Proc. Nati. Acad. Sci. USA Vol. 88, pp. 2505-2509, March 1991 Biochemistry

Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide (myristoylated alanine-rich C kinase substrate/myristoylation/phosphorylation/signal transduction/actin)

JOHN T. SEYKORA*, JEFFREY V. RAVETCHt,

AND

ALAN ADEREM*

*The Rockefeller University, 1230 York Avenue, New York, NY 10021; and tThe Dewitt Wallace Research Laboratory, Program of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021

Communicated by Zanvil A. Cohn, December 28, 1990

ABSTRACT We have isolated and characterized a cDNA clone encoding the murine macrophage 68-kDa protein kinase C substrate, which is homologous to the 80- to 87-kDa protein identified by the acronym MARCKS (myristoylated alaninerich C kinase substrate). The murine MARCKS cDNA done encodes an acidic protein of 309 amino acids with a calculated molecular weight of 29,661. Transfection of the murine MARCKS gene into TK-L fibroblasts produced a myristoylated protein kinase C substrate that migrated on SDS/PAGE with an apparent molecular mass of 68 kDa. Peptide mapping studies indicated that MARCKS produced by the transfected gene was indistinguishable from the endogenous murine macrophage protein. Comparison of the murine macrophage sequence with the previously published chicken and bovine brain sequences revealed two conserved domains: an N-terminal membrane-binding domain and a phosphorylation domain that also contains calmodulin and actin binding sites. In murine peritoneal macrophages, bacterial lipopolysaccharide increased MARCKS mRNA levels by >30-fold. Multiple MARCKS transcripts were observed and could be accounted for by differential polyadenylylation and incomplete processing. Genomic Southern blot analysis suggested a single MARCKS gene per haploid genome.

Protein kinase C (PKC) transduces signals by phosphorylating substrate proteins that then act as cellular effector molecules (1). The myristoylated alanine-rich C kinase substrate (known by the acronym MARCKS) is a specific PKC substrate that has been implicated in cellular responses as diverse as neurosecretion (2-4), growth factor-dependent mitogenesis (5, 6), and macrophage and neutrophil activation

(7, 8).

Although MARCKS has been characterized at the biochemical (3, 9) and molecular (10, 11) level, its precise function is obscure. In the presence of Ca2W, MARCKS binds calmodulin in a phosphorylation-regulated manner; the nonphosphorylated protein binds calmodulin whereas the phosphorylated protein does not (12, 13). In macrophages the protein is found predominantly in punctate structures at the substrate-adherent surface of filopodia where it colocalizes with vinculin, talin, and PKC (14). Upon activation of PKC, MARCKS is phosphorylated, and this modification results in its displacement from the punctate structures and its translocation from the plasma membrane to the cytosol (14). Dephosphorylation results in the reassociation of cytosolic myristoylated MARCKS with the plasma membrane (M. Thelen, A. Rosen, A. Nairn, and A.A., unpublished data). MARCKS binds to filamentous actin (J. Hartwig, A. Rosen, P. Janmey, M. Thelen, A. Nairn, and A.A., unpublished The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2505

data) and it therefore represents a candidate molecule through which PKC might regulate the reversible association of the actin cytoskeleton with the substrate-adherent plasma membrane. Motile and inducible effector cells, such as macrophages and neutrophils, offer a model system for the dissection of MARCKS function. For example, bacterial lipopolysaccharide (LPS) regulates host defenses, in part, by priming macrophages for enhanced PKC-dependent responses such as those leading to the activation of cellular phospholipases and the release of arachidonic acid metabolites (15). Concomitant with priming, LPS also induces the synthesis and cotranslational myristoylation of MARCKS and potentiates PKC-dependent phosphorylation of the protein (7, 16). As a first step in defining the role of MARCKS in effector responses, we have cloned, sequenced,* and characterized the cDNA encoding murine macrophage MARCKS. MATERIALS AND METHODS Cloning the Murine MARCKS Gene. A Agtll cDNA library from the P388D1 murine macrophage cell line (provided by A. Ezekowitz, Harvard University) was screened according to standard protocols (17) with an affinity-purified rabbit polyclonal antibody directed against murine MARCKS purified from brain (16). One positive phage was plaquepurified, and its DNA was isolated (17). This positive phage clone was termed MM1; DNA from MM1 was used to rescreen the library and isolate clones MM2 and MM3 (Fig. 1B). DNA Sequencing. Double-stranded DNA sequencing reactions were performed on the various clones in pUC18 by using the dideoxynucleotide chain-termination kit from United States Biochemical. Prevalent compressions (due to a G+C-rich coding region) were resolved using dideoxyinosine reactions. Artifactual bands (bands extending across all four lanes) were resolved by adding a long deoxynucleotide chain with terminal deoxynucleotidyl transferase to those reaction products not terminated with a dideoxynucleotide (19). The entire coding region of MM3 was sequenced on at least one strand by using dideoxyinosine reactions. The bovine MARCKS cDNA (kindly provided by P. Blackshear) (10) was sequenced on both strands from nucleotide 2292 to 2502 (20). Northern and Southern Blot Analyses. Total cellular RNA was isolated by the method of Chirgwin et al. (21). RNA was fractionated on a 1% agarose gel containing 2.2 M formaldehyde and transferred to nitrocellulose (22). High molecular Abbreviations: LPS, lipopolysaccharide; MARCKS, myristoylated alanine-rich C kinase substrate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate. MThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M60474).

