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. M. Harlan, J.M.G., D.J.S., and P.J.B, unpub- otide sequencing. Double-stranded plasmid DNAs and sin- lished data). Therefore, the map of the MARCKS cDNA gle-stranded DNAs (obtained using the VCS-M13 helper shown in Fig. 1 contains a portion of the 5' untranslated phage as described by Stratagene) were sequenced by the region that was found only in pBB80K-3, while the intron was dideoxynucleotide chain-termination method (16). Due to from pBB80K-1; the rest of the sequence mapped in Fig. 1 problems associated with G+C-rich regions and secondary was identical in pBB80K-1 and pBB80K-3. structure, sequencing was performed with several enzymes The full-length MARCKS cDNA consisted of 4363 bases, including the Klenow fragment of DNA polymerase I (Inter- including 11 of the >50 adenine residues in the poly(A) tail national Biotechnologies), reverse transcriptase (Promega), (Fig. 1). Three potential polyadenylylation signals were pre- Sequenase (United States Biochemical), and T7 DNA poly- sent at positions 3896, 4333, and 4343 (corresponding to merase (Pharmacia). positions 2102, 2539, and 2549 in the spliced mRNA, Fig. 1). Transient Expression in Eukaryotic Cells. A 1.2-kb Xho II However, the two major mRNA species seen on Northern fragment of pBB80K-3 containing the entire MARCKS cod- blots (see below) were not due to alternative use of poly- ing sequence as well as 30 bases of 5' untranslated sequence adenylylation signals since a synthetic oligonucleotide com- and 177 bases of 3' untranslated sequence was subcloned into plementary to the region between the polyadenylylation pBC12/CMV (17) between the BamHI and Sma I sites. TK-L cells from R. D. National Institute (a generous gift Klausner, 6) N OD NIoo of Child Health and Human were plated the V - Development) I N day before transfection at 1.85 x 10 cells per 100-mm plate V on- N 6)iN e CD a.2 U-~ -)-ATAAA in Dulbecco's modified Eagle medium (DMEM) containing I.., t x 1794 bp intronii -.x x4 Z L 10% (vol/vol) fetal calf serum. The cells were then trans- ''''".§1, = | (A)n3' fected by the DEAE-dextran method (18) using DEAE- ATb 52 60 65 TAA dextran (200 ,g/ml) and 10 ,ug of plasmid DNA that had been ID ALPHA HELIX acceptance consensus sequences (23). The second open -42 reading frame was 903 bp long and was followed by a 3' 0 100 200 300 untranslated region of 1197 bp. The entire mRNA sequence, including 50 adenine residues in the 3' poly(A) tail, would Residue number therefore consist of 4402 bases; following processing of the FIG. 3. Bovine MARCKS hydropathy and secondary structure intron, the mature mRNA would be 2608 bases long. These profiles. The values for the hydropathy plot were calculated essen- sizes correspond exactly to the mRNA sizes determined by tially by the method of Kyte and Doolittle (26); positive values Northern blotting (see below); it seems likely, therefore, that indicate hydrophobicity. The secondary structure was predicted by the two mRNA species noted on these blots represent the Chou-Fasman method (27). Both predictions were made using unprocessed and spliced forms, respectively. the PEP program from BIONET. The open reading frame encoded by the processed mRNA predicts a protein 335 amino acids long (Mr, 31,949; Fig. 2). subjected to two-dimensional electrophoresis and autora- It contains an amino-terminal sequence [(Met)-Gly-Ala-Gln- diography, using acidic ampholytes to maximize separation Phe-Ser-Lys-Thr-Ala], which agrees with the consensus se- ofacidic proteins (20). Neither the nontransfected TK-L cells quence recognized by N-myristoyltransferase (24, 25). Be- nor those transfected with vector alone contained detectable ginning at amino acid residue 151, there was a 25-amino acid amounts of the 32P-labeled MARCKS protein (Fig. 4). How- domain of an extremely basic pl value (calculated to be 12.2, ever, the cells transfected with pCMV8OK1.2 expressed a compared to a calculated value of 5.46 for the complete 32P-labeled protein of the characteristic two-dimensional protein). This contained all of the known protein kinase C electrophoretic mobility and morphology of the MARCKS phosphorylation sites, determined by direct sequencing of protein (20, 29). The