Protein Kinase C alpha -mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C beta 1 in Swiss 3T3 Cells*

Aimin XuDagger , Yu Wang§, Lance Yi XuDagger , and R. Stewart GilmourDagger

From the Dagger  Liggins Institute, School of Medicine, and § School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

Received for publication, October 6, 2000, and in revised form, January 16, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies from several independent laboratories have demonstrated the existence of an autonomous phosphoinositide (PI) cycle within the nucleus, where it is involved in both cell proliferation and differentiation. Stimulation of Swiss 3T3 cells with insulin-like growth factor-I (IGF-I) has been shown to induce a transient and rapid increase in the activity of nuclear-localized phospholipase C (PLC) beta 1, which in turn leads to the production of inositol trisphosphate and diacylglycerol in the nucleus. Nuclear diacylglycerol provides the driving force for the nuclear translocation of protein kinase C (PKC) alpha . Here, we report that treatment of Swiss 3T3 cells with Go6976, a selective inhibitor of PKC alpha , caused a sustained elevation of IGF-I-stimulated nuclear PLC activity. A time course study revealed an inverse relationship between nuclear PKC activity and the activity of nuclear PLC in IGF-I-treated cells. A time-dependent association between PKC alpha  and PLC beta 1 in the nucleus was also observed following IGF-I treatment. Two-dimensional phosphopeptide mapping and site-directed mutagenesis demonstrated that PKC promoted phosphorylation of PLC beta 1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC beta 1 mutant in which the PKC phosphorylation site Ser887 was replaced by alanine, or a dominant-negative PKC alpha , resulted in a sustained activation of nuclear PLC following IGF-I stimulation. These results indicate that a negative feedback regulation of PLC beta 1 by PKC alpha  plays a critical role in the termination of the IGF-I-dependent signal that activates the nuclear PI cycle.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phospholipase C beta  isoforms (beta 1, beta 2, beta 3 and beta 4) at the plasma membrane are regulated by G protein-coupled seven-transmembrane receptors which activate heterotrimeric Galpha beta gamma protein complexes upon ligand stimulation (1-3). The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2)1 by PLC beta s generates two well recognized second messengers, inositol 1,4,5-trisphosphate and DG. Inositol 1,4,5-trisphosphate evokes release of Ca2+ from intracellular stores, while DG alone, or in concert with Ca2+, activates some isoforms of PKC (4). Mounting evidence suggests that an analogous phosphoinositide (PI) signaling, which is distinct from classic PI signaling at plasma membrane, exists in the nucleus (5-7). The presence of such a nuclear PI cycle has been demonstrated in several different cell lines (8-11, 12), and has been shown to be important for both cell proliferation (13) and differentiation (14).

The key enzyme for the initiation of nuclear PI signaling is phospholipase C (PLC) beta 1, which is the major isoform present in the nucleus of 3T3 cells (15) as well as other cell lines (16-19). It exists as two alternatively spliced subtypes, PLC beta 1a (150 kDa) and PLC beta 1b (140 kDa) which differ only in a short region of their carboxyl termini (20). Recent studies suggest that the beta 1b form predominantly localizes in the nucleus while the beta 1a form distributes equally between the nucleus and plasma membrane (21). A nuclear localization motif has been mapped to a cluster of lysine residues (between 1055 and 1072) which is common to both subtypes (22).

It has recently been demonstrated that ablation of PLC beta 1 with antisense abolished the mitogenic response of Swiss 3T3 cells to IGF-I, indicating the pivotal role of this enzyme in cell proliferation (13). Overexpression of both subtypes of PLC beta 1 alone, even in the absence of growth factors, is sufficient to elevate expression of cyclin D3 and cdk4 (23). This in turn leads to hyperphosphorylation of retinoblastoma protein (pRb) and activation of E2F-1 transcription factor, thus enhancing cell cycle G1/S progression. The importance of this enzyme in the cell cycle is further strengthened by a recent finding that in Saccharomyces cerevisivae nuclear PLC1 (homologous in function to the mammalian PLC beta 1), and two inositol polyphosphate kinases constitute a nuclear signaling cascade that is directly involved in RNA transport and transcriptional regulation (24, 25).

One of the critical downstream targets for nuclear PLC beta 1 is protein kinase C, which is involved in progression through the G1/S and G2/S checkpoints of the cell cycle (26, 27). Nuclear DG has been shown to be critical for the activation of nuclear PKC beta II during G2/M phase transition (28). It also acts as a chemoattractant to induce nuclear translocation of PKC alpha  by a mechanism which is not clearly understood (9, 29). In Swiss 3T3 cells, the IGF-I evoked production of nuclear DG, nuclear translocation of PKC alpha  and DNA synthesis were completely blocked by the selective phospholipase C inhibitor Et-18-0-CH3, but not by a PLD inhibitor (30).

