May 15, 2018 - Northern Blot Analysis-Total cellular RNA (5-20 pg) or poly(A)- selected mRNA (0.1-10 pg) was denatured and fractionated on a 1% agarose ...
Vol. 268, No. 14, Issue of May 15,pp. 10627-10635,1993 Printed in U.S. A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology. Inc.
Selective Regulation of Expression of Protein Kinase C p Isoenzymes Occurs via Alternative Splicing* (Received for publication, October 27, 1992, and in revised form, January 7, 1993)
Gerard C. Blobe, Wasiuddin A. Khan, Andrew E.Halpern, LinaM. Obeid, and Yusuf A. HannunS From the Divisions of HematologylOncology and Geriatrics, Departments of Medicine and Cell Biology, D u k University Medical Center, Durham, North Carolina-27710
The mechanisms involved in regulating the selectivetheir expression is modulated during differentiation. For exexpression of protein kinase C (PKC) isoenzymes are ample, the PKC gene is highly expressed in the brain and poorly understood. Two human B lymphoblastoid cell in lymphoid and hematopoietic cells (3). The expression of lines, IM-9 and BJA-B, exhibited differential expres- the PKCfi gene is also developmentally regulated. Low levels sion of the two alternatively spliced products of the are observed in the fetal spleen and during the first 2 weeks PKC /3 gene, PKC @Iand BII. The IM-9 cell line ex- of life after which a rapid increase in expression occurs. In pressed 3-4-fold more PKC BII protein than the BJA- the thymus, maximal levels occur shortly after birth with a B cell line, whereas the BJA-Bcell line expressed 2decrease in expression thereafter (3). Finally, expression of %fold more PKC 61 protein. This differential expres- the PKC p gene can be increased by inducers of differentiation sion was found to be regulated at the mRNA level. in the human promyelocytic leukemia cell line, HL-60 (4,5). Comparison of PKC BI and BII messages in poly(A)+ Little hasbeen done to determine the mechanisms by which mRNA and total cellular RNA revealed that selective polyadenylation was not involved. The messages for differential expression of PKC isoenzymes occurs. We have PKC @Iand BII had comparable half-livesin both cell shown that transcriptional activation of PKC p expression lines, ruling out differential message stability. In ad- results in increased PKC activity without changes in dlacyldition, similar ratios of PKC 01 and BII messages in glycerol levels (4). We have hypothesized that thismechanism cytosolic and nuclear fractions suggested that differ- of activating PKC may be an important means for induction ential mRNA transport was not involved. In the IM-9 of cellular differentiation by PKC. Thus, the mechanisms cell line, thepredominance of mature PKC BII message that regulate the expression of PKC isoenzymes may emerge as well as thatof a larger message spliced to PKC BII as critical pathways for regulating PKC activity in tissueprovided evidence that the differential expression of specific functions, development, and differentiation. PKC BII was regulated at the level of mRNA splicing. In ongoing studies we have found that the expression of In the BJA-B cell line, equal amountsof mature PKC PKC PI and 011, the two alternatively spliced products of the BI and BII message and the absence of the larger mes- PKC p gene, is modulated during lymphoid differentiation. sage suggested that the splicing of the PKC B gene Moreover, we have found that PKC PI and PKC PI1 undergo product can be regulated to produce altered ratios of selective translocation to the cytoskeletal compartment folPKC @Iand BII. Implications of these studies on the lowing stimulation of lymphocytes by phorbol esters.’ These differential expression of PKC isoenzymes and their results suggested that these two isoenzymes may mediate roles inbiology are discussed. distinct functions, and therefore regulation of their expression may have important biological consequences. For these reasons we chose to define mechanisms responsible for the differential expression of PKC PI and PII. In two Protein kinase C(PKC),’a phospholipid-dependent, di- B lymphoblastoid cell lines, the IM-9 and BJA-B cell lines, acylglycerol-activated serine/threonineprotein kinase, is PKC PI and PI1 are differentially expressed. Analysis of this known to play an important role in signal transduction, tumor differential expression revealed a novel mechanism for the promotion, and cell regulation. Recent cloning and molecular selective expression of PKC isoenzymes. analysis has revealed that PKC is actually composed of a family of closely related isoenzymes. These isoenzymes can MATERIALSANDMETHODS be classified into two groups: the calcium-dependent isoenCell Culture-The IM-9 cell line was obtained from ATCC. The zymes, PKC a, PI, PII, and y; and the calcium-independent BJA-B cell line was the generous gift of Dr. Peter Cresswell, Departisoenzymes, PKC 6, c, {, q, and 8 (for review, see Refs. 1 and ment of Microbiology/Immunology, Duke University Medical Center. 2). PKC isoenzymes are known to be expressed in a tissuespecific and developmentally regulated fashion. In addition,
* This work wassupported inpart by National Institutes of Health Grant HL-43707.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “odvertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ To whom correspondence should be addressed Division of Hematology/Oncology, Box 3355,Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2449; Fax: 919-681-8253. The abbreviations used are: PKC, protein kinase C; PCR, polymerase chain reaction; UTR, untranslatedregion; kb, kilobase(s).