Proc. Natl. Acad. Sci. USA 88 (1991)

Biochemistry: Seykora et al.

2506

A

10

30

20

50

40

60

80

70

90

100

110

120

,GGGAGATTTCTCCATTTTCCTCTTGTCTACAGTGCGGCTACAAATCTGGGATTTTTTTTTATTACTTCTTTTTTTAAAAAAACTACACTTGGG

CTTTTTCCCTCCTTCCTGCGCTGCTGCTTTTTTGATCTCTTCGACTAAAAATTTTTTATCCGGAGTATTTAATCGGGTCTCTTCTGTCCTCCT

120 240

TCTTTGTTGAAGAAGCCAGCATGGGTGCCCAGTTCTCCAAGACCGCAGCGAAGGGAGAAGCCACCGCCGAGAGGCCCGGGGAGGCGGCTGTGGCCTCGTCGCCTTCCAAAGCAAATGGGC

480

PTGTGTGCCGCCGCCGCTGTTGCTGCTGCTGCTGCTCGCCCCGTCGTTACACCAACCGAAGGCTCTTTGTTTCCTCTCTTGGATCTGTTGAGTT M G A Q r S K r a A K G E A T A E R P G E A A V A S S P S K A N G

Q E N G H V K V N G D A S P A A A E P G A K E E L Q A N G S A P A A D K E E P A

AGQAGAATGGCCACGTMAAAGTQAACGGGGACGCGTCTCCCGCCGCCGCCGAGCCGGGCGCCAAGGAGGAGCTGCAAGCCAACGGCAGCGCCCCGGCCGCCGACAAGGAGGAGCCCGCQA T

P

A A A

E K D E A A A A T E P G A G A A D K E A A E A E P A E P

G A A

A

P

0

S

G

S A A

G

G

E

S S

360 33

73

600 113

GCGGCAGTGCCGCGACCCCCGCCGCGGCCGAAAAGGATGAGGCTGCCGCGGCCACCGAGCCGGGCGCCGGCGCGGCCGACAAGGAGGCTGCGGAGGCCGAGCCCGCCGAGCCCAGCTCCC 720 P A A E A E G A SA S S T S S P K A E D G A A P S P S S E T P K K F K X R FJ 8 r 153 CGGCCGCCGAGGCCGAkGGGCGCGTCCGCCTCCTCCACGTCGTCGCCCAAGGCGGAGGACGGGGCCGCGCCGTCGCCCAGCAGCGAGACCCCGAAAAAAGAAGCGCTTTTCCTTCA 840 K K 5 I R L 8S a T 8 G E G A E A E G A T A E G A K D E A A A A A 193 1t K KX X Z AGAAGTCCTTQAAGCTGAGCGGCTTCTCCTTCAAGAAGAGCAAGAAGGAGTCGGGCGAGGGCGCTGAAGCAGAGGGAGCGACCGCGGAAGGCGCQAAGGACGAGGCTGCAGCCGQAGCGG 960 G

E

A

G

G

E P R E A E A A E P E Q P E 0 P E

S E A A G E K A E E

P

A P

G

G

A

G A

E

G A A

GCGGCGAGGGGGCCGCGGCCCCCGGGGAGCAGGCAGGCGGGGCGGGCGCCGAGGGCGCGGCGGGCGGAGAGCCCCGGGAGGCCGAGGCGGCGGAGCCCGAkGCAGCCGGAGCAGCCGGAGC