Nuclear PLC beta 1 is under separate control from that of PLC beta s at plasma membrane. Bombesin, a G-protein coupled-receptor agonist, only activates PLC beta  isoforms at the plasma membrane and has no effect on nuclear PLC activity (9). Conversely, stimulation of cells with IGF-I, which acts through a tyrosine kinase receptor, caused a rapid, 2~3-fold increase in enzyme activity of nuclear PLC beta 1, while the PLC activity at plasma membrane was not affected (15). The IGF-I-stimulated activation of nuclear PLC beta 1 is transient and the enzyme activity returns to basal level within 30 min (15, 31). However, the molecular mechanisms for the termination of this activation signal are currently unclear. Here, we demonstrated that a negative feedback regulation of nuclear PLC beta 1 by PKC alpha  accounts for the termination of IGF-I induced activation of this enzyme. PKC alpha  was shown to interact with PLC beta 1 in the nucleus, and evoke phosphorylation at serine 887, a putative PKC phosphorylation site which is located within the long, characteristic carboxyl tail of PLC beta 1. Overexpression of either a PLC beta 1 variant (S887A) or dominant-negative PKC alpha  caused a sustained elevation of nuclear PLC activity following IGF-I stimulation.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Polyclonal antibodies against PKC alpha  or gamma , a monoclonal antibody which recognizes PKC alpha , PKC beta 1, and PKC beta 2, PKC inhibitor calphostin C, Cy3-conjugated goat anti-rabbit IgG, aprotinin, and leupeptin were obtained from Sigma. Isotopes ([gamma -32P]ATP, [32P]orthophosphate, and [3H]PIP2) were purchased from ICN. Protein kinase C substrate peptide QKRPSQRSKYL was obtained from Upstate Biotechnology Inc. LipofectAMINE Plus, G418, Dulbecco's modified Eagle's medium and phosphate-free Dulbecco's modified Eagle's medium were the product of Life Technologies Inc. MEK inhibitor PD98059 was purchased from New England Biolabs, Inc. Protein kinase C alpha  (human, recombinant, Spodoptera frugiperda), PKC alpha  inhibitor Go6976, and PKC delta  inhibitor rottlerin were from Calbiochem. The parental mammalian expression vector pCIN4 which encodes a neomycin resistance gene, and pCIN4 DN PKC alpha , which expresses dominant-negative PKC alpha  (DN PKC alpha ), are the generous gifts from Dr Albert Descoteaux (Université du Québec, Canada) (32). pCIN DN PKC alpha  contains a dominant-negative version of the gene in which the conservative lysine residue (Lys338) in the ATP-binding domain was replaced by aspartic acid.

Site-directed Mutagenesis and Construction of Expression Vector-- The cDNA corresponding to wild-type PLC beta 1 (33) was amplified by PCR with forward and reverse primers containing BamHI and EcoRI restriction sites, respectively. Following digestion with the restriction enzymes, the DNA fragment was ligated into the pcDNA3.1 eukaryotic expression vector, which contains a cytomegalovirus promoter. Construction of the vector expressing histidine-tagged PLC beta 1 (referred to as PLC beta 1 (His)6) is similar to the above except that the upstream primer contains a DNA fragment encoding six histidine residues.

Mutation of the putative PKC phosphorylation site S887A was performed by sequential PCR mutagenesis as follows. A PCR reaction was performed using primer I (5'-CAGCATATGAGGAAGGAGGCAAATTTATTG-3') which spans a unique NdeI site at nucleotides 2236-2252) and primer II (5'-TGCCTTCACAGCCCCTGGAGCAGG-3') which includes the S887A mutation in non-coding strand) as a forward and a reverse primer, respectively. Another PCR reaction utilized primer III (5'-CAGGGGCTGTGAAGGCACCCGCCA-3'), which partially overlaps with primer II and also contains the Ser887 (TCT) to alanine (GCT) mutation in coding strand and primer IV (5'-CATCTGCAGCTTGGGCTTCTCATCCAGGAT-3'), which spans a unique PstI (in bold) site at nucleotides 3424-3430) as a forward and reverse primer. The resultant product of these two PCR reaction was purified, annealed, and used as template for a second round of PCR with primer I and primer IV as a forward primer and reverse primer. The 1191-base pair PCR product was then digested with NdeI and PstI and inserted into the corresponding sites in the wild-type PLC beta 1 expression vector. The resultant clone was confirmed by DNA sequencing and is referred as pcDNA.PLC beta 1 S887A.

Cell Culture, Transfection, and in Vivo 32P Labeling-- Swiss 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected with the above expression vectors using LipofectAMINE according to the vendor's instruction. Stable transfectants were generated by selection with 0.6 mg/ml G418. For in vivo 32P labeling, the cells were starved in phosphate-free Dulbecco's modified Eagle's medium for 1 h to deplete ATP metabolic pool, and subsequently incubated with 0.2 mCi/ml [32P]orthophosphate for 4 h. Cells were then subjected to different treatments as indicated.

