A Dominant Negative Protein Kinase C 4 Subspecies ... - Europe PMC

Vol. 13, No. 8

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1993, p. 4770-4775

0270-7306/93/084770-06$02.00/0 Copyright X 1993, American Society for Microbiology

A Dominant Negative Protein Kinase C 4 Subspecies Blocks NF-vdB Activation MARIA T.

DIAZ-MECO,l EDURNE BERRA,'

ISABEL DOMINGUEZ,' VICTOR

MARIA M. MUNICIO,1 LAURA SANZ1' JOSE

LOZANO,1

DIAZ-GOLPE,1 MARIA T. LAIN DE LERA,2 JOSE ALCAMi,2

CARLOS V. PAYA,3 FERNANDO ARENZANA-SEISDEDOS,4 JEAN-LOUIS VIRELIZIER,4 AND JORGE MOSCAT1*

Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas- Universidad Aut6noma de Madrid, Canto Blanco, 28049 Madrid, and Servicio de Microbiologia, Hospital 12 de Octubre, 28041 Madrid,2 Spain; Department of Immunology, Mayo Clinic, Rochester, Minnesota 559053; and Laboratoire d'Immunologie Virale, Institut Pasteur, 75724 Paris, France4 Received 4 March 1993/Returned for modification 5 April 1993/Accepted 19 May 1993

Nuclear factor KB (NF-KB) plays a critical role in the regulation of a number of genes. NF-KB is a heterodimer of 50- and 65-kDa subunits sequestered in the cytoplasm complexed to inhibitory protein IcB. Following stimulation of cells, IKB dissociates from NF-KB, allowing its translocation to the nucleus, where it carries out the transactivation function. The precise mechanism controlling NF-KB activation and the involvement of members of the protein kinase C (PKC) family of isotypes have previously been investigated. It was found that phorbol myristate acetate, (PMA) which is a potent stimulant of phorbol ester-sensitive PKC isotypes, activates NF-cB. However, the role of PMA-sensitive PKCs in vivo is not as apparent. It has recently been demonstrated in the model system ofXenopus laevis oocytes that the PMA-insensitive PKC isotype, 4PKC, is a required step in the activation of NF-KB in response to ras p21. We demonstrate here that overexpression of VPKC is by itself sufficient to stimulate a permanent translocation of functionally active NF-KB into the nucleus of NIH 3T3 fibroblasts and that transfection of a kinase-defective dominant negative mutant of CPKC dramatically inhibits the KB-dependent transactivation of a chloramphenicol acetyltransferase reporter plasmid in NIH 3T3 fibroblasts. All these results support the notion that tPKC plays a decisive role in NF-KB regulation in mammalian cells.

cytokines that stimulate progressively better-characterized transmembrane signalling mechanisms prompted the investigation of the precise role played by these signal transduction pathways in NF-KB activation (12, 14, 35). Thus, for example, the contribution of phorbol myristate acetate (PMA)-sensitive protein kinase C (PKC) isotypes to the stimulation of this parameter has previously been addressed (8). From those results, it is clear that although phorbol esters may be able to activate NF-KB (8), the role of PMA-sensitive PKCs in vivo is not as clear (14, 23). Interestingly, we have recently demonstrated, using the model system of Xenopus laevis oocytes, that the PMA-insensitive PKC isotype, tPKC, is a required step in the activation of NF-KB in response to ras p21 (5). This finding is particularly challenging in light of the role of ras in lymphocyte stimulation (6). We demonstrate here that the overexpression of tPKC is by itself sufficient to activate NF-KB in NIH 3T3 fibroblasts. Furthermore, expression of a dominant negative mutant of tPKC dramatically inhibits the KB-dependent transactivation of a chloramphenicol acetyltransferase (CAT) reporter plasmid.