Q P

A A E E

P Q A E

E Q

G A T A G D A S

S A A

G P

E Q

233

1080 273

AGCCCGCGGCGGAGGAGCCGCAGGCGGAGGAGCAGTCGGAGGCGGCGGGGGAGAAGGCGGAGGAGCCCGCGCCCGGCGCCACCGCGGGCGATGCGTCCTCCGCCGCAGGGCCTGAGCAGG 1200 E A P A A T D E A A A S A A P A A S P E P Q P E C S P E A P P A P T A E *

309

AGGCGCCCGCTGCCACCGACGAGGCCGCGGCGTCCGCAGCCCCCGCCGCGTCGCCGGAGCCGCAGCCCGAGTGCAGTCCGGAGGCGCCCCCCGCGCCAACGGCCGAGTAAGCTCCAGAGC 1320

CCTCACGCAATTCAAGAACTTTTCCCCCCAGTTTGTTTGTTGGAGTGGTGECCAGGTACTGGTTTTGGAGAACTTGTCTACAACCAGGGATTGATTTTAAGATTTTTTTTTAAATTTCAC 1440 ATTTTTTTTAAGCAGCAAATTTTTTGTTTTTTGTTTTTTTTAAGCCCCCTTCCCCACAGATCCCATCTCAGA'TAGTTGTTTCCQCCATTCCGACAGGCCGAGGACGTGTTAGACAGCTTC 1560 CTCTGCCTTCTTTCTTTACTTTTACTTTTTTTTTTTTTTTTTTGGCATCAGTATTAATGTTTTTTGCATACTTTGCATCTTTATTAAzAAAGTGTAAACTTTCTTTGTCAGATCTATAGA 1680 CATACCCATATATGAAGGAGATGGGTGGGTCAAAAGGAATAACAAATGAAGTGATAGGGGCCACTATGGGAAATTGAAGCAGTGCATAAC-ATTGCCAAGATAATATGCCACTAAAATGGT 1800 GGTGGGTGTAAAGGCTTAGGGTTCTTGTCCTTTCT-TTCTTTCmT1CTTTCTTTCTTTCTTTCTTTCTTTCTTTTTTTTTTTAAAGAAAAATTATTACGATGTATTTTGTGAGGCAGGTTT1920 ACAACACTACACGTTTTGAATAAGAAGGAAAGAGAAAAxAAMTAAAMCACCAATACCCAGATTT AAAAAA AGTCATAGTCTTAGGAGTTQT6TGTAACCATAGGAACTTCC 2040 TGCTTATCTCATGTTAGCTGTACCAAGTCAGTGATTAAdTAXC-TAACAAGTTGTATAGGCTTTATTGTTTATiTGCTGGTTTATGAIGGTTAATAAAGTGTAATTATGTATTACCAGCAGG 2160 GTGTTTTTAACTGTGACTATTGTATAAAAACAAATCTTGATATCCTTCAGAAGCACATGAGTTTGCAAGTCTCCACCCTGCCCATTTTTTMrCTGCAGTCATCTTGGACCTTTTAA 2280 ACACAAAATTTTAAACTCAACCAAGCTGTkGATACGTGGAATGGTTACTGTTTATACTGTGGTATGTTTTTTGATTACAGCAGACAATGCTTTCCTTTCCAGTTGCTTTdAGAATAAAGG 2400 AGAAACAGATCTTCAGATGCAATGGTTTTGTGTAGCATCTCGTCTCTCGTGTTTTGTAAATACTGGAGGAGCTTTGACCAATTTGACATA ,GAGCTGGAAGTAACGTTGCTTCAGA 2520 GCTATTCQACTCCTGCTTAAGGTGTTCTAATCTTCTGTGAkGCACACTAAAAGCAAAAAvAATAAATGTGATAAATGTAAAAAA 2603 5'

B Murine

MARCKSI

ATG

x|

'G M

p I

TAA X

1

1

Bg 1

An 1

P 1

Phos.