Isolation of Nuclei and Analysis of PLC Activity-- Nuclei were purified as previously described (15). Briefly, 5 × 106 cells were lysed in 400 µl of nuclear isolation buffer (10 mM Tris-HCl, pH 7.8, 1% Nonidet P-40, 10 mM mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin and leupeptin, 10 µg/ml soybean trypsin inhibitor, 15 µg/ml calpain inhibitor-1 and 2 (Roche Molecular Biochemicals), 2.0 mM Na3VO4, and 5 mM NaF) for 3 min on ice. 400 µl of MilliQ water were then added to swell cells for 3 min. The cells were sheared by 8 passages through a 23-gauge hypodermic needle. Nuclei were recovered by centrifugation at 400 × g and 4 °C for 6 min, and washed once in 400 µl of washing buffer (10 mM Tris-HCl, pH 7.4, 2 mM MgCl2, plus protease and phosphatase inhibitors as above). This method has been shown to yield nuclei which lack both inner and outer nuclear membranes and are essentially free of cytoplasmic contamination (13, 15).

The activity of nuclear PLC was analyzed as outlined previously (15). 10 µg of nuclear proteins were incubated with 100 mM MES buffer, pH 6.7, plus 150 mM NaCl, 0.06% sodium deoxycholate, 3 nmol of [3H]PIP2 (specific activity 30,000 dpm/nmol) for 30 min at 37 °C. Hydrolysis was stopped by adding chloroform/methanol/HCl, and the amount of inositol 1,4,5-trisphosphate in the upper phase was quantified by liquid scintillation counting.

In Vitro Nuclear PKC Activity Assay-- The PKC activity in isolated nuclei was measured as previously outlined by Neri et al. (30). Briefly, 10 µg of nuclear proteins were incubated at 30 °C for 10 min with 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 µM ATP, 5 µCi of [32P]ATP, 1.2 mM CaCl2, 40 µg/ml phosphatidylserine, 3.3 mM dioleoylglycerol, plus 10 µg of PKC substrate peptide. The reaction was stopped by adding 15 µl of acetic acid and spotted onto Whatman p81 paper. The filter paper was then washed with 0.75 mM H3PO4 and counted for radioactivity assay.

Immunoprecipitation and Purification of PLC beta 1 (His)6 from Nuclei-- Purified nuclei were solubilized in lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA and EGTA, 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, plus protease inhibitor mixture as above) for 20 min at 4 °C with shaking. Cell debris was removed by centrifugation at 12,000 × g and 4 °C for 5 min. The supernatants were incubated with 50 µl of a 50% slurry of protein A/G-agarose beads for 1 h. The cleared lysates were then incubated with 5 µg of mouse anti-PLC beta 1 antibody (34) for 16 h. The immunocomplexes were recovered by adding 50 µl of protein A/G-agarose beads for another hour, and released by boiling in 50 µl of 1 × SDS buffer for 5 min. Samples were then separated by 8% SDS-PAGE, and the protein phosphorylation was analyzed by autoradiography and quantified by ImageTM software (Amersham Pharmacia Biotech).

To purify nuclear PLC beta 1 (His)6 or PLC beta 1 (His)6 mutant S887A, nuclei were purified from cells transfected with the corresponding expression vectors constructed as above, and solubilized in lysis buffer (50 mM Tris-HCl, pH 8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1% Triton X-100, plus protease and phosphatase inhibitor mixture as above). The lysates were centrifuged at 10,000 × g and 4 °C for 10 min. The supernatant was applied to a pre-equilibrated column packed with Ni-NTA resin. After washing the column, PLC beta 1 (His)6 was eluted using lysis buffer plus 100 mM imidazole and 10% glycerol. The elutes were pooled, concentrated, and analyzed by SDS-PAGE and autoradiography.

Phosphorylation of Nuclear PLC beta 1 by Recombinant PKC alpha -- Equal amounts of PLC beta 1 (His)6 purified as above were incubated with 50 ng of recombinant PKC alpha  or PKC delta  in the presence of 2.5 µCi of [gamma -32P]ATP in a total volume of 30 µl of reaction mixture (20 mM HEPES, pH 7.4, 100 mM CaCl2, 10 mM MgCl2, 50 µg/ml phosphatidylserine, 40 µg/ml diacylglycerol, 0.03% Triton X-100) at 30 °C for the times specified. The reactions were terminated by adding 6 × Laemmli sample buffer and boiling for 5 min. Following separation by 10% SDS-PAGE, the phosphoprotein was visualized and analyzed by phosphorimaging.

