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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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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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Results |
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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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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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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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Discussion |
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From immunoprecipitation experiments of endogenous DGK and PKC
, we estimated, using densitometry, that
1020% of cellular DGK
coimmunoprecipitated with
1020% 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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Materials and methods |
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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). GSTBD was generated by cloning a PCR fragment comprising nucleotides 14001906 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 antiFLAG-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 greenconjugated antimouse and Texas redconjugated antirabbit 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
DGKFLAG was expressed in HEK293 cells and immunoprecipitated with antiFLAG-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. GSTDGK
, GSTBD, and GST proteins were expressed in bacterial strain BL21 and purified by incubation with glutathionesepharose 4B according to the manufacturer's instructions. The glutathionesepharose 4Bbound 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
ATPFLAG 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 ßglycerol 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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