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Molecular Cloning and Identification of a Serine/Threonine Protein Kinase Of Proc. Nati. Acad. Sci. USA Vol. 88, pp. 4171-4175, May 1991 Biochemistry Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily (protein phosphorylation/signal transduction/homology to protein kinase A and protein kinase C/MCF-7 and WI38 cells) PAMELA F. JONES, TERESA JAKUBOWICZ, FERNANDO J. PITOSSI*, FRANSISCA MAURER, AND BRIAN A. HEMMINGSt Friedrich Miescher-Institut, Postfach 2543, CH-4002 Basel, Switzerland Communicated by Martha Vaughan, February 11, 1991 ABSTRACT A partial cDNA was isolated that encoded a YPK1 (8). We present data on both the expression and protein kinase, termed rac Crelated to the A and C kinases). activity of this protein kinase. This cDNA was subsequently used to screen libraries derived from the human cell lines MCF-7 and W138 and led to the MATERIALS AND isolation of full-length cDNA clones. DNA sequence analysis METHODS identified an open reading frame of 1440 base pairs encoding Molecular Cloning and Sequence Analysis. Human cDNA a protein of 480 amino acids (Mr, 55,716). This result was libraries derived from MCF-7 cells and W138 fibroblasts supported by the synthesis of a Mr 58,000 protein in an in vitro (Clontech and Stratagene) were screened as described (9) translation system that used RNA transcribed from cloned using cDNA probe (ALLC-PK1rac4B-5) at 1-2 x 106 cpm/ml cDNAs with SP6 RNA polymerase. The predicted protein radiolabeled by the random-priming method (10). Washing contains consensus sequences characteristic of a protein kinase was for 2 hr in 0.15 M NaCI/15 mM sodium citrate, pH 7.0 catalytic domain and shows 73% and 68% similarity to protein (lx SSC)/0.1% SDS at 60°C. Positive clones were plaque kinase C and the cAMP-dependent protein kinase, respec- purified, and the EcoRI inserts were subcloned for further tively. Northern (RNA) analysis revealed a single mRNA analysis. cDNA sequences were determined by the dideoxy- transcript of 3.2 kilobases that varied up to 300-fold between nucleotide chain-termination method (11) by using Seque- different cell lines. Specific antisera directed towards the nase according to the manufacturer's protocols with the carboxyl terminal of the rac protein kinase were prepared and universal or reverse-sequencing primers or specific oligonu- used to identify a protein of Mr 59,000 by immunoblotting. A cleotides. All clones were sequenced on both strands with specific protein kinase activity was identified that phosphoryl- dGTP or with dITP for regions of high G+C content. Se- ated several substrates in immunoprecipitates prepared with quences were analyzed using the University of Wisconsin the rac-specific antisera. Genetics Computer Group software package (12). Northern (RNA) Analysis. Total RNA was isolated as Protein phosphorylation is a fundamental process for the described (13). For Northern analysis, 20 ,ug of total RNA regulation of cellular functions. It is the coordinated action of was fractionated on a 1% formaldehyde-agarose gel, trans- both protein kinases and phosphatases that controls the ferred to Zetaprobe (Bio-Rad) membrane, and hybridized to phosphorylation levels and, hence, the activity of specific the 2.3-kilobase (kb) insert from AMCFrac8 (specific activity, target proteins (for review, see refs. 1 and 2). One of the 109 cpm/,ug) with 2 x 107 cpm of radiolabeled probe as predominant roles of protein phosphorylation is in signal described (13). After hybridization the blot was washed at transduction, where extracellular signals are amplified and 650C in 1x SSC/0.1% SDS for 2 hr. propagated by a cascade of protein phosphorylation and In Vitro Transcription and Translation. The EcoRI insert dephosphorylation events. Two of the best-characterized from AWI38rac7l was subcloned in both orientations into signal transduction pathways involve the cAMP-dependent pGEM-4Z (Promega), linearized with BamHI, and used as protein kinase (cAMP-PK) and protein kinase C (PKC) (1). templates for in vitro transcription by SP6 polymerase. The Each pathway uses a different second-messenger molecule to capped RNA (41 ,ug) was translated in vitro by using a rabbit activate the protein kinase, which, in turn, phosphorylates reticulocyte lysate (Stratagene) for 1 hr at 300C in the specific target molecules. Extensive comparisons of kinase presence of 20 ,uCi of [35S]methionine (1 Ci = 37 GBq). sequences have defined a common catalytic domain, ranging Samples (2.5 ,ul) were analyzed by 12% SDS/PAGE (14) and from 250 to 300 amino acids (3, 4). This domain contains key subjected to autoradiography for 2 hr at -70°C with two amino acids that are conserved between kinases and are intensifying screens. thought to play an essential role in catalysis. Immunization and Purification of rac-Specific Antisera. Recently many protein kinase sequences have been iden- Rabbits were immunized by s.c. injection with 1 mg of tified either by low-stringency hybridization (5) or by using rac-specific peptide (FPQFSYSASSTA) coupled to keyhole PCR to amplify sequences between two conserved regions in limpet hemocyanin (15). Antisera were purified by precipi- the kinase domain (6). Using a 500-base-pair (bp) cDNA tation with 50% (NH4)2SO4 followed by affinity chromatog- probe encoding amino acids 193-349 of the a catalytic raphy on rac-peptide coupled to Affi-Gel 15 (Bio-Rad). The subunit of the porcine cAMP-PK (7), we isolated several rac-specific antisera were eluted from the column by using cDNA clones encoding a member of the serine/threonine protein kinase family.