In-gel Trypsin Digestion and Two-dimensional Phosphopeptide Mapping-- In vivo 32P-labeled PLC beta 1 (His)6 was purified from nuclei as above, separated by SDS-PAGE. The bands corresponding to this protein were excised from the gels, minced, and in-gel digested as previously described (35). The tryptic mixtures were lyophilized and solubilized in 10 µl of electrophoresis buffer (1% pyridine, 10% acetic acid, pH 3.5) and applied to the middle of a thin layer chromatography (TLC) plate. The samples were then subjected to first dimensional electrophoresis, followed by second dimensional chromatography as previously detailed (36). The tryptic phosphopeptides were visualized by autoradiography.

Immunoblotting, Immunostaining, and Confocal Imaging Microscopy-- Nuclear proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 10% fat-free milk, and incubated with the various primary and secondary antibodies as described in the text. The immunoreactive proteins were detected using ECL reagents according to the manufacturer's instructions.

For immunostaining, The cells grown on coverslips were starved for 24 h in serum-free medium, stimulated without or with 40 ng/ml IGF-I, or IGF-I plus 50 µM calphostin C as above. Cells were then stained for PKC alpha  using rabbit anti-PKC alpha  (1:1000) antibody, followed by goat anti-rabbit antibody conjugated with Cy3 (1:250). The specimens were then examined using a Leica TCS 4D confocal laser scanning microscopy (Lasertechnik, Heidelberg, Germany) fitted with a mercury vapor lamp and a mixed gas krypton-argon laser.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Effect of Protein Kinase C Inhibitors on IGF-I-dependent Activation of Nuclear PLC-- Several recent studies have found that PKC can regulate PLC activity in vitro or at the plasma membrane (37-40). Thus, we investigated the effect of PKC on the activity of nuclear PLC using several selective PKC inhibitors. As shown in Fig. 1, the nuclear PLC activity in Swiss 3T3 cells was significantly increased following IGF-I treatment, in agreement with a previous report (31). After 5 min of stimulation, the enzyme activity increased 3.1-fold over the basal level. This level of activity persisted for up to 15 min, and then began to decrease, reaching basal level after 30 min. By contrast, treatment of cells with Go6976, a specific inhibitor for alpha  isoform of PKC (41), caused a sustained activation of nuclear PLC activity. Under this treatment the enzyme still retained maximal activity at 30 min after IGF-I stimulation. It remained 1.9-fold higher after 60 min and only returned to basal level after 2 h (data not shown). The IGF-I dependent activation of nuclear PLC was also sustained by treatment of the cells with 40 µM calphostin C (a specific inhibitor of PKC (42, 43)), but not by 15 µM rottlerin (a specific inhibitor for delta  isoform of PKC (44)) (data not shown). This result indicates that PKC alpha  is probably involved in the attenuation of the IGF-I-evoked activation of nuclear PLC.


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Fig. 1.   Treatment of cells with Go6976 causes sustained elevation of nuclear PLC following IGF-I stimulation. Quiescent Swiss 3T3 cells were pretreated without or with 0.5 µM Go6976 for 30 min, and then incubated with 40 ng/ml IGF-I for the indicated time periods. The purified nuclei were assayed for PLC activity as described under "Experimental Procedures." The results are from three independent experiments and expressed as mean ± S.D.

A previous study showed that among the several PKC isoforms detected in Swiss 3T3 cells only PKC alpha  is present in the nucleus (29). In response to IGF-I stimulation, PKC alpha  was selectively translocated into the nucleus from the cytosol (30). This nuclear translocation process occurs within the same time frame for the activation of nuclear PI cycle by IGF-I. Western blot analysis of the nuclear proteins from Swiss 3T3 cells revealed that the concentration of PKC alpha  in the nucleus starts to increase after 15 min of IGF-I stimulation, and reaches a maximal level after 30 min (Fig. 2A). In agreement with this result, stimulation of cells with IGF-I also caused a progressive increase in nuclear PKC activity which reached a maximum at 30 min (Fig. 2B). Significantly, the activity of nuclear PKC inversely correlates with the activity of nuclear PLC, further suggesting the involvement of PKC alpha  in the termination of nuclear PI cycle signaling.


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Fig. 2.   Time course analysis for nuclear translocation of PKC alpha  and nuclear PKC activity in IGF-I-treated cells. A, cells were treated without or with 40 ng/ml IGF-I. Nuclear proteins were separated by 8% SDS-PAGE and probed with an anti-PKC alpha  polyclonal antibody. B, aliquots of the same nuclear samples were analyzed for PKC activity as described under "Experimental Procedures." The results are from three independent experiments and expressed as mean ± S.D.

We next investigated the effect of Go6976 on nuclear translocation of PKC alpha  and nuclear PKC activity in cells treated with IGF-I. Immunofluorescent analysis revealed that pretreatment of cells with Go6967 does not affect the IGF-I induced nuclear translocation of PKC alpha  (Fig. 3). However, the IGF-I-induced increase in nuclear PKC activity was completely inhibited under these conditions (data not shown).


