Eur. J. Biochem. 210,521 - 529 (1992) 0FEBS 1992

Identification, purification and characterization of a novel phosphatidylinositol-specific phospholipase C, a third member of the p subfamily Amanda J. CAROZZI', Ron W. KRIZ', Catherine WEBSTER' a n d , Peter J. PARKER' Imperial Cancer Research Fund, Lincoln's Inn Fields, London, England Genetics Institute, Cambridge, USA (Received July 6/August 28, 1992)

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EJB 92 0960

The partial sequence of a novel PtdIns-specific phospholipase C of the p subfamily (PtdInsPLCP,) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns-PLCP3 is membrane associated; purification ( N 1000fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 pmol . min-' . mg-', with PtdIns 4,5-bisphosphate as substrate. Substrate specificity and Ca2 dependence of this purified Ptdlns-PLC are characteristic of the PtdIns-PLCB subfamily. +

The eukaryotic PtdIns-specific phospholipascs C (PtdInsPLC) are phosphodiesterases which hydrolyse the glycerophosphate bond of inositol lipids, in particular phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)P2] to generate the two second messengers, sn-1,2-diacyglycerol and inositol 1,4,5trisphosphate, (reviewed in [l, 21). A number of these enzymes have been purified from different sources and from sequence data obtained, they can be subdivided, at present, into three distinct subfamilies: p, 1' and 6 (reviewed in [2, 31). The different subfamilies of PtdIns-PLC share two regions of quite striking similarity which have been strongly implicated as encoding the active site of the enzyme [4], although the overall similarity between subfamilies is low. The y subfamily, whose genes encode proteins of about 145 kDa, is distinguished by the presence of SYC homology (SH) domains betwcen the two conserved regions; these have been shown to be involved in mediating the interaction of the y subfamily with certain activated tyrosine kinase receptors [5; 61. Neither the p nor 6 subfamily contains these SH domains, but can be dislinguished from each other on the basis of size, the j l family members ( "N 150 kDa) being much larger than the 85-kDa 6 members. The fi subfamily have an extended C-terminal region, which in the case of the protein has been implicated in its well-documented interaction with the heterotrimeric GTPbinding protein class, Gq [7- 91. Each subfamily has more than one member, of which some have been purified and sequenced ; others have been discovered by low-stringency screening of cDNA libraries

using probes for the conserved regions of PtdIns-PLC. Using the latter method, full-length clones have been obtained for 6, and Pz, and the existence of a third /l-family member has been reported [lo]. Sequence comparison indicates that members do not differ widely within a subfamily. Here we describe the isolation of a 8, cDNA. The derived sequence has been employed to obtain p,-selective antisera which have been used to identify the protein in cell lines. A suitable source is identified that has permitted the purification and characterization of this novel p form.

Correspondence to P. J. Parker, Imperial Cancer Research Fund, PO Box 123, Lincoln's Inn Fields, London WC2A 3PX, England Ahhreviations. PtdIns(4,5)P2, phosphatidylinositol-4,5bisphosphate; PtdIns-PLC, PtdIns-specific phospholipase(s) C ; HEDTA, N (2-hydroxyethyl)ethylenediaminetriacetic acid. Enzyme. Phospholipase C (EC 3.1.4.10). Note. The novel nucleotide sequence data published here have bccn submitted to the EMBL sequence data bank(s) and are available under accession number(s) 21643 1.

cDNA isolation

MATERIALS AND METHODS Materials Sources of chemicals and radioisotopes are as described previously [ll. 121.

EDNAlibrary construction A cDNA library for the human fibroblast cell-line, W1-38, was constructed essentially as described previously [13] with the following exceptions : the oligonucleotides comprising the adapter, blunt-end ligated to the double-stranded cDNA, had the sequence 5'-AATTCCGTCGACTCTAGAG-3', 5'-CTCTAGAGTCGACGG-3', and the vector used was 1 Zap-I1 (Stratagene).

Approximately lo6 recombinants of the WI-38 cDNA library were screened using a DNA fragment representing amino acid residues 290-464 of bovine PtdIns-PLCP1 [12]. The fragment was random-primer labelled to give a specific activity of about 500 cpm/pg. Hybridization conditions used were 5 x NaCl/Cit (NaCl/Cit: 120 mM NaCl and 0.12 mM sodium citrate, pH 7.0), 5 x Denhardt, 0.1 % SDS, 50 pg/ml

522 yeast total RNA at 55°C for 36-72 h with probe at 5 x l o 5 cpm/ml. Subsequent washings were carried out at 55°C with 4 x NaCl/Cit and 0.1 YOSDS. Clones with varying hybridization intensities were subcloned and partially sequenced. Several clones, which had hybridized strongly, were subsequently confirmed to be human Ptdlns-PLCpl partial cDNA. One clone (clone 50) exhibited weaker hybridization and, upon partial sequence analysis, was shown to represent a potentially novel PtdIns-PLC gene. The 1.4-kb insert was isolated from this clone and used for screening an additional lo6 WI-38 clones to identify additional cDNA. Two clones identified also represent partial cDNA with inscrt sizes of 1.3 kb and 2.4 kb. The DNA sequence for two of these clones was determined for both strands using a Bu131 nuclease-deletion series protocol [14] with subsequent subcloning into appropriate M13 vectors [I 51, followed by sequence determination, as described by Sanger et al. [16].