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Cells were maintained in RPMI1640medium (GIBCO) supplemented with 10%fetal calf serum (GIBCO) in 5% COzat 37 “C in a humidified atmosphere. The cell lines were passaged every 48-72 h to maintain logarithmic growth. Western Blot Analysis-Cell extracts from IM-9 and BJA-B cells were prepared by suspending cells in ice-cold homogenizing buffer (20 mM Tris-HC1, pH 7.5, 250 mM sucrose, 10 mM EGTA, pH 7.4, 2 mM EDTA, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 0.02% leupeptin, 0.1% Triton X-100) at lo’ cells/ml. The cells were then lysed by sonication, an aliquot taken for protein determination, and the remainder mixed (1:l)with 2 X sodium dodecyl sulfate sample buffer and boiled for 5 min. Samples (2.5-150 pg) werethen analyzed
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* G . Blobe, L. Obeid, and Y. Hannun, unpublished observations.
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Regulation of Expression of PKC PI and PI1
by Western blot analysis as described previously (6). RESULTS RNA Isolation-RNA was prepared by the single-step RNA isolaCharacterization of PKC @I and PII Protein Expression in tion procedure (7). Briefly, for total cellular RNA, cells were centrifuged and lysed directly in denaturing solution (4 M guanidinium f M - 9 and BJA-B Cell Lines-In two B lymphoblastoid cell thiocyanate, 25 mM sodium citrate, 0.1 M #?-mercaptoethanol,0.5% lines, IM-9 and BJA-B, PKCPI and PI1 proteins were differN-lauroylsarcosine), at 1 ml/107 cells. For nuclear and cytoplasmic entially expressed. Although both the IM-9 and BJA-B cell RNA, 4 X 10’ cells were washed twice in ice-cold 1 X Dulbecco’s PI and PII, the IM-9 cells expressed more phosphate-buffered saline and then lysed in 6 mlof ice-cold lysis lines expressed PKC PI1 than the BJA-B cells, whereas the BJA-B cell line exbuffer (50 mM Tris, pH 7.5,lOO mM NaCI, 5 m M MgC12, 0.5%Nonidet P-40). Nuclei were centrifuged at 1,000 X g for 5 min at 4 “C and pressed more PI than the IM-9 cell line. Since P K C PI and lysed in 5 ml of denaturing solution. The supernatant (cytoplasm) PI1 are derived from the same PKC P gene and transcribed was mixed with an equal volume of 2 X denaturing solution. These from the same promoter ( l a ) , the differential expression of various fractions in denaturingsolution were then phenol/chloroform these two isoenzymes suggested a novel mechanism for the extracted and precipitated with isopropyl alcohol. The RNA pellet selective expressionof P K C isoenzymes. Therefore we sought was resuspended in a0.3 volume of denaturing solution and reprecipthe itated. Nuclear RNA represented approximately 20% of total cellular to determinethemechanismsinvolvedinregulating expression of P K C PI and PI1 in these cell lines. RNA in both IM-9 and BJA-Bs,consistent with the proportion Initially, the expression of P K C PI and PI1 protein was obtained from other cell lines (8).Poly(A)+mRNA wasobtained from total cellular RNA using an oligo(dT)-cellulose column under condi- characterized. First, the specificity of the P K C PI a n d PI1 tions described by the manufacturer (GIBCO-BRL). Poly(A)+mRNA immunoreactive bands on Western blot analysis was deterrepresented about 1-3% of total cellular RNA in IM-9 and BJA-Bs. mined by preadsorbing the isoenzyme-specific antibody with Samples for half-life determination were treated with actinomycin D the peptide against which it was raised. In both cell lines a a t 10 pg/ml, and aliquots were harvested over a 24-h period. cDNA Cloningof PKC PI- and PII-specificProbes-cDNA synthesis band specific for PKC PI was present as a single band at 77 of PKC PI- and PII-specific probes involved reverse transcription of f 0.2 kDa, and two bands specific for PKC PI1 appeared at a single-stranded DNAcopyfrom mRNA using a synthetic DNA 77 f 0.6 kDa and 74 +. 