Great effort is being invested in elucidating the mechanisms responsible for the transmission to the nucleus of signals generated by cytokine-receptor interactions at the plasma membrane. Activation of transcription factors is a critical step in these pathways (21). Thus, nuclear factor KB (NF-KB) is involved in the regulation of a large number of genes, including the enhancer of human immunodeficiency virus (HIV) (11, 20, 24, 33). NF-KB is a heterodimer of 50-kDa (p5O) and 65-kDa (p65) subunits (1, 2) located in the cytoplasm in an inactive form, bound to the inhibitory protein IKB through the p65 molecule (1, 2). Upon stimulation of cells, IKB dissociates from NF-KB, allowing its translocation to the nucleus, where it carries out the transactivation function (1, 2). The cDNAs of p50 and p65 have recently been cloned, and their amino acid sequences have been deduced (2, 9, 13, 17, 30). Interestingly, they belong to the same gene family as do c-rel and the Drosophila maternal morphogen dorsal (2, 9, 13, 17, 30). The p50 subunit is synthesized as a p105 precursor whose C-terminal portion controls the subcellular localization of p65 and c-rel, apparently in an analogous way to IKB (13, 16, 29). The pathways that regulate NF-KB translocation to the nucleus are presently unclear. Two potential mechanisms have been proposed: (i) inactivation by phosphorylation of IKB, which prevents its ability to complex and retain NF-KB into the cytosol, thus allowing its translocation to the nucleus (8, 34); and (ii) release of p65 or c-rel as heterodimers with p50 upon cleavage of its precursor, p105 (29). The fact that NF-KB activation takes place following stimulation with *

MATERUILS AND METHODS Plasmids. The cDNA of tPKC from X. laevis oocytes subcloned into plasmid pBluescript (pBluescripttPKC) (4) was excised with XbaI and ApaI and ligated into plasmid pRcCMV (Invitrogen) to give pRcCMVt. To obtain the kinase-defective mutant of tPKC, the following strategy was carried out. pBluescripttPKC was excised with XbaI and SalI and ligated to plasmid pSELECT previously cut with the same enzymes. Afterwards, single-stranded template

Corresponding author. 4770

ROLE OF

VOL. 13, 1993

rescued and annealed both to an ampicillin repair oligonucleotide (Promega) and to a mutagenic oligonucleotide (5'-AATCTATGCAATGTGGGTTGTCAAGAAGGAG C-3'), and the mutant strand was synthesized with T4 DNA polymerase and ligated. Mutants were selected and screened by direct sequencing, to give plasmid pSELECT1mut. This plasmid was used to obtain pRcCMVtrut according to the following protocol. Plasmid pSELECT1mut was digested with SailI, blunt ended, and digested again with XbaI. Plasmid pRcCMV was digested with ApaI, blunt ended, and cut again with XbaI. The two fragments were ligated to give

was

pRcCMV;mut.

To make plasmid pRcCMV;del, a fragment corresponding tPKC (nucleotides 900 to 1867) was obtained by polymerase chain reaction (PCR) using pBluescripttPKC (4) as the template and primers 5'-ATGCG GGTTATCGGAAGAGGCAGCT-3' and 5'-ATCTCTAGAT TATACACATTCI1TCG-3' (which has an XbaI site). This PCR product was digested with XbaI and ligated to pRcCMV previously cut with HindIII, blunt ended, and digested again with XbaI. Isolation of stable transfectants. NIH 3T3 fibroblasts were grown in Dulbecco modified Eagle medium containing 10% fetal calf serum, and subconfluent cultures were transfected with pRcCMVt or with control pRcCMV by the calcium phosphate precipitation method (GIBCO BRL). Cell mass populations expressing transfected genes were selected for the ability to grow in the presence of geneticin. CPKC RNA levels in different transfectants. Total RNA from overexpresser or transiently transfected cells was isolated by the RNAzol B method (Cinna/Biotecx Laboratories Inc., Houston, Tex.) according to the manufacturer's instructions. Approximately 1 ,ug of total RNA each from pRcCMV, pRcCMVt4, and pRcCMV;6 or from NIH 3T3 cells transfected with either plasmid pRcCMV, plasmid pRcCMV;, or plasmid pRcCMV;mut was reverse transcribed into first-strand cDNA and subsequently used for PCR amplification with primers 1 (5'-ACAATAGCCGGAA TCT-3') and 2 (5'-GCTAGATGGGCTC`I"I`T-3') or primers 3 (5'-ATGGATCCGCAAGTAG-3') and 4 (5'-TTCTTGGGAG AlTTTG-3'), respectively, depending on the experiments. Primers 1 and 2 and primers 3 and 4 specifically amplify a 162-bp fragment corresponding to the D3 domain of Xenopus and mouse tPKC, respectively (4). The reaction was cycled 35 times at 94°C for 1 min, at 56°C for 2 min, and at 72°C for 3 min. Half of the PCR mixture was electrophoresed on 4% NuSieve 3:1 agarose (FMC). Samples were visualized by ethidium bromide staining. Reverse transcriptase control containing every component but the reverse transcriptase was treated identically. Immunoblot analysis of different transfectants. Seventy micrograms of cellular protein from the different cell lines was resolved in sodium dodecyl sulfate (SDS)-9% polyacrylamide gels. Afterwards, they were electrophoretically transferred into an Immobilon membrane (Millipore) and incubated with a specific anti-tPKC peptide antibody (GIBCO BRL). The bands were visualized with the AuroProbe BL system (Amersham International). Isolation of PC-PLC from Bacilus cereus. Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) was isolated from cultures of B. cereus SE-i essentially as described previously (19). Following this protocol, the enzyme preparation was purified to complete homogeneity, as confirmed by SDS-polyacrylamide gel electrophoresis and silver staining. The specific activity of the purified enzyme was 1.5 U/l,g. to the catalytic domain of