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Fig. 3.   The effect of Go6976 on IGF-I-induced nuclear translocation of PKC alpha . Quiescent cells grown on coverslips were pretreated without or with 0.5 µM Go6976 for 30 min, and then incubated with 40 ng/ml IGF-I for another 30 min. Cells were then fixed, stained for PKC alpha , and analyzed using confocal laser scanning microscopy.

Time-dependent Association of PLC beta 1 and PKC alpha  within the Nucleus following IGF-I Stimulation-- It has previously been shown that PLC beta 1, a major isoform of PLC family residing in the nucleus, was responsible for the initiation of nuclear PI cycle (13). To investigate the direct involvement of PKC alpha  in the regulation of nuclear PLC beta 1 activity, nuclear proteins from cells overexpressing PLC beta 1 were subjected to immunoprecipitation using an anti-PLC beta 1 antibody. The precipitated immunocomplex was separated by SDS-PAGE and probed with either anti-PLC beta 1 or anti-PKCalpha . A direct interaction between PLC beta 1 and PKC alpha  within the nucleus was detected at 15 min after stimulation with IGF-I (Fig. 4). This association reached a maximum after 30 min, and persisted for up to 1 h. Again, the time course of interaction between these two enzymes strongly correlates with the decrease in nuclear PLC activity (Fig. 1). A similar analysis failed to detect association between PLC beta 1 and several other isoforms of PKC (beta 1, beta 2, gamma 1, and gamma 2) (data not shown), suggesting that the interaction between PLC beta 1 and PKC is specific for the alpha  isoform.


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Fig. 4.   Time course of interaction between PKC alpha  and PLC beta 1 in the nucleus following IGF-I stimulation. Nuclear proteins from cells overexpressing PLC beta 1 were immunoprecipitated with an anti-PLC beta 1 monoclonal antibody. The immunocomplexes were separated by 8% SDS-PAGE and then probed with either anti-PLC beta 1 or anti-PKC alpha .

PKC alpha  Evokes Phosphorylation of PLC beta 1 at Serine 881 within the Nucleus-- Previous in vitro studies have shown that PKC can phosphorylate PLC beta 1 at serine 887 (45). However, the physiological relevance of this phosphorylation is uncertain. Here, we tested whether or not the PKC-mediated PLC beta 1 phosphorylation on serine 887 is the direct consequence of IGF-I stimulation. To this end, Swiss 3T3 cells were transfected with a plamid which express either PLC beta 1 (His)6, or PLC beta 1 (His)6 mutant S887A. These proteins were then radiolabeled with 32P in vivo and purified from nuclei as described under "Experimental Procedures." Two-dimensional phosphopeptide mapping analysis of 32P-labeled nuclear PLC beta 1 (His)6 revealed a single, prominent tryptic phosphopeptide for PLC beta 1 overexpressed in quiescent 3T3 cells, indicating that a constitutive phosphorylation of PLC beta 1 occurs within this peptide (Fig. 5A). Stimulation of cells with IGF-I for 30 min caused the production of two extra tryptic phosphopeptides, referred to as phosphopeptides 2 and 3, respectively (Fig. 5B). The IGF-I-dependent production of phosphopeptide 2 was inhibited by MEK inhibitor PD98059 (Fig. 4D), indicating that MEK/MAP kinase is involved in phosphorylation of this peptide. The significance of this observation is currently under separate investigation.


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Fig. 5.   PKC-mediated phosphorylation of PLC beta 1 at serine 887 occurs in the nucleus following IGF-I stimulation. Cells were transiently transfected with a plasmid expressing His6-tagged PLC beta 1 (A-D) or PLC beta 1 mutant S887A (E and F) and rendered quiescent by starvation in serum-free medium after 24 h of transfection. Cells were then radiolabeled with 32P for 4 h, and then treated without (A and E), or with 40 ng/ml IGF-I (B-D and F) for another 30 min. In C and D, cells were pretreated with 0.5 µM Go6976 or 50 µM PD98059 for 30 min, respectively, before adding IGF-I. His6-tagged PLC beta 1 or PLC beta 1 mutant S887A protein were then purified from nuclei as described under "Experimental Procedures." Proteins were separated by 8% SDS-PAGE and visualized by autoradiography. The bands corresponding to 32P-PLC beta 1 (His)6 or 32P-PLC beta 1 (His)6 mutant S887A were excised from the gel, in-gel digested with trypsin, and analyzed by two-dimensional phosphopeptide mapping as described under "Experimental Procedures." Note that IGF-I-dependent phosphopeptide 3 was absent in samples treated with Go6976 or overexpressing PLC beta 1 (His)6 mutant S887A.

Phosphopeptide 3 could not be detected in cells treated with PKC alpha  inhibitor Go6976 (Fig. 5C), or cells overexpressing PLC beta 1 (His)6 mutant S887A in which the phosphorylation site serine 887 was replaced by alanine (Fig. 5, E and F). The production of phosphopeptide 3 was also inhibited by calphostin C, but not rottlerin (data not shown). This result demonstrates that PKC-mediated phosphorylation at serine 887 occurs in vivo within the nucleus in response to IGF-I.