phosphorylation of this protein was tryptic phosphopeptides (21); two of these were contained stimulated by exposure of the cells to PMA (Fig. 4 e and f). within a repeated motif, Phe-Ser-Phe-Lys-Lys. The amino In addition, the 32P-labeled protein from the PMA-stimulated, acid sequences of the three tryptic peptides from which the transfected TK-L cells comigrated exactly in both dimen- cloning oligonucleotides were derived were all identified sions with the endogenous 32P-labeled protein from fetal within the predicted protein sequence (Fig. 2). bovine fibroblasts (Fig. 4, g-i). A protein which migrated Hydrophobicity analysis did not show any major hydro- identically on two-dimensional gels was also obtained after phobic regions (Fig. 3). The major peak of hydrophilicity cell-free transcription/translation of a construction in Blue- represented the amino-terminal boundary of the phosphoryl- scribe containing the 1.2-kb Xho II fragment of pBB80K-3 ation site domain, at residues 151-156. Chou-Fasman anal- (data not shown). ysis suggested extensive segments of a-helix, with consider- Northern Blot Analysis. By using pBS80K-4, a 1.7-kb able stretches of a-turn but no /3 sheet (Fig. 3). cDNA probe derived from pBB80K-1 (between Pst I sites at A search of the NBRF-PIR (release 18) and SWISS-PROT positions 531 and 2229 in Fig. 1), the distribution of mRNA (release 9) protein sequence data bases (February 25, 1989) for the MARCKS protein was analyzed in a variety ofbovine using the FASTA program (ref. 28; BIONET, IntelliGenetics) tissues (Fig. 5). Two mRNA species of -2.6 and -4.4 kb failed to reveal any biologically significant regions ofidentity were noted, which correspond to the sizes of the processed or similarity with known sequences. and unprocessed mRNA species. The mRNA levels were Expression of the Bovine MARCKS Protein in Mammalian greatest in spinal cord, spleen, lung, and brain and were Cells. To determine whether the spliced cDNA, pBB80K-3, barely detectable or undetectable in skeletal muscle and liver. was capable of directing the synthesis of the complete The 4.4-kb hybridizable band is a specific mRNA species, MARCKS protein, a 1.2-kb Xho II fragment of pBB80K-3 because (i) it can be seen readily in poly(A)-containing RNA (between positions 324 and 1538 in Fig. 1, without the intron) (Fig. 5, lanes 1-6); (ii) in tissues where there was essentially was cloned into the eukaryotic expression vector pBC12/ no 2.6-kb hybridization (Fig. 5, rat and bovine liver RNA), CMV (17). This plasmid construction, pCMV80K1.2, was there was also no detectable hybridization to the 4.4-kb band; then transfected into TK-L cells (which lack endogenous and (iii) the pattern of expression of the 2.6-kb species MARCKS protein and mRNA; data not shown). The cells paralleled that of the 4.4-kb species. Fig. 5 also demonstrates were then labeled with 32P; and exposed to control conditions that the bovine brain cDNA probe, pBS80K-4, hybridized to or PMA for 15 min, and the 32P-labeled proteins were two mRNA species from rat, chicken, human, and mouse that

1 MGAQFSKTAA KGEATAERPG EAAVASSPSK ANGQENGHVK VNGDASPAAA EPGAKEELQA NGSAPAADKE EPAAAGSGAA SPAAAEKDEP 91 AAAAPDAGQP VEKEAPVEGG AAEPGSPTAA EGEAASAASS SSSPKAEDGG HALAQQRDAE KKKKKRFSFK KSFKLSGFSF KKNKKEAGEG 181 GEAEGAAGAS AEGGKDEASG GAAAAAGEAG AAPGEPTAAP GEEAAAGEEG AAGGDPQEAK PEEAAVAPEK PPARRGAKAV EEPSKAEEKA 271 EEAGVSAAGA AGCEAPSAAG PGCPRAGGAP REEAAPPRAS SACSAPSQEA QPECSPEAPP AEAAE FIG. 2. Predicted amino acid sequence of the MARCKS protein. Sequenced peptides 52, 60, and 65 are underlined. The 25-amino acid basic domain that contains the phosphorylation sites has been double underlined. Arrowhead indicates the site of intron processing. The single-letter amino acid code is used. Downloaded by guest on September 28, 2021 Biochemistry: Stumpo et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4015

C. L Cells Control WZecto c jerA e l e .: itry mRNA Bovine Tissues: Total RNA &~r, i I 0 amx .c o co < -0 O .c 0 :: - U a- '4 E 0 f I a Z Z s_ CD 0 O. r 0 - e - .c : , o D2Iu - 0 -0 0 > .- .-QL--w o IW I of U CDal ~~~~~~~~a Ln Cl) b.CeA. V O2

b. L Cells: PMA d Vector alone PMA f. pCMV80K 12 PMA RX ....E -28s

^ !1 \*, ~~~~~~18s- -18s

~~~~~~~28s-;i l A O Ot