with the addition of 10 p1 lipid mix. The reaction mixture was incubated at 37°C for 10 min then terminated by the addition of 260 p1 CHC13/MeOH/concentrated HCI (100: 100:0.6) with vortexing followed by the addition of 75 pl 1 M HCl. The tubes were vortexed thoroughly and the phases separated by centrifugation in a minifuge at maximum speed for 5 min. A volume of the upper aqueous phase (150 pl) was taken for liquid scintillation counting. In order to ensure the linearity of the assay, enzyme preparations were diluted until not more than 30% of the substrate head group was liberated into the aqueous phase during the 10-min period. 1 U activity is equal to 1 pmol substrate hydrolysed/min under standard assay conditions. Purification of PtdIns-PLCj3

Between 5 x lo9 and 10'' HeLa S3 cells, maintained in suspension culture at 5 x lo5 cells/ml in RPMI, 10% (by vol.) foetal calf serum, 2% (mass/vol.) bicarbonate, were harvested Immunoblotting of PtdIns-PLC by centrifugation at 450 x g for 10 min at 4°C then washed in Antisera selective for PtdIns-PLCB,, p2, /I3, y1 and y 2 were a total of 2 1 cold NaCl/KCI/P, (137 mM NaCI, 2.7 mM KC1, raised against synthetic oligopeptides based upon C-terminal 10.1 mM Na2HP04, 1.8 mM KH2P04,pH7.4), prior to being sequences of these proteins. The sequences employed were: lysed and processed in a manner similar to that outlined in GENPGKEFDTPL [12], QDPLIAKADAQESRL [17], [21]. Briefly, cells were resuspended in 5 vol. hypotonic buffer GADSESQEENTQL (see below), RTPRRTRVNGDNRL (10 mM Hepes pH 7.9,750 pM spermidine, 150 pM spermine, [I 81 and VNENHSSCTRRNATRG [19], for PtdIns-PLCP1, 100 pM EDTA, 100 pM EGTA, 1 mM m-dithiothreitol, b2,/j3, y1 and y 2 , respectively. Peptides were coupled to key- 10 mM KC1,lO mM benzamidine, 5 pg/rnl leupeptin, 5 pgjml hole-limpet haemocyanin using glutaraldehyde, and rabbits aprotinin, 50 pg/ml phenylmethylsulfonyl fluoride), placed on were immunised with this conjugate over a period of three ice for 10 min, subjected to centrifugation at 450 x g for 10 min months. Sera were titred against peptide -bovine-serum-albu- at 4 "C, then resuspended in 2 vol. hypotonic buffer. They were min conjugates (glutaraldehyde coupled) and subsequently then lysed on ice with three strokes of a Dounce homogenizer tested in Western blotting. Where indicated, sera were affinity (pestle A) and were immediately supplemented with 0.1 vol. purified on peptide-Actigel (Sterogene) columns coupled ac- sucrose restoration buffer [67.5% (massjvol.) sucrose, 50 mM cording to manufacturer's procedures. Hepes, pH 7.9, 750 pM spermidine, 150 pM spermine, Western blotting was performed as detailed below. The 0.2 mM EDTA, 10 mM KCl: 1 mM DL-dithiothreitol and prodilutions for the primary unpurified antisera used for Western- tease inhibitors], before being subjected to Dounce homoblot analysis were: 1:5000 for p1 and y l , and 1 :2500 for p2, genization again with two strokes of a B pestle and subjected p3 and y 2 ; affinity-purified anti-/?, serum was used at 1 : 30 to centrifugation at 12000 x g for 30 s at 4°C to pellet nuclei dilution. All incubations of sera with blots were performed and unbroken cells. The resultant supernatant was subjected overnight at 4°C in the presence of 5% (mass/vol.) skimmed to centrifugation at 125OOOxg at 4"C, and the pellet was milk power. taken for extraction of PtdIns-PLCp3. All subsequent procedures were performed at 4':C and all columns were coupled to the Pharmacia FPLC system. The pellet was resuspended PtdIns-PLC activity assay in 20ml 20mM Tris/HCl pH7.5, 5 m M EDTA, 10mM The activity assay is essentially as detailed previously [20]. EGTA, 2% (mass/vol.) sodium cholate, 10 mM benzamidine, In brief, the standard assay was carried out in a final volume 0.3% (by vol.) 2-mercaptoethanol, 5 mg/ml leupeptin, 5 mgj of 50 p1 in a reaction mix containing (final concentrations) ml aprotinin and 50 pg/ml phenylmethylsulfonyl fluoride 20 mM Tris/maleate, pH 7.0, 100 mM NaC1, 400 pg/rnl bov- (buffer A with cholate), and extracted with stirring for 1 h. ine serum albumin, 0.6% (mass/vol.) sodium cholate, 5 mM The extract was then clarified by ultracentrifugation at 2-mercaptoethanol and a Ca2+/N-(2-hydroxyethyl)ethylene- 90000 x g for 1 h, and the supernatant was then loaded at diaminetetraacetic acid (HEDTA) solution buffered to give a 0.5 ml/min onto an 8-ml heparin Sepharose CL-6B (Pharmafree-calcium concentration of 10 pM [solutions of 0.158 M cia) column, equilibrated in buffer B [20 mM Tris/HCl, HEDTA, 0.158 M HEDTA/CaC12 and H20 were mixed in a pH 7.5,1 mM EDTA, 1"/0 (mass/vol.) sodium cholate, 10 mM ratio (5: 25.2: 169.8) and diluted 1 :10 into final assay volume]. benzamidine, 0.3% (by vol.) 2-mercaptoethanol]. The column The PtdIns(4,5)P2 substrate, which was at a final concen- was washed until the A280 had returned to base line, and tration of 180 pM in the assay, was prepared by mixing 2 pl/ developed at 0.5 ml/min with a linear gradient of 0- 1 M assay tube of a stock of unlabelled PtdIns(4,5)Pz at 4.4 mg/ NaCl over 20 ml, then 1 -2 M NaCl over 10 ml. The resultant ml in CHC13/MeOH (1 : I ) and 2 pl/assay tube of [3H] 0.5-ml fractions were assayed for PtdIns-PLC activity and PtdIns(4,5)Pz (Amersham 10 pCl/ml), drying down under N2 immunoblotted; fractions eluting at 16.5 and 21.5 ml were and resolubilizing the lipids in 10 pljassay tube of a double- pooled on the basis of activity and immunoreactivity. The conccntrated reaction mix. This mixture, with a specific ac- pool was diluted 1 : 5 in buffer C [20 mM Tris/HCI, pH7.5, tivity of approximately 2500 cpm/nmol, was sonicated on ice, 1 mM EDTA, 1% (by vol.) Triton X-100, 0.3%0 (by vol.) twice for 10 s at half-maximum power on a Soniprep 150 2-mercaptoethanol, 10 mM benzamidine] prior to rechroma(MSE). Each assay tube received 15 p1 of double reaction mix, tography on a 1-ml packed heparin-Sepharose column (Phar10 pl HzO, 10 p1 enzyme solution and the reaction was begun macia) equilibrated with buffer C. The column was washed at