To further establish that nuclear PLC beta 1 is the direct target of PKC alpha , nuclear PLC beta 1 or PLC beta 1 mutant S887A purified as above was subjected to in vitro phosphorylation by recombinant PKC alpha . In the presence of PKC alpha , phosphorylation of nuclear PLC beta 1 was observed and increased with time of incubation (Fig. 6). Maximal phosphorylation was achieved within 30 min. Phosphorylation was not detected in the absence of recombinant PKC alpha , demonstrating that PLC beta 1 purified from nuclei was free of contamination of other potential kinases. Recombinant PKC alpha  did not cause phosphorylation of the mutated PLC beta 1 S887A within the time frame observed, further confirming S887 to be the phosphorylation site of PKC alpha .


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Fig. 6.   Recombinant PKC alpha  phosphorylate nuclear PLC beta 1 at serine 887 in vitro. PLC beta 1 (His)6 or PLC beta 1 (His)6 mutant S887A was purified from nuclei of transiently transfected cells as described in the legend to Fig. 5. An equal amount of these proteins was incubated without or with 50 ng of recombinant PKC alpha  and 2.5 µCi of [gamma -32P]ATP for different periods as described under "Experimental Procedures." The reaction was terminated by adding 6 × Laemmli loading buffer. Equal aliquots of samples were separated by 8% SDS-PAGE and analyzed by phosphorimaging or probed with anti-PLC beta 1 antibody.

The Sustained Responsiveness of PLC beta 1 Mutant S887A to IGF-I Stimulation-- To evaluate the effect of PKC-mediated PLC beta 1 phosphorylation on enzyme activity, stable transfectants expressing wild type PLC beta 1 or PLC beta 1 mutant S887A were generated as described under "Experimental Procedures." The clones in which the level of immunoreactive PLC beta 1 was significantly higher than that of endogenous PLC beta 1 were selected by immunoblotting, and used for further experiments. As shown in Fig. 7A, a similar level of immunoreactive PLC beta 1 was detected in the nuclei of cells overexpressing wild type PLC beta 1 and cells overexpressing PLC beta 1 mutant S887A, indicating that the nuclear localization of PLC beta 1 mutant S887A was not affected. Quantitative analysis showed that the level of immunoreactive PLC beta 1 in both of these cell lines is about 3-fold above that of control cells.


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Fig. 7.   Mutation of S887A abolishes PKC-mediated inhibition of nuclear PLC beta 1 in IGF-I-treated cells. Stable transfectants which overexpress wild type PLC beta 1 or PLC beta 1 mutant S887A were selected as described in the text. 40 µg of nuclear proteins from control cells or cells overexpressing wild-type PLC beta 1 or PLC beta 1 mutant S887A were separated by 8% SDS-PAGE and probed with anti-PLC beta 1 polyclonal antibody. B, quiescent cells overexpressing PLC beta 1 or PLC beta 1 mutant S887A were treated without or with 40 ng/ml IGF-I for the indicated period. The nuclei from these cells were analyzed for PLC activity (n = 4, expressed as mean ± S.D.). The figure shows the result of a typical experiment, and similar results were obtained from another two separate experiments using independent stable transfectant which express wild type PLC beta 1 or PLC beta 1 mutant S887A.

Consistent with the above result, analysis of nuclear PLC activity revealed a similar basal level of enzyme activity between cells overexpressing wild type PLC beta 1 and cells overexpressing S887A, both of which were about 3-fold higher than that of control cells. In both of these cell lines the nuclear enzyme activity increased by about 2.7-fold after 5 min of IGF-I stimulation (Fig. 7B). In cells overexpressing wild-type PLC beta 1, the level of enzyme activity decreased by 37.4% after 20 min and returned to basal level after 30 min. In contrast, the elevated activity of nuclear PLC from cells overexpressing S887A was sustained for a much longer period following IGF-I stimulation. It was still maximal after 30 min (Fig. 7B), only decreasing about 43% after 60 min, and returning to basal level by 150 min (data not shown). This result suggests that PKC-mediated PLC beta 1 phosphorylation at serine 887 is critical for the attenuation of nuclear PLC activity induced by IGF-I.