0.5 kDa (Fig. 1A). These bands were oligonucleotide primer and synthesis of double-stranded cDNA n o longer present with addition of competing peptide. The through the combined action of RNase H and DNA polymerase I size of the P K C PI and PI1 proteins is consistent with that using the riboclone system (Promega). The double-stranded DNA predicted by the sequence of P K C PI and PII. The two bands was then used as a template to amplify a specific fragment of DNA PI1 antibodyinbothcelllines are by PCR which was then ligated into an appropriate vector. Specifi- detectedbythePKC cally, 10 pg of total cellular RNA (from BJA-B for PI and IM-9 for referred to as upper and lower P K C PII, respectively, and may PII) was hybridized to 0.5pgof3’ antisense primer-adapter with reflect a post-translational modification such as phosphorylsequence specific to PI (5’-GGG AAT TCC ACA TGT TGA ATG ation. Notably, the upper PKC PI1 band was not subject to CCA-3’) or PI1 (5’-GGG GAT CCA AAC TAC CTG AGG ACA-3’) down-regulationbylongtermphorbolestertreatmentin and a convenient terminal restriction site (EcoRI and BamHI, re- either cell line (data not shown). The significance and relaspectively). The hybridization, synthesis of the first strand cDNA with avian myeloblastosis virus reverse transcriptase, and second tionship of these forms of P K C PI1 remain tobe explored. The relative quantity of P K C PI and PI1 in the IM-9 and strand cDNA synthesis were performed under conditions described by the manufacturer. The ends were then flushed with 2 p~ T4 DNA BJA-B cell lines was then determined by Western blot analypolymerase. The cDNA was used as a template for PCR. Primers for sis. Since the polyclonal antibodies for PKC PI and P K C PI1 PCR were: ( a ) a 1 p~ concentration 5’ sense primer-adapter with have distinct affinities for their respective antigens (45 and sequence specific to PI (5’-GGA AGC TTG AGACAAGAGAGA 10.5 WM, respectively), it was not possible to compare quanCA-3‘) or PI1 (5’-GGA AGC TTGTGG GCG AAATGC TG-3’) and titatively the absolute amount of P K C PI a n d PI1 protein a convenient terminal restriction site (HindIII); and ( b ) a 1 p M concentration of the appropriate 3’ antisense primer-adapters from within a cell line. However, a determination of the relative above. The PCR products were digested with the appropriate restric- expression of P K C PI or PI1 between the two cell lines was tion enzymes and cloned into the pBS- vector (Stratagene). The possible. As shown in Fig. l B , the BJA-B cell line expressed constructs were then verified by sequencing and named pBSPI and approximately %fold more PKCPI than the IM-9 cell line. I n pBSPII for vectors containing PKC PI and PI1 sequence, respectively. contrast, the IM-9 cell line expressedat least 4-fold as much RNase Protection Analysis with PKC Isoenzyme-specific Probesof the lower PKC PI1 as the BJA-B cell line but equivalent An antisense PKC 01 or 011 RNA probe was transcribed from vectors amounts of the upper PKC PII. pBSPI or pBSOI1 with T7 polymerase and labeled with [ L ~ - ~ ~ P ] U T P Determination of PKC PI a n d PII mRNA Levels-To corusing the Stratagene RNA transcription kit under conditions described by the manufacturer. The DNA template was degraded with