523 0.5 ml/min until the A z s o had returned to base line, then developed at the same flow rate with a linear gradient of 0 1 M NaCl over 40ml. The activity in fractions eluting at 14.5- 20 ml was pooled and diluted 1 :5 in buffer D [20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 0.5% (by vol.) Triton X100, 10 mM benzamidine, 0.3% (by vol.) 2-mercaptoethanol] before being applied at 0.5 ml/min to a 3 -ml Mono Q HR5/5 anion-exchange column (Pharmacia) equilibrated in buffer D. Activity was eluted with a linear gradient of 0-0.5 M NaCl over 20 ml, then 0.5 - 1 M over 5 ml at 0.5 ml/min. Activity eluting at 11.5 - 13.5 ml was pooled and diluted 1 : 5 in buffer E [SO mM Mes, pH 5.5, 1 mM EDTA, 0.1% (by vol.) Triton X-100, 0.3% (by vol.) 2-mercaptoethanol, 10 mM benzamidine] and its pH was checked to ensure it had dropped to 5.5 before loading at 0.5 ml/min onto a 1 ml Mono S cationexchange column (Pharmacia) equilibrated with buffer E. This column was developed with a multistep gradient, 0 - 0.25 M NaCl in 2.5 ml, which was maintained for 1.5 ml, then 0.250.75 M NaCl over 16 ml, and finally a steep gradient of 0.75 1 M over 2.5 ml, all at 0.5 ml/min. The resultant peak activity fractions eluting from this column were used for subsequent characterization.

Calcium curves The dependence of the enzyme on calcium was assessed under standard assay conditions with the calcium concentration being manipulated using Ca2+/EGTA buffers; free Ca2+ was calculated using the CHELATE programme. PtdIns hydrolysis Assays monitoring the ability of PLCPJ to hydrolyse PtdIns used standard assay conditions, the only distinction being that 200 FM PtdIns (specific activity w 2500 cpm/nmol) was used instead of PtdIns(4,5)P2. When PtdIns hydrolysis was monitored as a function of calcium, the same buffered calcium solutions were used as for the PtdIns(4,5)P2 determinations. Protein determinations were performed, unless stated otherwise, using the Bio-Rad dye-binding reagent and bovine serum albumin as a standard.

RESULTS Isolation of PtdIns-PLCj3 cDNA

Western-blot analysis Samples and 8 % polyacrylamide resolving gels with 5% stackers were prepared in the presence of SDS, as outlined by Lacmmli (1970) [22]. After electrophoresis, proteins were transferred to nitrocellulose (Schleicher & Schull) using a semi-dry Semi-phor TE70 blotting apparatus (Hoefer Scientific) and a three-buffer transfer system, as described previously [23]; the only deviation being the omission of the methanol from the buffers. Transfers were performed according to manufacturer’s specifications for 1.5 h at 210 mA and constant voltage. After transfer, the nitrocellulose was stained with 2% (mass vol.) Ponceau S in 3% (by vol.) HC104 and destained in 10% (by vol.) acetic acid to visualize samples and high molecular mass markers (Bio-Rad) before being rinsed in NaCl/KCI/Pi and blocked for 2 h in 5% (mass/vol.) skimmed milk powder solubilized in NaC1/KC1/Pi. Blots were then incubated overnight with antisera or with affinitypurificd anti-PLCB, sera in 5% (mass/vol.) skimmed milk dissolved in NaCl/KCl/Pi containing 0.05% (by vol.) Tween 20. Blots were then washed with approximately 200ml of NaCliKCljP, plus 0.05% Tween 20, then NaCljKCl:’Pi plus 0.05% Tween 20 with 0.5 M NaCl, then again with NaCl/ KC1/Pi A plus 0.05% Tween 20, before being incubated with a secondary donkey anti-(rabbit IgC) serum conjugated to horseradish peroxidase (Amersham), at 1 :5000 in 1% (mass/ vol.) skimmed milk in NaCl/KCl/Pi plus 0.05% Tween 20 for 1 h at room temperature. The blot was then washed as above and developed using the ECL detection system (Amersham). PtdIns(4,5)P2 dependence Substrate dependence was performed at 1OpM free calcium at pH 7. The concentration of PtdIns(4,5)P2 was varied by making a stock of radiolabelled lipid in organic solvent at a specific activity of 2500 cpm/nmol and pipetting various amounts into separate tubes, drying under N2 and sonicating these ditkrcrcnt amounts of lipid into a uniform volume of double reaction mix. Assays werc then performed as for the standard phospholipase assay. No-enzyme controls were performed for each concentration of PtdIns(4,5)P2.