tPKC IN NF-KB REGULATION

4771

Gel mobility shift assay. Nuclear extracts were obtained as described previously (26). Cells at 75% confluence in 100mm-diameter culture dishes were made quiescent by serum starvation for 24 h, after which they were either untreated or stimulated with tumor necrosis factor alpha (TNF-a) for 4 h, according to the experiments. Cells were lysed for 6 min at 4°C in lysing buffer (26) and centrifuged at 6,500 rpm for 15 min at 4°C, and nuclear extract was prepared from the pellet. For mobility shift assays, 5 to 7 ,g of nuclear protein extract was incubated with 15,000 cpm of a 32P-labeled (5) doublestranded synthetic oligonucleotide probe corresponding to the HIV enhancer containing the NF-KB binding site at 22°C for 15 min in 10 ,ul of buffer as described previously (26). The binding reaction was analyzed by electrophoresis in nondenaturing 6% polyacrylamide gels. DNA-binding competition was assessed by preincubating the extracts with a 40-fold excess of unlabeled oligonucleotide. When indicated, either 3 i±l of rabbit anti-pSO or 5 >±l of rabbit anti-p65 antiserum (5) or 15 ng of recombinant IKB (MAD-3; generously provided by Ron T. Hay) was added to the standard reaction mixture 10 min prior to the addition of radiolabeled probe. The anti-pSO antiserum was generously provided by Alan Israel and does not cross-react with p65 or c-rel. The anti-p65 antiserum was generously provided by Ron T. Hay and does not cross-react with c-rel. The oligonucleotide used in the binding reactions corresponded to the wild-type NF-cBbinding sequence described previously (5, 26). Transfections. Subconfluent cultures of NIH 3T3, pRcCMV, pRcCMVt4, and pRcCMVt6 cells were transfected with 5 ,ug of either plasmid ConACAT or plasmid 3EConACAT by the calcium phosphate precipitation method. After 4 h, the DNA-containing medium was removed and cells were incubated with low (0.2%)-serum medium for 36 h. Afterwards, extracts were prepared and CAT activity was determined as described previously (5). Similarly, subconfluent cultures of NIH 3T3 fibroblasts were transfected with 5 p,g of either ConACAT or 3EConACAT in addition to 5 ,ug of either pRcCMVtmut, pRcCMVt, pRcCMVtdel, or pRcCMV, after which cells were stimulated with different agents following 36 h in low-serum medium, according to the experiments.

RESULTS AND DISCUSSION To demonstrate that CPKC is by itself sufficient and necessary to activate NF-KB, we chose a mammalian system like NIH 3T3 fibroblasts. We have previously cloned the cDNA of tPKC from a library of X. laevis oocytes (4); this cDNA, which is highly homologous (72% identity at the amino acid level) to the rat brain enzyme (4), was subcloned into plasmid pRcCMV to give pRcCMVt. This plasmid has enhancer/promoter sequences from the immediate/early gene of human cytomegalovirus and provides a high level of expression of the cloned gene in eukaryotic cells. Consequently, subconfluent NIH 3T3 fibroblasts were transfected with pRcCMVt or with control pRcCMV by the calcium phosphate precipitation method. Both plasmids have a neo selectable marker that conferred resistance to geneticin (G418). Cell mass populations expressing transfected genes were selected for the ability to grow in the presence of

geneticin.