roborate these Western blot findings and to determine the RQ1 DNase, andthe labeled probes weregel purified ona 4% mechanism of the differential expressionof P K C PI and PII, polyacrylamide gel. Probes (1.2-5 X lo6cpm/hybridization) were then the relative levelsof P K C PI a n d PI1 mRNA were determined. hybridized with 5-20 pg of total RNA or 0.1-10 pg of poly(A) selected For this purpose it was necessary to design probes specific for mRNA from IM-9 or BJA-B cells and analyzed using the RPA I1 ribonuclease protection assay kit (Ambion). Hybridizations were at P K C PI and PII. These isoenzymes diverge over the last 503‘45 “C overnight. Nonprotected probe was digested with 0.5 unit/ml 52 amino acids of their coding sequence and in their 2). To take maximal RNase A and 100 units/ml RNase T1 at 37 “C for 30 min. The size untranslated regions (UTRs) (see Fig. of the protected fragment was determined by electrophoresis on a advantage of this divergence, oligonucleotides were synthedenaturing polyacrylamide gel (6% acrylamide, 7 M urea) with a sized corresponding to the antisense strand of the 3’-UTR sequencing ladder runas size markers. PKC PI- and 011-specific region of P K C @Ia n d PII, respectively. These oligonucleotides bands were subject to laser densitometry for quantification. Northern Blot Analysis-Total cellular RNA (5-20 pg) or poly(A)- were then used as primers to synthesize cDNA for PKC PI selected mRNA (0.1-10 pg) was denatured and fractionated on a 1% a n d PI1 from the IM-9 and BJA-Bcell lines. This cDNA was agarose gel containing formaldehyde as described (9). RNA was then used as a template for PCR of P K C PI- and PII-specific analyzed by Northern blot analysis as described previously (4). PKC sequences extending from their divergent coding region into PI- and PII-specific bands were subject to laser densitometry for the 3’-UTR. These sequences were cloned and used to make quantification. P K C PI- a n d 011-specific riboprobes for RNase protection and Sequence Analysis-Sequences for PKC PI and PI1 were analyzed Northern blot analysis (see “Materials and Methods” Fig. and utilizing the TFASTA and FASTA programs of the GCG package, Release 6.1on the Duke Comprehensive Cancer Center Microvax 2 ) . Using RNase protection, a protected band of the expected (10). FASTA and TFASTA use the method of Pearson and Lipman size was detected for both PKC PI and PI1 in total cellular (11).
Regulation of Expression of PKC PI and PII
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FIG.1. PKC j3I and j3II protein expression in IM-9 and BJA-B cell lines. Total cellular homogenate from IM-9 and BJA-B cells was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis was performed using PKC isoenzymespecific antibodies. Arrows point to the isoenzyme specific bands. Lines indicate nonspecific bands for reference. Panel A, PKC PI and PI1 protein expression. Profrom IM-9 and +peptide tein extracts +peptide(100 pg) -peptide BJA-B cells were analyzed by Western blot analysis in the presence and absence of competing antigenic peptide (40 pg). Panel B, quantitative Western blot analysis. Protein extracts (2.5-150 pg) from IM-9 and BJA-B cells were analyzed by Western blot analysis. Intensity of staining over the linear range was compared to determine the relative expression of a particular isoenzyme between cell lines.
IM-9 BJA-B
IM-9
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-peptide
PKC B,
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B A T pg protein
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