The Effect of DN PKC alpha  on IGF-I-dependent Activation of Nuclear PLC-- To confirm further that IGF-I-induced activation of nuclear PLC beta 1 is terminated by PKC alpha , a kinase-deficient mutant of this isoenzyme was introduced into Swiss 3T3 cells by transfecting with pCIN DN PKC alpha . Such a catalytically inactive mutant has been shown to act as a dominant-negative molecule by competing with the corresponding endogenous isoenzyme (32, 46). Following selection with 500 ng/ml G418, Western blot analysis was performed for clones selected from each independent population of stable transfectants to evaluate the expression level of PKC alpha . Two clones (termed DN PKC alpha  A and DN PKC alpha  B), in which the immunoreactive PKC alpha  is significantly higher than endogenous expression level, were chosen for further experiment (Fig. 8A). The expression of DN PKC alpha  in these two clones was also verified by taking advantage of the fact that PKC degradation and down-regulation in response to tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is dependent on an active kinase (47). Following TPA treatment for 24 h, PKC alpha  in control cells decreased to an undectable level. In contrast, the level of PKC alpha  in the two clones expressing DN PKC alpha  was reduced only slightly, consistent with the expression of the kinase-deficient DN PKC alpha  (Fig. 8A).


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Fig. 8.   Expression of DN PKC alpha  inhibits PKC-mediated nuclear PLC beta 1 phosphorylation at serine 887 and causes sustained activation of nuclear PLC in IGF-I-treated cells. A, Swiss 3T3 cells were transfected with pCIN DN PKC alpha  or empty vector pCIN4. Following selection with 500 ng/ml G418, 80 µg of cell lysates from two clones (DN PKC alpha  A and DN PKC alpha  B) expressing the dominant-negative PKC alpha  mutant and two control clones (pCIN4 A and pCIN4 B) were analyzed for the level of immunoreactive PKC alpha  before and after treatment with TPA (400 nM, 24 h). TPA treatment causes degradation of the endogenous wild-type PKC alpha , but not the kinase-deficient mutant (47). B, the clones selected in A were transiently transfected with a plasmid expressing (His)6-tagged PLC beta 1. Following in vivo 32P labeling and IGF-I treatment (40 ng/ml, 30 min), 32P-PLC beta 1 (His)6 was purified from nuclei and the tryptic peptide mixtures was analyzed by two-dimensional phosphopeptide mapping as described in the legend to Fig. 5. Note that phosphopeptide 3 was not detected in two clones overexpressing DN PKC alpha . C, the clones selected above were treated with IGF-I and the nuclei from there cells were subjected to analysis for PLC activity as described in the legend to Fig. 1. The results are from three independent experiments and expressed as mean ± S.D.

In both clones expressing DN PKC alpha , the PKC-mediated phosphorylation of PLC beta 1 at serine 887 was not detected in IGF-I-treated cells, suggesting PKC alpha  is the major isoform responsible for this phosphorylation (Fig. 8B). A time course analysis for nuclear PLC activity revealed a sustained activation of nuclear PLC in both cell lines that express DN PKC alpha  (Fig. 8C). At 30 min after IGF-I stimulation, the activity of nuclear PLC is still maintained at the maximally activated level. It decreased only by 43% after 60 min and returned to basal level after 150 min (data not shown). We thus conclude that alpha  isoform of PKC is the major isoenzyme responsible for the termination of IGF-I dependent activation of nuclear PLC.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Agonist-induced desensitization is an important regulatory process in the phosphoinositide signaling pathway (48). Compelling evidence suggests that PKC activation attenuates agonist-stimulated PLC activity, thus providing a negative feedback regulatory mechanism to control the magnitude and duration of the signal transmitted (37, 40, 49-51). Treatment of a variety of cells with the PKC activator phorbol 12-myristate 13-acetate inhibits both Galpha q- and Galpha i-coupled receptor stimulated PtdIns 4,5-P2 hydrolysis (49, 51, 52). This phorbol 12-myristate 13-acetate-dependent inhibitory action was prevented by prior incubation of cells with PKC inhibitors. PKC has also been shown to decrease the catalytic activity of PLC beta  isoforms in several in vitro reconstitution assays (38, 53).

The targets of PKC involved in the desensitization of agonist-stimulated PIP2 hydrolysis include G-protein-coupled receptors, G proteins functionally coupled with PLC, or PLC itself (for review, see Ref. 54). Direct phosphorylation of several subtypes of PLC beta  isoforms by PKC has been reported, although the functional consequences of these observations remains to be established (37, 38, 53, 55). A recent study by Filtz and co-workers (38) revealed that PLC beta t, a turkey PLC beta  isoform which shares highest homology with mammalian PLC beta 2, is phosphorylated by conventional PKCs (37, 53, 55). This PKC-mediated phosphorylation appears to decrease both basal activity of PLC beta t and the activity stimulated by GTPgamma S. A close correlation between PLC beta 3 phosphorylation and PKC-mediated inhibition of PIP2 hydrolysis has been observed (37). Yue et al. (56) have recently demonstrated that conventional PKCs induce phosphorylation of PLC beta 3 at serine 1105 both in vivo and in vitro. Mutation of serine 1105 to alanine completely abolishes the PKC-mediated inhibition of Gq-stimulated PLC beta 3 activity.