In a preliminary report [lo], the expression of a novel PtdIns-PLC of the p class was noted. In order to further define this gene product, a number of cDNA obtained from the WI38 human fibroblast cell line have been sequenced (Fig. la). The predicted open reading frame (Fig. lb), derived from these overlapping cDNA, yields a protein sequence which shows substantial similarity to the other two members of the PtdIns-PLCB subfamily (Fig. lc). No other cDNA species extending further 5’ has been isolated from this cDNA library, nor from HeLa cDNA libraries. As shown in Fig. lc, it would appear that about 600 bp (i.e. about 200 amino acids) of the open reading frame are missing from the compiled sequence (it is unlikely that the encoded protein is significantly different in length from either PI or B2, given the apparent size of the protein; see below). Localization In the absence of a full-length cDNA for PtdIns-PLCP3, it has not been possible to express the protein for the purpose of characterization. Thus, in order to determine the properties of this protein, a source was identified using antisera selective for p3 (see below). Although the partial PtdIns-PLCP3 cDNA was identified in W1-38 cells, these were not considered suitable for bulk culture and PtdIns-PEP3 purification. However, immunoblot of other cell lines revealed the expression of this enzyme in HcLa S3 cells (not shown). In order to assess the extractability of Ptdlns-PLCP3 from HeLa S3 cells, 5 x lo6 cells were washed in NaCl/KC1/Pi and lysed by Dounce homogenization in either buffer A, buffer A with 1”/0 (mass/ vol.) sodium cholate, or buffer A with 1% (by vol.) Triton X100, and the resultant soluble and particulate fractions were separated by ultracentrifugation. Western analysis of these fractions (not shown) indicated that there was very little PtdIns-PLCP, in the supernatant from extractions with buffer A or buffer A with Triton X-100, the enzyme remaining predominantly membrane bound. However, in the cholate extract, approximately 70% of the immunoreactive material was present in the supernatant. This indicates that in proliferating subconfluent HeLa S3 cells, the majority of PtdIns-PLCP, is

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. Sequence analysis of PtdIns-PLCfi3 cDNA clones. (a) An alignment of three Ptdlns-PLCb3 cDNA is shown (clones 50, 14B and 3),

juxtaposed to a schematic representation of the open reading frame (boxed). (b) A translation of the open reading frame compiled from the overlapping cDNA. The sequence is incomplete at the 5' end (see text). (c) An alignment of the three PtdIns-PLCP subfamily sequences is shown. plcbl b, PtdIns-PLCP1; P1; PLCB2H, PtdIns-PLCP, ; plcb3h, P3 sequence. The stars below the sequences indicate identical amino acids; the dots indicate conservative changes. It is evident that some 200 amino acids are missing at the N-terminus of thc P3 scqucncc.

tightly associated with the membrane, from which it can be extracted by anionic detergents. Fractions derived from HeLa S3 cells were also analysed for the presence of other PtdInsPLC species. No evidence for p2 expression was obtained, y I was prcsent predominantly in the soluble fraction and while low immunoreactivity was observed in HeLa S3, no immunoreactivity was observed in the purified PtdIns-PLCP, preparation (not shown).

Purification Using the ability of cholate to solubilize PtdIns-PLCB,, the enzyme was extracted from 2 x 10" HeLa S3 cell membranes, as outlined in Materials and Methods. The extract was then chromatographed, in the presence of 1 O h (mass/vol.) sodium cholate, on a n 8-ml heparin-Sepharose column. As can be seen from Fig. 2, the PtdIns-PLC activity bound tightly to this matrix, eluting between 350 m M and 550 m M NaC1,

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........................ Sepharose Identificationof Ptdlns-PUJp3 after chromatography on heparin in the presence of sodium cholate. Fig. 2.

(a) A membrane fraction from HeLa S3 cells was extracted with cholate and applied to an 8-ml heparin-Sepharose column, equilibrated in a cholate-containing buffer (Materials and Methods). The column was eluted with a linear PRI.FWNPIGC(XmmLNFO~~I~YEYNGKSGY~KPE~MRRPDKHFDPFTEGIV gradient of 0- 1 M NaCl over 20 ml, then 1 -2 M NaCl over 10 ml, t~~tt.~.*t~.*tt*tt~+~~~. **-*-**.*** . the column having a void volume of approximately 4 ml. An aliquot (10 pl diluted 1 :40) of each fraction was assayed for PtdIns-PLC DGI~LSVKIISGQFLSDKKGTYVEVDMFGLPVDTRRKAFKTKTXS-NAVNPIWEE activity ( 0 ) using PtdIns(4,5)P2 as substrate. The absorbance at DVWATTLSITVISGQFLSERSVRTnLFGLPGDPKRR-YRTKLSPS'SINPWKE 280 nm ( - ) was monitored by an in-line detector. (b) An aliquot of DGIVANAWlVKVISMFLSDRKVGIYVEVDMFGLPVDTRRK-YRTRTSQG-NSFNPVWDE each fraction (40 pl) was mixed with SDS sample buffer and subjected E P 1 V F ~ S L R C L R ~ V Y E E U ; K F I G H R I L P ~ A I R P G Y H Y I C L R N E R N Q P L M L P A L to SDS/polyacrylamide-gel electrophoresis prior to Western-blot EPFVFEKILMPELASLRVAWEGNKFEHIUIPINALNSGYHHLCLHSESNMPLTMPAL analysis as outlined in Materials and Methods. The affinity-purified EPFDFPKVVLPTWLRIAAFEEGGKFVGHRILPVSAIRSGYHYVCLRNEANOPLCLPAL anti-peptide sera specific for PtdIns-PLCB3 was used at 1 : 30 dilution to probe the blot which was developed using the ECL detection FWIEVKDYVPDTYADVIEALSNPIRYVNLMEQRAKQLARLTLEDEEEVKKEADPGETPS FIFLEMKDYIPGAWADLlPIKFFSAHDTKSVKLK-----------EAMZ system. The molecular mass markers (Bio-Rad) indicated on the left LIYTEALDYIPDDHQDYAEALINPIKHVSMJQRARQLAALIGESEAQAGVETCQDWSC in descending order are as follows: 200 kDa; 116.3 kDa; 97.4 kDa; ... 66.2 kDa and 45 kDa. On the basis of immunoreactivity, fractions EAPSEARPTPAENGVNH'ITSLTPKPPSQALHSQPAPGSVKAPAKTEDLIQSVLTEVEAQT CLPEKPFPLASPVASQV?iGALAP------TSNGSPAAFZAGPREEAMKEA--AEPRTAS eluting between 16.5-21.5 ml were taken as a pool of PtdIns-PLCB3 QLI;MPSSNPTPSPLDASPR--RPPGPT-TSPASTSLSSPWRDDLIASII,SEV~TP ........ . . . . . . . . . . . . . . . . . activity for further chromatography. K F E S F E I S K K R M S F E M S S R T T K G l . ~ Q l . T K S P V E F V E Y N ~ l . S RPKCl I Y RVUSSNYM KFVSFEFSAQKNRSWISSETELKAYDI.I.SKASVQFVDYNKH(;MSHIY PKCl HMIlSSNYM KFKSFEAARKRNKCFEMSSFT~,VLTKSPMEI'Vt:YNKWI.Sl(IYPKGlUVIlSSNYM