We next examined the expression of the transfected tPKC in these cells by using reverse-transcribed RNAs and PCR analysis with specific primers for the D3 domain of Xenopus or mouse tPKC (Fig. 1). Results from Fig. 1A reveal that cultures of pRcCMVt4 and pRcCMVt6 displayed dramati-

MOL. CELL. BIOL.

DIAZ-MECO ET AL.

4772

A

2

3

4

6

5

8

7

9

1 2 3 4

10 11

kDa 106 80

B

C

1

2

2

3

3

4

4

5

7

6

5

6

8

9

10

11

50

7

FIG. 1. Expression of tPKC RNA in transfected cells. Approximately 1 p,g of total RNA each from pRcCMV (A and B; lanes 3 to 5), pRcCMVt4 (A and B; lanes 6 to 8), pRcCMVt6 (A and B; lanes 1 and 9 to 11), or NIH 3T3 cells transfected with either plasmid pRcCMV (C; lanes 2 and 3), plasmid pRcCMVt (C; lanes 4 and 5), or plasmid pRcCMV;mut (C; lanes 6 and 7) was reverse transcribed into first-strand cDNA and subsequently used for PCR amplification with primers 1 (5'-ACAATAGCCGGAATCT-3') and 2 (5'-GCTA GATGGGCTCTTT-3') (A and C) or primers 3 (5'-ATGGATCCG CAAGTAG-3') and 4 (5'-TTCTTGGGAGATlTTlG-3') (B) as described in Materials and Methods. Primers 1 and 2 and primers 3 and 4 specifically amplify a 162-bp fragment corresponding to the D3 domain of Xenopus and mouse tPKC, respectively (4). Samples were visualized by ethidium bromide staining. The following controls were carried out: PCR with primers 1 and 2 (panel A, lane 1; panel C, lanes 2, 4, and 6) or 3 and 4 (panel B, lane 1) and every component but the reverse transcriptase; reverse transcription-PCR with primers 1 and 2 (panel A, lane 2; panel C, lane 1) or 3 and 4 (panel B, lane 2) with no template; PCR only with primer 1 (panel A, lanes 4, 7, and 10) or 3 (panel B, lanes 4, 7, and 10); and PCR with only primer 2 (panel A, lanes 5, 8, and 11) or 4 (panel B, lanes 5, 8, and 11). This is a representative experiment of three with identical results.

cally increased levels of tPKC RNA compared with control (pRcCMV) and NIH 3T3 (not shown) cells, which did not show any detectable band. As a control, the PCR was carried out with primers specific for the D3 domain of the endogenous tPKC. Equal amounts of RNA for this enzyme were found in the different cell lines (Fig. 1B). With this information in hand, pRcCMVt4 and pRcCMVt6 cells were used for further study. Figure 2 shows an immunoblot analysis performed in extracts from the different cell lines with an antipeptide antibody specific for tPKC. From this analysis, we demonstrate increased levels of tPKC protein in both pRcCMVt4 and pRcCMVt6 cells compared with the controls, NIH 3T3 and pRcCMV cells. It is noteworthy that the increase in the protein levels of tPKC is not as dramatic as that in RNA levels in the overexpresser cell lines because the antibody used does not discriminate between the transfected and the native mouse tPKC. Interestingly, an immunoreactive band of approximately 35 kDa is detected in the extracts from the tPKC overexpresser cell lines (Fig. 2). The possible significance of this fragment is discussed below. An immunoreactive band seen around 65 kDa is also increased, although to a lesser extent, in the transfectants. This protein has also been described previously, and its relationship to tPKC remains to be fully clarified (36). The origin of a higher-molecular-mass band (around 90 kDa) that reacts with the antibody and that is seen in the overexpresser cell lines is unclear.