MM1

MM2

MiM3

L

i

1l0o bp

weight genomic DNA was isolated from the murine T-cell line L5178Y according to standard procedures (17). DNA was fractionated and transferred to nitrocellulose. The nitrocellulose blots were hybridized to 32P-labeled random-primed DNA probes (generated using a 1.7-kb EcoRI-Bgl II fragment from MM3 as a template) and washed under high-stringency conditions (17), and autoradiography was performed as described above. The autoradiograms of murine macrophage MARCKS mRNA were quantitated by densitometry. Treatment of Macrophages with LPS. Macrophages were cultured overnight at 2.5 x 107 cells per 60-mm dish, washed twice with calcium- and magnesium-free phosphate-buffered saline (PD), and incubated for the indicated times in a modified minimal essential medium containing no addition or Salmonella abortus equfi LPS (100 ng/ml; gift of C. Galanos, Freiberg, F.R.G.) (15). At the end of the incubation, the cells were washed once with PD and RNA was isolated as described above. Expression and Characterization of Transfected MARCKS. A 1.15-kb Xho II fragment from MM3 was isolated and ligated into the BamHI site of the InVitrogen I/NEO mammalian expression vector. The construct with the murine MARCKS in the orientation to code for the correct protein was termed pI/NEOc, and the construct with the MARCKS gene in the opposite orientation was called pI/NEOi. TK-L cells, cultured overnight to 70% confluency, were transfected with pI/NEOc and pI/NEOi using standard calcium phosphate procedures (17). After 4 weeks of selec-

gene

FIG. 1. (A) cDNA nucleotide sequence and predicted amino acid sequence for the 3' murine MARCKS gene. N-terminal myrisAn toylation consensus (18) is shown in bold I italic letters. The phosphorylation domain is denoted by bold letters. The termination codon is designated with an asterisk. Polyadenylylation processing signals are underlined. (B) Schematic diagram and partial restriction map of individual murine MARCKS cDNA clones. The coding region is represented by the open box. An, utilized polyadenylylation signals; Bg, Bgl II site; M and cross-hatched box, myristoylation consensus

sequence; Phos. and stippled box, phosphorylation domain; P, Pst I site; X, Xho II site.

tion with G418 (GIBCO), 20 clones expressing MARCKS isolated and characterized. The data reported were obtained with clone TK-LM7. Myristoylation and phosphorylation of transfected and endogenous murine MARCKS were assessed by fluorography or by autoradiography after the radiolabeled protein was immunoprecipitated from [3H]myristis acid or 32P-labeled TK-LM7 cells (7, 16, 23). Staphylococcus aureus V8 protease digestion of MARCKS was performed as described (7). were

RESULTS Cloning the cDNA Encoding the Murine Macrophage MARCKS Gene. A Agtll P388D1 macrophage cDNA library was screened with an affinity-purified rabbit polyclonal antibody directed against murine brain MARCKS (16) and a single presumptive clone, termed MM1, was isolated and sequenced (Fig. 1B). Overlapping clones MM2 and MM3 were isolated by rescreening the cDNA library. Clone MM3 contained the complete coding region comprising 930 base pairs (bp) plus a 5' untranslated region of 380 bp and a 3' untranslated sequence of 674 bp. Fig. 1A shows the entire sequence of the nurine macrophage MARCKS transcript including the compiled 1.3 kb of 3' untranslated region obtained from clones MM1, MM2, and MM3. The murine macrophage MARCKS gene encodes a 309amino acid protein with a calculated molecular mass of 29.661 kDa and a theoretical pI of 4.1. The "68-kDa" murine

Biochemistry: Seykora et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

U/

200-

200

2600

Murine MARCKS FIG. 2. Self-comparison of the murine MARCKS cDNA seusing PLFASTA software with a k-tuple of 6 (highest stringency) (24). The axes represent the compiled nucleotide sequences of the murine MARCKS cDNA clones. quence