E7SKS Cells Control FIG. 5. Expression ofMARCKS mRNA in various tissues. Total g. h E7SKS Cells PMA i. f +h RNA (15 ,ug) isolated from various bovine tissues and 4 ,ug ofpoly(A) *t ds. RNA isolated from tissue culture cells and various tissues were fractionated on a 1.2% formaldehyde/agarose gel, transferred to Nytran, and hybridized with 32P-labeled pBS80K-4 (1.7-kb Pst I 0 fragment ofpBB80K-1). The 28S and 18S markers show the positions of the major species of rRNA on the stained gel. The blot was gs.W 0 intentionally overexposed to show the paucity ofhybridizing mRNA in rat and bovine liver. 1321-Ni refers to poly(A) RNA from human astrocytoma cells, exposed either to control conditions or to 16 hr of 16 AM PMA, which causes depletion ofcellular protein kinase C (8). The last lane contains total cellular RNA from murine 3T3-L1 FIG. 4. Phosphorylation of the MARCKS protein in transfected fibroblasts. murine TK-L cells and bovine E7SKS cells. TK-L cells were transfected with no DNA (a and b), 10 jLg of pBC12/CMV vector (c Similar differences have been observed with a variety ofother and d), or 10 ,Ag ofpCMV80K1.2 (e andf). Transfected cells and fetal bovine fibroblasts (E7SKS) were incubated in serum-free medium for proteins, including the growth-associated protein GAP43 and 18 hr, labeled with 32p, for 2 hr, and exposed for 15 min to 0.01% the human heavy neurofilament subunit (NF-H) (31-33). dimethyl sulfoxide (control) (a, c, e, and g) or 1.6 ,uM PMA in 0.01% Whatever the causes ofthis anomalous migration on SDS gels, dimethyl sulfoxide (PMA) (b, d, f, and h). Cellular supernatant it seems unreasonable to continue to refer to this protein as the fractions were subjected to two-dimensional electrophoresis, and the "80- to 87-kDa" protein. In the absence ofa known enzymatic resulting autoradiographs are shown. Trichloroacetic acid-precipi- or other function for the protein, we propose the name table radioactivity (2.13 x 105 cpm) was loaded onto each gel (a-f), myristoylated alanine-rich C kinase substrate (MARCKS). 7.45 x 104 cpm was loaded (g and h), and 7.5 x 104 cpm of each Another interesting aspect of the predicted amino acid sample shown infand h was loaded together (i). Arrows indicate the sequence is the amino acid composition ofthe protein. There 32P-labeled MARCKS protein. is an extreme preponderance of alanine (28.4 mol %) as well were of similar sizes to the bovine mRNAs. Northern blot as a relative abundance ofglycine (12.8 mol %), proline (10.1 mol %), and glutamate (13.9 mol %), the last amino acid analysis of various bovine brain tissues showed that the of the of the two mRNA was greatest largely accounting for the acidic isoelectric point expression species in the protein (pI 5.46). These values are in good agreement with the cerebral cortex, brain stem, and hypothalamus (data not amino acid composition ofthe purified protein (7). There are shown). no methionines, other than the amino-terminal methionine, Southern Blot Analysis of Genomic DNA. Genomic South- which accounts for our repeated inability to label the protein ern blot analysis of bovine DNA with the pBS8OK4 cDNA probe revealed a simple pattern that was compatible with a ~'- gene of low complexity. A single strongly hybridizing band 0 c E was obtained with Q mr) HindIII-, BamHI- and Bgl II-digested LI mc bovine DNA, while digestion with EcoRI yielded one strongly hybridizing band and one weakly hybridizing band 23.1 Kb (Fig. 6). 9.4 Kb- 6.7 Kb- DISCUSSION FIG. 6. Genomic Southern The primary sequence of the bovine "80- to 87-kDa" protein blot analysis. Bovine high molec- is interesting in several respects. One striking finding is that ular weight DNA (15 gg) was di- 2.3 Kb - gested with EcoRI, HindIII, the predicted molecular weight of the protein is 31,949. 