.................................................... *t~t.tttt.* * . A * .

plcblb P1;PLCBZH plcb3h plcblb Pl;PLCB2H plcb3h plcblb P1;PLCBZH plcb3h plcblb P1;PKBZH plcb3h

..................................................... ..................................................... .....................

plcblb P1;PLCBZH plcb3h

I EELKCQKSFVKLQKKHYKELVKPJ~HKKTTDLIKEHTTKYNFlQND--YLRRRAALE LEELRELKGWKLQRRHEKELRELERHW\RRWEELLQRGAAQlAEI.GPPCVGGVCACKLG

plcblb P1;PLCBZH plcb3h

KTAKKDNKKKSEPSSPDHVSSTI MDUVUDAEH?n~MLKDK(MCQI.LNI.WEQYYSE --PCKCSRKKRSLPREESAWVIPGEGPEGVDG---~RVReI.KDRLEI.EI.I.RQCF EQYECV

plcblb P1;PLCBZH plcb3h

LDELRGHKAI.VKLRSRPERDLRELRKKHORKn~LTRRLLDGIAQAAEGRCRI.RPW\IC

..*'.

4.

'a'....

.....a

. . . . .' . . . .

GAADVEDTKECE-----------DEAKUYQEFQN~QVQSI.1.FI.RFI\QMAE

. . . *. .

.................

......

*.

*'

.:

......................

plcblb P1;PLCBZH plcb3h

KSOMEEE-KTEMIRSY I Q E W Y I KRLEEROSKWEKLVEKIIKE IHool LDEKI'KIUW. TDK1.V\QERI.KREINNStlIQEWVI KQkTENLERHQEKLEt'KOAACl.EQ1 WWr(QI'QKE KHKL(EAE-LTEINRK11ITESVNSI RRLEEI\OKQRHDRl,VI\(;~VIQOIAEE~~I'KI.IJ\O

plcblb P1;PLCBZH plcb3h

LFaEYODKFKRLPLEILEFMEAMKGKISEDSNHSSAPP~SDSGKLN3KPPSSEELEG

plcblb P1;PLCBZH plcb3h

ENP-GKEFDTPL LIAKPDPQESRL ADSESQEENTOL

Fig. 1

.....*

*. ...* * *. *....*.

......' . . .

.*.... . . . . . . . . . . . .......

.....

.*.

.....

....

despite the fact that more than 50% of the Azso-absorbing material eluted in the breakthrough. Furthermore, as seen from the Western-blot data (Fig. 2), the PtdIns-PLCP3 immunoreactivity eluted with the majority of the PtdIns-PLC activity. The major activity peak was pooled, then rechromatographed on a packed heparin-Sepharose column in the presence of 1YO(by vol.) Triton X-100 (Fig. 3), which not only prepared the sample for anion exchange chromatography, but also achieved further purification. Interestingly, it was found that this two-step heparin-Sepharose chromatography with detergent exchange proved to be essential for complete purification. The activity binding to this column was then loaded onto a Mono Q anion-exchange column. Again, a substantial proportion of the Azso-absorbing material eluted in the breakthrough, whereas all the PtdIns-PLC activity bound to the column and eluted as a single peak at 300 mM NaCl (Fig. 4).

526 800

500 600 0.2

N, 0.1

B

e

.-x

51

B

3 c

9

200

0.0

0

10

20 0

Eluoon volume (ml)

Fig. 3. Chromatography of Ptdlns-PLCB, on heparin-Sepharose in the presence of Triton X-100. Pooled activity from the first heparinSepharosc column was diluted in a Triton X-100-containing buffer (Materials and Methods) then applied to a 1-ml packed heparinSepharose column equilibrated in the same buffer. The column was eluted with a linear gradient of 0- 1 M NaCl over 40 ml, and PtdInsPLC activity ( 0 )was assessed in aliquots (I0 pI diluted 1 :20) of each fraction, using Ptd1ns(4,5)P2 as substrate. Absorbance a1 380 nrn () was monitored by an in-line detector. Activity eluting at 14.5-20 ml was pooled.