1*

-1032

FIG. 2. Expression of tPKC protein in transfected cells, demonstrated by immunoblot analysis of different transfectants. Seventymicrogram samples of cellular protein from NIH 3T3 (lane 1), pRcCMV (lane 2), pRcCMVt6 (lane 3), and pRcCMVt4 (lane 4) cells were resolved in SDS-9% polyacrylamide gels, electrophoretically transferred into an Immobilon membrane (Millipore), and incubated with a specific anti-^PKC peptide antibody. The bands were visualized with the AuroProbe BL system (Amersham International). This is a representative experiment of three with identical results.

To determine whether overexpression of this kinase leads to activation of NF-KB, nuclear extracts from the various cell lines were obtained, and gel shift assays were carried out with an oligonucleotide corresponding to the wild-type NFKB-binding sequence of the HIV long terminal repeat (LTR).

Results in Fig. 3A show the lack of NF-KB activity in nuclear extracts from either unstimulated NIH 3T3 cells or the control cell line pRcCMV. However, nuclear extracts from

pRcCMVt4 and pRcCMVt6 cells showed dramatically increased levels of NF-KB activity. Interestingly, pRcCMVt6, whose tPKC levels are higher than those of pRcCMVt4 (Fig. 1 and 2), also displayed higher NF-KB activity. As a positive control, nuclear extracts from NIH 3T3 cells treated with TNF-a, a classical stimulant of NF-KB, also displayed increased activity of this parameter (Fig. 3A). To characterize the NF-KB activity induced as consequence of tPKC overexpression, the following experiments were carried out. Nuclear extracts from pRcCMVC6 and NIH 3T3 cells stimulated with TNF-a were analyzed in a band shift assay as shown in Fig. 3B. The results indicate that the band induced in nuclear extracts of pRcCMVt6 is specific, since it is competed for by a 40-fold molar excess of unlabeled oligonucleotide (Fig. 3B, lane 2), in a similar way to the band induced by TNF-aL in NIH 3T3 cells (lane 7). Interestingly, that band is completely abolished when the nuclear extracts were incubated with recombinant IKB (MAD-3) (generously provided by Ron Hay) (lane 6). The presence of IKB also dramatically reduced the NF-KdB activity of pRcCMVt6 (lane 3); the band is not completely abolished, probably because of the massive NF-KB activity observed in nuclear extracts from pRcCMVt6 cells compared with TNF-a-activated NIH-3T3 cells. Actually, when the experiment was repeated with as low as 1.5 ,ug of pRcCMVC6 nuclear extracts, the NF-KB activity was completely abolished (not shown). Preincubation of nuclear extracts with antibodies raised against either the p50 or the p65 subunit decreased specific DNAbinding activity, promoting a characteristic upshifting of the NF-KB band (Fig. 3B, lanes 4 and 5). Incubation with the preimmune serum did not produce any effect (not shown).

ROLE OF PKC IN NF-KB REGULATION

VOL. 13, 1993

B

4773

LL

z

I-

A

be

Q0 C.) et

t

.0

4-,

z

U-.

+ C

c'

_

_

> u>

a:

z z CQ

c: cc CQ

CQ

---

-+- -+-

-

Comp. Anti p50 Anti p65 IkB

4-, El

U

Twal

-

+

-

+

-

+

-

+

pWZ4 picONf pAoW vecexpression ConACAT enhancer the KB FIG. 4. Activity of tor in different transfectants. Subconfluent cultures of NIH 3T3, pRcCMV, pRcCMV44 (pRcCMVZ4), and pRcCMVC6 (pRcCMVZ6) cells were transfected with 5 pg of either plasmid ConACAT or plasmid 3EConACAT by the calcium phosphate precipitation method. After 4 h, the DNA-containing medium was removed and cells were incubated with low (0.2%)-serum medium for 36 h. Afterwards, extracts were prepared and CAT activity was determined as described in Materials and Methods. The control ConACAT activity was not affected by any condition. As positive controls, cells were incubated in the presence of 500 U of TNF-a per ml. Results are the means ± standard deviations of four independent experiments with incubations in duplicate.

aD-3T3

,ii

t I v

I

I

i i

-.

1 2345678 2 3 4 5 FIG. 3. Induction of NF-KB in different transfectants. (A) NF-K