2.0 Kb- Expression studies confirmed that a 1.2-kb fragment of the BamHI, and Bgl II, separated on a transferred to which contained a 335-amino acid open 0.8% agarose gel, spliced cDNA, single Nytran, and hybridized with 32p- reading frame, encoded the entire protein. The discrepancy labeled pBS80K-4 (1.7-kb Pst I between its predicted and apparent molecular masses on SDS fragment of pBB80K-1). The gels presumably stems from factors such as its acidic pI and numbers at the left represent the general hydrophilicity, allowing for incomplete binding ofSDS HindIll-digested A DNA size (30), as well as its apparent rod-like helical structure (7). markers. Downloaded by guest on September 28, 2021 4016 Biochemistry: Stumpo et al. Proc. Natl. Acad. Sci. USA 86 (1989) with [35S]methionine in intact cells. The amino terminus synthesis of oligonucleotide probes. We also thank Dr. John Knopf consists of a sequence, (Met)-Gly-Ala-Gln-Phe-Ser-Lys-Thr- for advice concerning oligonucleotide synthesis, Dr. Tatsuya Kuri- Ala, which closely resembles the consensus sequence for hara for the cDNA library, Dr. Bryan R. Cullen for the expression protein myristoylation (24, 25). This modification of the vector, Dr. Angus C. Nairn for frequent helpful advice and for protein reviewing the manuscript, Dr. Richard M. Levenson for computer has been found in studies of murine macrophages, assistance, Elizabeth Kennington and D. McNeill Haupt for techni- where it is thought to regulate association of the protein with cal support, and Lessie Detwiler and Ginger Marshall for secretarial membrane structures (5). In addition, myristoylation of the assistance. We are especially grateful to Dr. Henrik Dohlman for amino terminus probably explains our repeated inability to protein data base searching and help with secondary structure sequence the native protein by Edman degradation. analysis. This work was supported in part by Grants 2T32-GM-07171 We have found that all of the sites known to be phospho- and MH-39327 from the National Institutes of Health and the rylated by protein kinase C in a cell-free system are contained Howard Hughes Medical Institute Laboratories Graduate Students within a 25-amino acid domain located between residues 151 Support Fund (J.M.G.). D.J.S. is an Associate and P.J.B. is an and 175 in the bovine MARCKS protein. This peptide, which Investigator of the Howard Hughes Medical Institute. is quite hydrophilic and has a calculated pI value of 12.2, is enclosed by blocks ofbasic residues. Based studies of the 1. Nishizuka, Y. (1988) Nature (London) 334, 661-665. on 2. Niedel, J. E. & Blackshear, P. J. (1986) in Phosphoinositides and intact protein and synthetic peptides, we believe that all four Receptor Mechanisms, ed. Putney, J. W. (Liss, New York), pp. 47- serines within this peptide are phosphorylated by protein 88. kinase C (21). Two of these serines are in a repeated motif, 3. Woodgett, J. R., Hunter, T. & Gould, K. L. (1987) in Cell Mem- Phe-Ser-Phe-Lys-Lys. The 25-amino acid phosphorylation branes: Methods and Reviews, eds. Elson, E. L., Frazier, W. A. & site domain is perfectly conserved at the amino acid level Glazer, L. (Plenum, New York), Vol. 3, pp. 15-340. 4. Rodriguez-Pena, A., Zachary, I. & Rozengurt, E. (1986) Biochem. among the bovine, human, and chicken proteins (J.M.G., Biophys. Res. Commun. 140, 379-385. D.J.S., D. M. Harlan, and P.J.B., unpublished data). This 5. Aderem, A. A., Albert, K. A., Keum, M. M., Wang, J. K. T., conservation ofthe phosphorylation site domain supports the Greengard, P. & Cohn, Z. A. (1988) Nature (London) 332, 362-364. hypothesis that protein kinase C-induced phosphorylation of 6. Albert, K. A., Wu, W. C. S., Nairn, A. C. & Greengard, P. (1984) the protein is of regulatory significance. Proc. Natl. Acad. Sci. USA 81, 3622-3625. Studies in intact cells have raised the 7. Albert, K. A., Nairn, A. C. & Greengard, P. (1987) Proc. Natl. possibility that at Acad. Sci. USA 84, 7046-7050. least one other phosphopeptide is present in the protein 8. Blackshear, P. J., Stumpo, D. J., Huang, J.-K., Nemenoff, R. A. & phosphorylated after exposure of cells to active phorbol Spach, D. H. (1987) J. Biol. Chem. 262, 7774-7781. esters (data not shown). Other studies have postulated even 9. Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170. greater numbers of phosphorylation sites (see, for example, 10. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. ref. 35); however, it is possible that this large number of 11. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, 266- 267. apparent sites reflects the numerous tryptic peptides that can 12. Kurihara, T., Lewis, R. M., Eisler, J. & Greengard, P. (1988) J. be generated from the protein, given that the 25 amino acid Neurosci. 