bsu construct, DGK–FLAG plasmid was digested with Bsu36I and then religated. The catalytically inactive DGK mutant (ATP), MARCKS motif deletion (M), and MARCKS motif mutants of DGK (S/N and S/D), in which all four serines in the MARCKS motif were altered, were generated as described previously (Topham et al., 1998). pGEX-5X plasmid containing wild-type DGK was a gift from Sarah Leibowitz (Rockefeller University, New York, NY). GST–BD was generated by cloning a PCR fragment comprising nucleotides 1400–1906 of human DGK into pGEX-5X. PKC and PKC were subcloned into pcDNA3/Amp plasmid (Invitrogen).
Cell culture and transfection
HEK293 and NIH 3T3 cells were maintained in DME containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. HEK293 cells were transiently transfected as previously described (Bunting et al., 1996). After 48 h, the cells were stimulated with 90 nM PMA or vehicle for 30 min. Where indicated, Gö 6983 (500 nM) was added for 10 min before PMA stimulation. A172 cells were cultured as previously described (Topham et al., 1998).
Preparation of rat brain extracts
Rat brain was homogenized by Dounce homogenizer in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) containing 1 mM DTT and then centrifuged at 100,000 g for 30 min. Cleared lysates were used as brain extracts.
Immunoprecipitation and immunoblotting
FLAG-tagged DGK was transfected along with PKC into HEK293 cells. After 48 h, the cells were harvested in lysis buffer containing phosphatase inhibitor cocktail (Sigma-Aldrich), allowed to lyse for 30 min on ice, and then centrifuged to remove debris. 1 ml of cleared lysate (3 mg of total protein) was incubated with 25 µl monoclonal anti–FLAG-M2 agarose affinity gel or normal mouse IgG coupled to agarose beads for 2 h. After centrifugation for 5 s at 10,600 g, the beads were washed with 500 µl wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) three times. Immunoprecipitates were used for SDS-PAGE. Polyclonal anti-PKC (Calboichem) was used to immunoblot for PKC, and DGK was detected with a previously described polyclonal DGK antibody (Topham et al., 1998). To immunoprecipitate endogenous DGK or PKC, A172 cell lysates and rat brain extracts were precleared with normal rabbit IgG together with protein A/G agarose for 30 min at 4°C, and then the supernatants were incubated with anti-DGK or anti-PKC (Santa Cruz Biotechnology, Inc.) overnight at 4°C. The immunocomplexes were collected using protein A/G agarose, washed three times with wash buffer, and then used for immunoblotting as described above.
Immunofluorescence and confocal microscopy
NIH 3T3 cells grown on glass coverslips were rinsed in PBS, fixed with 4% formaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with PBS containing 5% BSA for 1 h. The cells were then incubated for 1 h with 1:50 rabbit anti-DGK antibody and 1:100 mouse anti-PKC antibody (Transduction Laboratories), followed by Oregon green–conjugated anti–mouse and Texas red–conjugated anti–rabbit IgG (Molecular Probes). After being washed with PBS, the immunofluorescently stained cells were imaged using a confocal microscope (Bio-Rad Laboratories).
In vitro binding assay
DGK–FLAG was expressed in HEK293 cells and immunoprecipitated with anti–FLAG-M2 agarose affinity gel. The immunoprecipitates were mixed with purified recombinant PKC (0.7 µg) and incubated for 2 h at 4°C. Then, the beads were washed with wash buffer three times and analyzed for PKC binding by immunoblotting. GST–DGK, GST–BD, and GST proteins were expressed in bacterial strain BL21 and purified by incubation with glutathione–sepharose 4B according to the manufacturer's instructions. The glutathione–sepharose 4B–bound GST fusion proteins were incubated with purified recombinant PKC (0.7 µg) for 2 h at 4°C. The beads were washed three times with wash buffer and then used for immunoblotting as described above.
Kinase activity assay
PKC or PKC was transfected along with a control vector, DGK–FLAG, or ATP–FLAG into HEK293 cells. After 48 h, the cells were harvested in 200 µl of lysis buffer containing phosphatase inhibitor cocktail. PKC activity in the cell lysate was determined by a PKC assay kit (Upstate Biotechnology) according to the manufacturer's instructions. To measure endogenous PKC activity, PKC proteins from lysates of HEK293 cells transfected with different DGK constructs were immunoprecipitated by polyclonal PKC antibody (Santa Cruz Biotechnology, Inc.). The PKC immunoprecipitates were collected using protein A/G agarose and washed three times with wash buffer and once in PKC assay buffer (20 mM MOPS, pH 7.2, 25 mM ß–glycerol phosphate, 1 mM sodium orthovanadate, 1 mM DTT, 1 mM CaCl2). The immunoprecipitate was used for PKC activity assays. The DGK kinase assay was performed as previously described using lysates of HEK293 cells transfected with DGK (Bunting et al., 1996). 1,2-dioleoyl-sn-glycerol was used as the substrate. The reaction was performed for 10 min in the presence of [-32P]ATP. Lipids were extracted and separated by TLC, and PA was visualized by autoradiography.
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Acknowledgments |
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This work was supported by the Huntsman Cancer Foundation.
Submitted: 20 August 2002
Revised: 23 January 2003
Accepted: 24 January 2003
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References |
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