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Molecular Cloning, Characterization, and Expression of a Cdna

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Molecular Cloning, Characterization, and Expression of a Cdna Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4012-4016, June 1989 Biochemistry Molecular cloning, characterization, and expression of a cDNA encoding the "80- to 87-kDa" myristoylated alanine-rich C kinase substrate: A major cellular substrate for protein kinase C (signal transduction/protein phosphorylation) DEBORAH J. STUMPO*, JONATHAN M. GRAFF*, KATHERINE A. ALBERTt, PAUL GREENGARDt, AND PERRY J. BLACKSHEAR*t *Howard Hughes Medical Institute Laboratories, and Section of Diabetes and Metabolism, Division of Endocrinology, Metabolism and Genetics, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, NC 27710; and tLaboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021-6399 Contributed by Paul Greengard, March 2, 1989 ABSTRACT We isolated and sequenced a cDNA clone protein kinase C. In this paper, we describe the molecular encoding the bovine "80- to 87-kDa" protein, a major cellular cloning of a cDNA§ encoding the bovine protein, and the use substrate for protein kinase C. An open reading frame of 1005 of this cDNA in studies of its mRNA expression. Since the base pairs predicted a protein of 335 amino acids (Mr, 31,949). predicted size of the protein bears little relationship to the Despite this predicted size, the protein migrated on SDS/ size (80-87 kDa) observed on SDS/polyacrylamide gels and polyacrylamide gels with an apparent molecular weight of 80- since amino acid analysis of the protein revealed nearly 30 87,000 after expression ofthe cDNA in cells lacking the protein. mol % of alanine (7), we propose the name myristoylated It was highly enriched in alanine (28.4 mol %), contained an alanine-rich C kinase substrate (MARCKS) for this protein. amino-terminal myristoylation consensus sequence, and in- cluded a 25-residue basic domain containing the known protein kinase C phosphorylation sites. Two mRNA species (2.6 and 4.4 EXPERIMENTAL PROCEDURES kilobases) were most highly expressed in brain, spinal cord, Protein Purification, Tryptic Digestion, Purification of Pep- spleen, and lung, in parallel with the distribution of immuno- tides, Amino Acid Sequencing, and Synthesis and Labeling of reactive protein. Genomic blot analysis indicated the likelihood Oligonucleotides and of cDNAs. The "80- to 87-kDa" protein of a single gene coding for this mRNA. We propose the name was purified from bovine brain by the method of Albert et al. myristoylated alanine-rich C kinase substrate (MARCKS) for (7). Approximately 100 ug of protein was digested with this protein. trypsin and the tryptic peptides were separated by reverse- phase HPLC as described (8). Peaks of absorbance were Protein kinase C, the Ca2+/diacylglycerol-dependent protein monitored at 219 nm, and samples from several of the peaks kinase, is a ubiquitous enzyme known to consist of a family were subjected to gas-phase sequencing on an Applied Bio- of related gene products (1, 2). Although protein kinase C systems model 470A sequencer in combination with a model itself is becoming better understood, little is known about its 120A phenylthiohydantoin analyzer. Three peaks yielded cellular substrates and how they are involved in the physio- useful peptide sequences (see Results). Six synthetic oligo- logical processes affected by protein kinase C activation. nucleotides were synthesized based on these amino acid One of the most prominent cellular substrates for the sequences and were purified using C18 SEP-PAK columns kinase, the "80- to 87-kDa protein", has received consider- (Millipore, Waters) as described in the Applied Biosystems able attention in the past several years (for review, see refs. DNA synthesizer user manual; these were labeled at the 5' 2 and 3). This protein is of particular interest for several end with [_y-32P]ATP (DuPont/NEN) using T4 polynucleotide reasons: (i) In a wide variety of cell types, it is phosphoryl- kinase (5'-DNA-end-labeling kit from Bethesda Research Lab- ated within seconds of protein kinase C activation by such oratories). Unincorporated [_y-32P]ATP was removed by two stimuli as growth factors in fibroblasts and neurotransmitters rounds of ethanol precipitation with ammonium acetate as in neuronal and glial cells, as well as by phorbol esters and described (9). The specific activities of the probes were >5 x cell-permeable diacylglycerols. This property has made it a 107 cpm/,tg of DNA. convenient marker for the activation of protein kinase C in Cloned cDNAs were cut with restriction endonucleases intact cells. (ii) It is rapidly dephosphorylated in response to and fragments were isolated from a 1.0 or 1.5% low- receptor blockade (4), suggesting a potential role in the acute melting-temperature agarose gel (Bethesda Research Labo- effects of protein kinase C activation. (iii) It is myristoylated ratories) as described (10, 11). Isolated inserts were labeled (5), a modification that might promote association of the with [a-32P]dCTP (DuPont/NEN) using the random primers protein to a more hydrophobic environment, presumably DNA labeling kit from Bethesda Research Laboratories to a allowing more intimate association with protein kinase C. (iv) specific activity of >109 cpm/,ug of DNA. It binds calmodulin, and this binding can be prevented by Colony Hybridization. A modified Okayama-Berg bovine protein kinase C-dependent phosphorylation of the protein caudate cDNA plasmid library (12) was screened with a (J.M.G., T. N. Young, and P.J.B., unpublished data). Fur- mixture of the labeled oligonucleotide probes, essentially as thermore, calmodulin can inhibit phosphorylation of the described (13). Positive colonies were purified through two protein by protein kinase C (6). successive screenings. A second screening of the bovine We undertook the present study to elucidate the structure ofthis protein and to characterize the sites phosphorylated by Abbreviations: MARCKS, myristoylated alanine-rich C kinase sub- strate; PMA, phorbol 12-myristate 13-acetate. tTo whom reprint requests should be addressed at: Duke University The publication costs of this article were defrayed in part by page charge Medical Center, Box 3897, Durham, NC 27710. payment. This article must therefore be hereby marked "advertisement" §The cDNA sequence has been deposited in the GenBank data base in accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession no. M23738). Downloaded by guest on September 28, 2021 4012 Biochemistry: Stumpo et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4013 caudate cDNA library was performed using the 1.7-kilobase sis and autoradiography as described (19). Bovine skin fibro- (kb) Pst I fragment of pBB80K-1 (pBS80K-4) as a hybridiza- blasts (E7SKS) known to contain the authentic MARCKS tion probe. The filters were hybridized as described for the protein (20) were studied in parallel. oligonucleotide hybridization except that the hybridization was done at 650C and denatured salmon sperm DNA (100 ,4g/ml) replaced the yeast tRNA. The filters were washed for RESULTS two 15-min periods with 2x SSC/0.1% SDS (lx SSC is 0.15 Isolation and Sequencing of cDNA Clones. Screening of the M NaCl/0.015 M sodium citrate, pH 7.0) at room tempera- bovine caudate cDNA library (106 recombinant colonies) ture, two 15-min periods with 0.1x SSC/0.1% SDS at room with a mixture of the six oligonucleotide probes yielded three temperature, and two 15-min periods with lx SSC/0.1% SDS plasmids that hybridized to oligonucleotide probes derived at 50'C. from all three peptides. Restriction enzyme analysis and Northern Blot Analysis. Total cellular RNA was isolated Southern hybridization revealed that two of the three inserts and analyzed on formaldehyde/agarose gels as described (8, were identical and were =5.1 kb (pBB80K-1), whereas the 14) with the following modifications. The formaldehyde/ third was 2.6 kb (pBB80K-2). Subsequent nucleotide se- agarose gels were transferred directly to Nytran in 1Ox SSC quence analysis demonstrated that pBB80K-2 was unrelated after staining with acridine orange and destaining. The baked to pBB80K-1. blot was prehydridized for 24 hrfollowed by hybridization for Sequencing of the insert from clone pBB80K-1 revealed 24 hr with 32P-labeled cDNA restriction fragments (106 cpm/ that it was =5170 base pairs (bp) long. It contained two open ml). Poly(A)-containing mRNA was isolated as described reading frames, with the first separated from the second, (15). longer open reading frame by numerous stop codons. Based Southern Blot Analysis. Genomic DNA (15 Ag) was di- on the cDNA sequencing of the equivalent chicken protein, gested overnight with restriction endonucleases and electro- which we had determined independently (J.M.G., D.J.S., phoresed on a 0.8% agarose gel. Prior to transfer to Nytran and P.J.B., unpublished data), we suspected that the two (Schleicher & Schuell) in Southern transfer buffer (3 M open reading frames in pBB80K-1 were separated by an NaCl/0.5 M NaOH), the gel was treated for two 15-min unprocessed intron. This was confirmed by rescreening the periods with 0.25 M HCl followed by a 30-min incubation in original cDNA library with a 1.7-kb Pst I fragment of Southern transfer buffer. The filter was hybridized at 42°C pBB80K-1 (pBS80K-4) as a probe. Of several positive clones with [32P]cDNA (2 x 106 cpm/ml) and washed as described obtained, two were identical; sequence analysis of one of above for Northern blot analysis. these clones (pBB80K-3; 2.6 kb) proved that the proposed Subcloning and Sequencing ofcDNA Fragments. Restriction site of splicing was correct. However, pBB80K-1 also con- fragments were isolated by electrophoresis in agarose gels tained an unrelated sequence located 5' to the sequence of followed by electroelution into troughs as described (13). The pBB80K-3, which we believe to be a cloning artifact, based isolated fragments were subcloned into the appropriate sites on comparison with sequences of human cDNA and genomic of Bluescribe (Stratagene) for restriction mapping and nucle- clones (D.
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