* Sequence comparisons revealed ho- Abbreviations: cAMP-PK, cAMP-dependent protein kinase; PKC, mology to the cAMP-PK, PKC, and the yeast protein kinase protein kinase C; MBP, myelin basic protein. *Present address: Institut fur Immunologie and Virologie der Uni- versitat Zurich, Gloriastrasse 30, CH-8028 Zurich, Switzerland. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M63167). 4171 Downloaded by guest on September 27, 2021 4172 Biochemistry: Jones et al. Proc. Natl. Acad. Sci. USA 88 (1991) 0.2 M glycine, pH 1.85, and dialyzed against phosphate- the porcine cAMP-PK (7) was used at low stringency to buffered saline. screen a cDNA library derived from the porcine kidney cell Immunoanalysis. Cell extracts were prepared by lysis in 50 line LLC-PK1. A partial clone (ALLC-PK1rac4B-5) isolated mM Tris-HCl, pH 7.5/1 mM EDTA/1 mM phenylmethylsul- by this method was sequenced (unpublished data) and shown fonyl fluoride/0.2% Triton X-100. Samples (40 tig of protein) to encode a serine/threonine protein kinase, termed rac were subjected to 12% SDS/PAGE and electroblotted (16) to (related to the A and C kinases; see below). The insert from Immobilon membranes (Millipore) for 75 min at 300 mA. The this clone was subsequently used to screen cDNA libraries membranes were then probed with the rac-specific peptide derived from the human epithelial cell line, MCF-7, and WI38 antisera and visualized using an 125I-labeled donkey anti- human lung fibroblasts. Several clones were isolated that rabbit second antibody (0.1 ,Ci/ml; Amersham) followed by autoradiography for 2 days at -70'C with two intensifying encoded the same protein as that identified from LLC-PK1 screens. cells. The human cDNAs used for DNA sequence analysis Immunoprecipitation and Assay of rac Protein Kinase. Cell and further characterization are shown in Fig. lA. extracts (-2 mg/ml), prepared as described above, were DNA Sequence Analysis of Human rac Protein Kinase. precleared by incubation for 1 hr with Pansorbin (Calbio- Complete sequence analysis of the overlapping EcoRI frag- chem) and then incubated with the affinity-purified rac- ments of AMCFracEP10 and AMCFrac8 gave a 2.6-kb pair specific antisera (3 ,ug) for 2 hr at 00C. Immunoprecipitates cDNA, with an open reading frame of 480 amino acids from were collected by using protein A-Sepharose (Pharmacia) nucleotides 199-1641. Sequence data from AWI38rac7l con- and extensively washed with lysis buffer. Finally, the immu- firmed that the cDNA encoded the same protein and was a noprecipitates were washed with 50 mM Tris HCI, pH 7.5/10 full-length coding clone. The sequence was identical to that mM MgCI2/1 mM dithiothreitol before performing kinase obtained from the two overlapping clones but contained only assays. The kinase incubation mixture (50 IlI) was 50 mM 85 bp of 5'-untranslated region and 127 bp of 3'-untranslated Tris HCI, pH 7.5/10 mM MgCI2/1 mM dithiothreitol/1 ,M region. The complete coding nucleotide sequence and the protein kinase inhibitor (PKI) peptide (17)/25 ,ug of histone predicted amino acid sequence ofrac kinase are shown in Fig. H1 (Sigma fraction IIIS) or 25 ,ug of myelin basic protein 1B. The molecular weight of the predicted polypeptide was (MBP, Sigma)/50 ,uM [y-32PPATP (800 cpm/pmol)/immuno- 55,716. The methionine initiator codon (nucleotides 199-201) precipitate from 50 ,l of cell-free extract, corresponding tor was in a favorable position for translation initiation (18). 100 ,ug of protein. Other substrates were used as indicated in AMCFrac8 lacked a polyadenylylated tail and also lacked a Table 1. After incubation at 30°C for 30 min samples were polyadenylylation signal, suggesting that the cDNA did not analyzed by 12% SDS/PAGE and then autoradiographed. contain the full 3'-untranslated region. For measurement of protein kinase activity the phosphoryl- The predicted protein encoded by the rac kinase cDNA ated substrate bands were excised from the dried gel, and appears to be a member of the serine/threonine protein radioactivity was counted by scintillation spectropho- kinase family, based on the following observations.
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