0.8

800

0.6 0.4

-@

8 I

P 0.2

P

3 0.0

0

0

10

20

Elution volume (ml)

Fig. 4. Chromatographyof PtdIns-PLCB, on a Mono Q anion-exchange column. The pooled activity from the second heparin-Sepharose column was diluted in buffer D (Materials and Methods) and applied to a Mono Q 5/5 column, cquilibrated in buffcr D. The column was washed then eluted with a linear gradient of 0-0.5 M NaCl Over 20 ml, then 0.5- 1 M NaCl over 10 ml. Aliquots of each fraction (1 0 pl diluted 1 :20) were assaycd for PtdIns-PLC activity ( 0 )using Ptd1ns(4,5)P2 as a substrate, and absorbance at 280 nm ( - ) was monitored using an in-line detector. Activity eluting at 11.5 - 13.5 ml was pooled.

Thc eluting activity was adjusted to pH 5.5 and applied to a Mono S cation-exchange column, to which, at the reduced pH, the Ptdlns-PLC activity bound quite tightly, eluting at about 500 mM NaCl (Fig. 5). This activity eluted with PtdInsPLCP3 immunoreactivity, as seen in Fig. 5. In some preparations, a second activity eluted with the majority of the A,,,-absorbing material at 250 mM NaCl. This early eluting activity did not react with PtdIns-PLCP, antisera, but, due to its variable nature (and the reported susceptibility of PtdlnsPLCpl to C-terminal cleavage) [I, it is likely that it corresponds to a C-terminal clipped fragment of PtdIns-PLCfi, (this would account for the non-immunoreactivity). When the major activity peak from the Mono S column was assessed for purity after electrophoresis on an 8%

Fig. 5. Isolation of PtdIns-PLCj, after Mono S cation-exchange chromatography at pH 5.5. (a) Pooled activity from the Mono Q step was diluted in buffer E (Materials and Methods), such that i t s pH was reduced to pH 5.5, and applied to a 1-ml Mono S column equilibrated in buffer E. The column was washed then eluted with a multistep gradicnt; 0-0.25 M NaCl over 2.5 ml, 0.25 M NaCl maintained for a further 1.5 ml to resolve a potential activity contaminant, 0.250.75 MNaClovcrl6 inl;O.75-1 MNaCIover2.5 ml. Aliquots(l0 pl diluted 1 :50) of each fraction were assessed for PtdIns-PLC activity ( 0 ) using PtdIns(4,5)P2 and absorbance at 280nm (-) was monitored using an in-line detector. (b) An aliquot (40 pl) corresponding to elution volumes of 10-14ml was mixed with SDS sample buffer and subjected to polyacrylamide-gel electrophoresis prior to Western-blot analysis, as outlined in Matcrials and Methods. The affinity-purified anti-peptide sera spccific for PtdIns-PLCB, was used at a 1: 30 dilution to probe the blot which was developed using the ECL detection system. The molecular mass markers on the left (BioRad) are, in descending order, as follows: 200 kDa; 116.3 kDa; 97.4 kDa; 66.2 kDa; 45 kDa.

Laemmli resolving gel, a single band of 158.5 kDa was detected after Coomassie-blue staining, which migrated to the same position as the protein detected by the anti-PEP, sera. When the same gel was stained with silver (Fig. 6), several minor contaminating bands were also noted. This simple four-step procedure thus results in a purification of more than 1000fold relative to the particulate fraction, yielding a near homogeneous preparation. A summary of the purification is given in Table 1. Characterization of Ptdlns-PLCf13 The purified activity obtained after Mono S chromatography was used to characterize PtdIns-PLCfi3 activity in vitro. Assays throughout the purification routinely used 200 pM PtdIns(4,5)P2 as substrate, sonicated in the presence of detergent and assayed at 10 pM free calcium, pH 7.0. When sub-

527

0.0J 0.00

0 01

I 0.02

0

0

1000

substrate concennation (pM)

Fig. 6. Silver-stained polyacrylamide gel of PtdIns-PLCb3-containing fractions eiuted from the Mono S column. Aliquots (40 p1) of fractions corresponding to elution volumes of 10.5-13 ml (given at thc top of the lanes) were mixed with SDS sample buffer and subjected to polyacrylamide-gel electrophoresis, as outlined in Materials and Methods. The gel was then staincd with Coomassie brilliant blue, and after extensive destaining was silver-stained. Molecular mass markcrs indicated on the left and right are in descending order: 200 kDa; 116.2 kDa; 97.4 kDa; 66.2 kDa; 45 kDa. From comparison of the relative migration of the immunoreactive material with molecular mass markers, the major band, indicated by the arrow, corresponds to iinmunoreactive PtdIns-PLCP3.

Fig. 7. Effect of varying Ptdlns(4,5)P2 concentration on the activity of PtdIns-PLCJ,. The activity of PtdTns-PLC[j3 was assayed at 10 pM free calcium, pH 7.0, and varying concentrations of PtdIns(4,5)Pz (maintained at a constant specific activity). As shown by the graph, the enzyme shows a typical hyperbolic substrate dcpendence, although at 1 mM there is evidence of substrate inhibition. When the data is expressed as a Lineweaver-Burk plot, generated by plotting the inverse of activity versus the inverse of substrate concentration (inset), the expected linear relation was obtained and a K , of 216 pM PtdIns(4,5)P2 was calculated.

300

I

Table 1. Purification of PtdIns-PLCbJ from HeLa S3 cells. Summary of a PtdIns-PLCj, purification from lo1' HeLa S3 cells. Details of column steps are in the text and Materials and Methods. Protein concentration at all stcps except after Mono S chromatography was determined by Bradford assay. After the Mono S step, protein contcnt was estimated from Coomassie-blue staining of bands on an SDS/ polyacrylamide gel that showed Ptdlns-PLCB3 immunorcactivity. PLC activity was assessed under standard conditions (Materials and Methods). Step

Cholate extraction 1st heparin Sepharose 2nd heparin Sepharose Mono Q Mono S

Volumc

Total Protein

Total activity

Specific activity

ml

mg

pmol PtdInsPz hydrolysed min

pmol PtdInsP, hydrolysed . min-l'mg

.