8, 508-517. domain containing the sites consists of 52% lysine and 13. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular arginine. It seems likely that the numerous isoelectric vari- Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold ants ofthe protein commonly seen in intact cell studies reflect Spring Harbor, NY). a combination of multisite phosphorylation and variable 14. Hovis, J. G., Stumpo, D. J., Halsey, D. L. & Blackshear, P. J. (1986) J. Biol. Chem. 261, 10380-10386. phosphorylation stoichiometry at each site. 15. Stumpo, D. J. & Kletzien, R. F. (1984) Eur. J. Biochem. 144, 497- Northern blot analysis oftotal cellular RNA from a variety of 502. bovine tissues revealed two characteristic mRNA species, of 16. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. -2.6 and ""4.4 kb, with the former predominating over the latter Sci. USA 74, 5463-5467. in a ratio of about 5:1. These two species most likely represent 17. Cullen, B. R. (1986) Cell 46, 973-982. processed and 18. Lopata, M. A., Cleveland, D. W. & Sollner-Webb, B. (1984) Nu- unprocessed mRNA, respectively, with the cleic Acids Res. 12, 5707-5717. latter form likely to be more prevalent in the nucleus; however, 19. Spach, D. H., Nemenoff, R. A. & Blackshear, P. J. (1986) J. Biol. we cannot yet exclude the possibility that the unprocessed Chem. 261, 12750-12753. mRNA exists in the cytoplasm. Levels of mRNA expression 20. Blackshear, P. J., Wen, L., Glynn, B. P. & Witters, L. A. (1986) J. were highest in brain and spinal cord, spleen, and lung; inter- Biol. Chem. 261, 1459-1469. mediate in many other tissues; low in skeletal muscle; and 21. Graff, J. M., Stumpo, D. J. & Blackshear, P. J. (1989) J. Biol. Chem., in press. essentially undetectable in liver. These levels correlate very 22. Kozak, M. (1986) Cell 44, 283-292. well with levels of the protein in bovine and rat tissues (20, 34). 23. Green, M. R. (i986) Annu. Rev. Genet. 20, 671-708. Within the brain, the cerebral cortex, brain stem, and hypo- 24. Towler, D. A., Eubanks, S. R., Towery, D. S., Adams, S. P. & thalamus expressed the greatest amount of mRNA. Glaser, L. (1987) J. Biol. Chem. 262, 1030-1036. Without exception, our studies to date have shown that the 25. Towler, D. A., Gordon, J. I., Adams, S. P. & Glaser, L. (1988) amount MARCKS mRNA in a cell or tissue Annu. Rev. Biochem. 57, 69-99. of hybridizable 26. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. is correlated directly with the amount of immunoreactive or 27. Chou, P. Y. & Fasman, G. D. (1978) Annu. Rev. Biochem. 47, 257- phosphorylated protein. These studies indicate that most of 276. the regulation of MARCKS protein levels is likely to occur at 28. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA the level of transcription or mRNA stability, with no evi- 85, 2444-2448. dence to date of a major role of translational regulation in 29. Blackshear, P. J., Witters, L. A., Girard, P. R., Kuo, J. F. & of Quamo, S. N. (1985) J. Biol. Chem. 260, 13304-13315. determining the observed tissue- and cell-specific patterns 30. Reynolds, J. A. & Tanford, C. (1970) Proc. Natl. Acad. Sci. USA expression of the protein. 66, 1002-1007. The MARCKS protein is a widely distributed, prominent 31. Karns, L. R., Ng, S.-C., Freeman, J. A. & Fishman, M. C. (1987) cellular substrate for protein kinase C, and it has been impli- Science 236, 597-600. cated in numerous physiological processes affected by acti- 32. Lees, J. F., Schneidman, P. S., Skuntz, S. F., Carden, M. J. & vation ofthis kinase. The structural data presented here should Lazzarini, R. A. (1988) EMBO J. 7, 1947-1955. 33. Banker, G. A. & Cotman, C. M. (1972) J. Biol. Chem. 247, 5856- help in the eventual determination of the physiological role of 5861. this protein in cellular biology. 34. Albert, K. A., Walaas, S. I., Wang, J. K.-T. & Greengard, P. (1986) Proc. Nat!. Acad. Sci. USA 83, 2822-2826. We are very grateful to Dr. Richard R. Randall, Mildred McAd- 35. Isacke, C. M., Meisenhelder, J., Brown, K. D., Gould, K. L., ams, and Kathy Theisen for peptide sequence analysis and for Gould, S. J. & Hunter, T. (1986) EMBO J. 5, 2889-2898. Downloaded by guest on September 28, 2021