Despite the existence of distinct stimulatory controls for PI turnover at the plasma membrane and in the nucleus (16), PKC appears to exert similar inhibitory effects on PIP2 hydrolysis at the two sites. In the case of the nucleus, it is responsible for the termination of IGF-I dependent nuclear signaling. This conclusion is supported by the following two findings. First, inhibition of PKC activity by either calphostin C or Go6976 causes sustained activation of nuclear PLC activity induced by IGF-I (Fig. 1). Second, within the same time frame of IGF-I stimulation, the nuclear PKC activity inversely correlates with the activity of nuclear PLC (Fig. 2B).

It was previously demonstrated that the PKC activator TPA induces phosphorylation of PLC beta 1 in vivo, and phosphorylation of bovine brain PLC beta 1 by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887 (45). However, the physiological relevance of these findings has not been reported. Our two-dimensional tryptic peptide mapping analysis for 32P-labeled nuclear PLC beta 1 revealed that stimulation of Swiss 3T3 cells with IGF-I for 30 min induced production of two "extra" phosphopeptides (Fig. 5). One of these tryptic phosphopeptides was not observed in either cells treated with PKC inhibitor Go6976, or the cells overexpressing a PLC beta 1 variant in which serine 887 was replaced by alanine. We thus conclude that PKC-mediated phosphorylation of PLC beta 1 at serine 887 occurs within the nucleus following IGF-I stimulation. The pivotal role of this phosphorylation in PKC-mediated attenuation of PIP2 hydrolysis was confirmed by the observation that mutation of serine 887 to alanine causes sustained elevation of nuclear PLC activity in IGF-I-treated cells (Fig. 7).

An explanation of how phosphorylation of PLC beta 1 at serine 887 modulates the enzyme activity in the nucleus remains to be defined. Phosphorylation of PLC beta 1 by PKC in vitro has no direct effect on the enzyme activity (45). A recent report demonstrates that PKC-mediated phosphorylation inhibits the stimulation of PLC beta 1 activity by Gbeta gamma subunits, but does not affect Galpha -stimulated enzyme activity (53). Whether or not the Gbeta gamma subunit plays a role in IGF-I stimulated PLC activity in the nucleus is currently unclear. Nevertheless, evidence does exist for growth factor-induced nuclear translocation of Gi (57, 58), which could in turn lead to nuclear localization of beta gamma subunit and subsequent activation of putative PLC beta 1 in the nucleus.

PKC-promoted phosphorylation of PLC beta s appears to be isoenzyme specific (56). Our results strongly suggest that the alpha  isoform of PKC is responsible for phosphorylation of nuclear PLC beta 1 in Swiss 3T3 cells. This notion is supported by the following observations. (i) PKC alpha  interacts specifically with PLC beta 1 in the nucleus of IGF-I-treated 3T3 cells (Fig. 4). (ii) Nuclear accumulation of PKC alpha  closely correlates with the attenuation of nuclear PLC activity (Fig. 2). (iii) IGF-I dependent activation of nuclear PLC beta 1 is sustained by treatment with the selective PKC alpha  inhibitor Go6976 (Fig. 1) and by introduction of DN PKC alpha  (Fig. 8C). (iv) Recombinant PKC alpha  can directly phosphorylate PLC beta 1 purified from nuclei (Fig. 6). On the other hand, expression of DN PKC alpha  blocked phosphorylation of nuclear PLC beta 1 at serine 887 (Fig. 8B). In agreement with this conclusion, a recent study has found that, among several PKC isoforms tested, PKC alpha  is most effective in promoting phosphorylation of PLC beta  in vitro (53). Furthermore, Neri and co-workers (29) have demonstrated that of four PKC isoforms (alpha , beta I, epsilon , and xi ) detected in Swiss 3T3 cells, only PKC alpha  is present in the nucleus. Therefore, PKC activity in the nucleus of Swiss 3T3 cells appear to be due solely to this isozyme.

In summary, the present study demonstrates for the first time the existence of a negative feedback control mechanism to desensitize IGF-I receptor-evoked activation of nuclear PI cycle, and that PKC alpha -promoted phosphorylation of nuclear PLC beta 1 at serine 887 plays a key role in this regulation process. However, our study could not exclude the possibility that PKC alpha  may also exert its inhibitory action on PLC beta 1 by phosphorylating other nuclear components involved in the regulation of its activity. The potential downstream nuclear targets of PKC alpha  are currently under investigation in our laboratory.

    ACKNOWLEDGEMENT

We thank Dr Albert Descoteaux for providing the vectors expressing dominant-negative PKC alpha .

    FOOTNOTES

* This work was supported by the Marsden Fund of the Royal Society of New Zealand and Health Research Council of New Zealand.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.

To whom correspondence should be addressed. Fax: 64-9-3737492; E-mail: s.gilmour@auckland.ac.nz.

Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M009144200

    ABBREVIATIONS

The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PI, phosphoinositide; DG, diacylglycerol; DN PKC alpha , dominant-negative protein kinase C alpha ; PLC, phospholipase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; IGF-I, insulin-like growth factor I; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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