16.5

89.2

9.09

0.10

24.0

8.4

2.33

0.28

23.0 9.9 1.5

1.6

0.09 0.05

1.42 1.02 0.68

0.89 11.33 136.0

strate dependence was examined under the same pH, calcium and detergent conditions, the substrate dependence curve (Fig. 7) was found to be consistent with Michaelis-Menten kinctics (at the highest concentration, slight substrate inhibition was observed; data for this concentration was omitted for K , determination). From a double-reciprocal plot (Fig. 7, inset) a K,,, of 216 pM PtdIns(4,5)P2 was obtained which is similar to that obtained previously for PtdIns-PLCP, under these assay conditions [20]. When PtdTns(4,5)P2 hydrolysis was examined as a function of free [Ca2+] at 180pM

log ca2+ concentration

Fig. 8. Effect of varying free-calcium concentrations on the activity of PtdIns-PLCb3. PtdIns-PLCB, activity was assayed at 180 pM PtdIns(4,5)P2, pH 7.0, with varying concentrations of free calcium adjusted using calculations for EGTA and CaCI2obtained from the CHELATE programme. There was no appreciable activity at zero calcium, and, as seen by the curve, activity increases sharply at 100- 1000 nM free CaZ+,being half-maximal at about 750 nM free Ca2+.The activity thenessentially plateaus until 1 mM Ca2+.10 mM Ca2+is inhibitory.

PtdIns(4,5)P2, pH 7.0, activity was found to show a bellshaped curve (Fig. X), with half-maximal activation occurring at 750 nM free calcium. Again, this dependence resembles PtdIns-PLCP1 and is in direct contrast to the activity pattern obtained when PtdIns is used as substrate instead of PtdIns(4,5)P2. The activity of PtdIns-PLCb3 against PtdIns increases with calcium concentration up to 10mM (not shown), but is at least one order of magnitude lower than that for PtdIns(4,5)P2, and, under standard conditions (200 pM substrate, 10 pM free calcium, pH 7.0), PtdIns-PLCB3 was at least 100-fold less active with PtdIns than PtdIns(4,5)P2.

528 DLSCUSSION

2. Meldrum, E., Parker, P. J. & Carozzi, A. (1991) The I'tdIns-PLC superfamily and signal transduction, Biochim. Biophys. Acta We report here the partial sequence of a novel PtdIns-PLC 11/92, 49 - 71. of the fl subfamily (designated PtdIns-PLCD3), and further3. Rhee, S. G., Suh. P.-G., Ryu. S.-H. & Lee, S. Y. (1989) Studies more, a relatively straightforward procedure for the purifiof inositol phospholipid-spccific phospholipase C, Science 244, 546 - 550. cation of this enzyme from a readily available source, i.e. 4. Bristol, A,, Hall, S. M., Kriz, R. W., Stahl, M. L., Fan, Y . S., HeLa cells. 'The identification of PtdIns-PLCf13in HeLa cells Byers, M. G., Eddy, R.-L., Shows, T. B. & Knopf, J. L. (1988) and subsequent monitoring of purification was achieved via Phospholipase C-148: Chromosomal location and deletion the use of polyclonal anti-peptide sera, specific for this mapping of functional domains, Cold Spring Harbor Symp. isoenzyme. The antisera was raised against a peptide correQuant. Biol. Ll11, 915-920. sponding to the amino acid sequence at the C-terminus of 5. Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S . M. T., Tonks, Ptdlns-PLCp3, on the premise that this region of the molecule N. K., Rhee, S. G. & Carpenter, G. (1990) Increase of the differs significantly between the three known fl subfamily catalytic activity of phospholipase C-yl by tyrosine phosmembers, and that the C-terminal sequences have proven to phorylation, Science 250, 1253- 1256. 6. Moran, M. F., Koch, C. A., Anderson, D., Ellis, C., England, L., be suitably antigenic. 'These antibodies, together with those Martin, G. S. & Pawson, T. (1990) Src homology region 2 raised against analogous sequences from fll and 8,. will prove domains direct protein-protein interactions in signal Lransducuseful tools, not only in establishing patterns of expression of tion, Proc. Natl Acad. Sci. USA. 87, 8622-8626. the different isoenzymes in different tissues, but also perhaps 7. Park,D., Jhon, D.-Y., Lee, C.-W., Lee, S. Y. & Rhee, S. G. (1992) in positively identifying many undefined PtdIns-PLC activities Activation of different forms of PLCpl by Gq, FASEB J. 6 , examined by groups investigating these enzymes in various 2199. contexts. 8. Taylor, S. J., Chae, H. Z., Rhee, S. G. & Exton, J. H. (1991) The final specific activity obtained for purified human Activation of the isozyme of phospholipase C by x subunits PtdIns-PLCP, of 136 pmol PtdInsP, hydrolysed . min- . of the G, class of G proteins, Nature 350, 516 -518. 9. Simon, M. I., Strathmann, M. P. & Gautam, N.(1991) Diversity mg-' is very similar to that obtained for PtdIns-PLCP1 of G proteins in signal transduction, Science 252, 802- 808. purified from bovine brain particulate fraction [20], endorsing the silver stained gel analysis that the end product obtained 30. Kriz, R., Idh-Ling, L., Sullzman, L., Ellis, C., Heldin, C.-H., Pawson, T. & Knopf, J. (1990) Phospholipase C isozymcs: from the purification procedure outlined is of a high level of structural and functional similarites, C f B A Found. S-vmp. 150, purity. The in vitro properties of the /I3 isoenzyme are also 112- 127. very similar to those reported for fl,. The enzyme shows an 11. Meldrum, E., Kriz, R. W., Totty, N. & Parker, P. J. (1990) A absolute dependence on Ca2 in a cholate-mixed-micelle assecond gene product of the inositol-lipid phospholipid-specific say, which is half-maximal between 100 nM and 1 pM free phospholipase C subclass, Eur. .J. Biochem. 196, 159-165. Ca2+ and also shows a marked preference for PtdInsP, over 12. Katan, M., Kriz, R. W., Tolly, N., Philp, R., Meldrum, E., PtdIns over all Ca2+ concentrations examined. Aldapc, R. A., Knopf, J. L. & Parkcr, P. J. (1988) Determination of the primary structure of PLC-154 demonstrates diverThe catalytic properties of PtdIns-PLCP, shows no unique sity of phosphoinositide-specific phospholipase C activities, features, and it is likely that the regulation of activity is what Cell54, 171 -177. will distinguish this isoenzyme, at least from the 6 and y 13. Toole, J. J., Knopf, J. L., Wozney, J. M., Sultzman, L. A., subfamily isoenzymes. Whether the different /? isoenzymes Beucker, J. L., Pittman, D. D., Kaufman, R. J., Brown, E. L., show differences in their in vivo regulation, however, remains Shoemaker, C., Om, E. C., Amphlett, G. W.. Foster, W. B., to be determined. It has been known for some time that Coe, M. L., Knutson, G. J., Fass, D. N. & Hewick, R. M. PtdIns-PLC activity in many cell types can couple to certain (1984) Molecular cloning of a cDNA encoding human anticell surface rcceptors via both pertussis-toxin-sensitive and haemophilic factor, Nature 312, 342- 347. insensitive heterotrimeric GTP-binding proteins (reviewed in 14. Poncz, M., Solowiejczyk, D., Ballantine, M., Schwartz, E. & Surrey, S. (1982) "on-random' DNA sequence analysis in [2]). Recently, it has been established that PtdIns-PLCP1 can bacteriophage M13 by the dideoxy chain-termination method, interact with, and be regulated by a recently purified and Proc. Natl Acad. Sci. USA. 79, 4298 -4302. sequenced class of guanine-nucleotide-binding-regulatoryJ. & Viera, J. (1982) A new pair of MI3 vectors for protein (G-protein) a subunits, the Gq class, of which there are 15. Messing, subcloning either strand of double-digest restriction rragmenls, at least five members [24]. It is conceivable that the different Gene (Amst.) 19,269 -276. fl isoen7ymes interact differentially with different Gq class 16. Sanger, F., Nicklen, S. & Coulsen, A. R. (1977) DNA sequencing members, particularly given reports that the C-terminus of with chain-terminating inhibitors, Pror. Nail Arad. S c i . USA PtdIns-PLCJ, is necessary for its interaction with Gaq [7]. 74, 5463 - 5467. The description of a third P-subfamily Ptdlns-PLC and its 17. Park, D., Jhon, D.-Y., Kriz, R., Knopf, J. & Rhee, S. G. (1992) Cloning, sequencing, expression and Gq-independent actiisolation will allow biochemical analysis of the interaction of vation of phospholipase C-P2,J . Biol. Chem., in thc press. another p member with potential regulatory components in an in vitro context, and thus facilitate further investigation 18. Suh, P.-G., Ryu, S. H., Moon, K. H., Suh, H. W. & Rhee, S. G. (1988) Inositol phospholipid-specific phospholipasc C : Cominto the specificity of regulation of PtdIns-PLC activity within plete cDNA and protein sequences and sequence similarity to the cell. tyrosine kinase-related oncogene products, Proc. Natl Acad. Sci. USA 85, 5419- 5423. Our thanks to Kalim Mir for technical assistance, to Maureen 19. Ohta, S . , Matsui, A,, Nazawa, Y. & Kagawa, Y. (1988) Complete Harrison and everyone at the Cell Production Unit for the bulk culture cDNA encoding a putative phospholipase C from transforrncd of HeLa S3 cells and to Mary Wallace for secretarial assistance. human lymphocytes, FEBS Lett. 242, 31 -35. 20. Katan, M. &Parker, P. J. (1987) Purification of phosphoinositidespecific phospholipase C from a particulate fraction of bovine REFERENCES brain, Eur. J . Biochem. 168, 413-418. 21, Shapiro, D. J., Sharp, P. A,, Wahli, W. W. & Keller, M. J. (1992) 1. Berridge, M. J. & Imine, R. F. (1984) Inositol trisphosphate, a A high-efficiency HeLa cell nuclear transcription extract, D N A novel second messenger in cellular signal transduction, Nature ( N Y ) 7,47-55. 312. 315-321.

'

+

529 22. Laemmli, U. K. (1970) Cleavagc of structural proteins during thc assembly of the head o f bacteriophage T4, Nature 227, 680 6R5. 23. Khyse-Anderson, J. (1984) Electroblotting of multiple gels: Simple apparatus without buffer tank for rapid transfer of proteins for polyacrylamide to nitrocellulose, J . Biochem. Biophy.y. Mt.thod,Y 10, 203 -~209.

24. Wilkie, T. M., Scherle, P. A., Strathmann, M. P., Slepak, V. Z. & Simon, M. I. (1991) Characterization of G-protein GI subunits in the Gq class: Expression in murine tissues and in stromal and hematopoietic cell lines, Proc. ,Vat/ Acnd. Sci. USA 88, 10049 - 10053.