macrophage protein migrates anomalously on SDS/PAGE (7) and we show below that the transfected cDNA of MM3 encodes a protein that migrates on SDS/PAGE with an apparent molecular mass of 68 kDa and a pI of 4.1 (see Fig. SA), confirming that MM3 encodes the full-length murine macrophage MARCKS protein. The myristoylation consensus sequence, starting with Gly-2 (18), and the phosphorylation domain, residues 145-171, are indicated in Fig. LA. The amino acid composition of murine MARCKS is unusual; alanine accounts for 28.8% of the residues and glutamate and proline constitute 16.5% and 11.0%o of the residues, respectively. The coding region of murine MARCKS has a repetitive internal structure (Fig. 2) as indicated by additional lines parallel to the diagonal as revealed by analysis with PLFASTA (24). Comparison of the Primary Protein Structure of Murine, Bovine, and Chicken MARCKS. Murine macrophage MARCKS contains two major regions of homology to the bovine brain (10) and chicken brain proteins (11). The N-terminal 73 amino acids of the murine and bovine proteins are identical, and the first 66 amino acids of these proteins are 90% identical to that of the chicken protein (Fig. 3). The phosphorylation domain of murine MARCKS (residues 145171) is identical to those of chicken and bovine MARCKS with two exceptions: residue 167 in the murine protein is serine whereas the corresponding residue in the bovine and chicken proteins is asparagine, and residue 171 in murine

2507

MARCKS is serine whereas the corresponding residue in the bovine and chicken proteins is alanine (Fig. 3). The presence of Ser-167 is important since it creates another potential protein kinase C phosphorylation site (FKKSKK) (Fig. 3). We were surprised to find that the 17 amino acids N-terminal to the phosphorylation domain in murine MARCKS were 76.5% identical to the corresponding residues of the chicken protein and only 41% identical to the bovine protein (Fig. 3 and refs.' 10 and 11). To resolve this apparent evolutionary dichotomy, we resequenced this region of the cDNA encoding the bovine protein (kindly supplied by P. Blackshear). Since the MARCKS gene is very G+C-rich in this region, it was necessary to use dideoxyinosine sequencing reactions to resolve the prevalent compressions and to use terminal deoxynucleotidyltransferase tailing to determine the specificity of artifactual bands. We determined the sequence of the cDNA clone between nucleotides 2292 and 2502 on both strands (20). The sequence we obtained for the bovine clone in this region yielded a predicted amino acid sequence in which' 13 out of 70 amino acids were different than the published sequence (10). Fig. 3 presents a' composite of the bovine MARCKS sequence derived from ref. 10 and the corrected sequence derived above. Comparison of this bovine sequence to chicken and murine MARCKS now results in >85% identity in the region of the protein immediately N-terminal to the phosphorylation site. In general, the C-terminal regions of the murine, bovine, and chicken MARCKS genes have diverged greatly, although the C-terminal 15 amino acids in bovine and murine MARCKS are 87% identical and the C-terminal 18 amino acids of the murine and chicken proteins are 67% identical. Genomic DNA Analysis. Southern blot analysis of murine genomic DNA gave a simple hybridization pattern that was consistent with MARCKS being encoded by'a gene with few introns (Fig. 4A). Digestion of murine genomic DNA with Xba I gave one strongly hybridizing band. Two strongly hybridizing bands were obtained with BamHI-, HindIII-, and Pst I-digested murine DNA. These bands can be explained by the presence of Pst I sites in the cDNA clone and by the predicted presence of a BamHI site and a HindIII site in the intron found 102 nucleotides 5' to the start codon'(10). This conclusion was supported by the observation that a probe 3' to the intron only hybridized to one band in each digest (data not shown). mRNA Analysis. The tissue distribution of the m.RNA encoding MARCKS was examined. Highest levels of MARCKS mRNA were found in brain and spleen, intermediate levels were found in kidney and heart, and very low levels of MARCKS mRNA were found in liver (data not

Myr. M-

FSKTAAGEATAERPFEAAVASSPSKANGQENGHVKVNGDASPAAAEPGAKEELQANGSAPAADKEEPAS-GS-AATPAAAEKDE-AAAATEPGAG

B-

-100 :MGQS:K:TA:KGEATAERPGEAAVASSPSKANGQENGHVKVNGDASPAAAEPGAKEELQANGSAPAADKEEPAAAGSGAASPAAAEKDEPAAAAPDAGAS 1 1 -GEAAAEKPGEA-VAASPSKANGENGHVKVNGDASPAAAEAG-KEEVQANGSAPA---EETG----------KEEAASSEP---AS -79 AADKEAA-EAEPAEPSSP-AAEAEGASA-SSTSSPKAEDGAAPSPSSET GEGAEAEG---ATAEGAKDEA--189 'SFKKSFKLSGFSFKKSKKE PVEKEAPVEGEAAEPGSPTAAEGEAASAASSTSSPKAEDGATPSPSNET SFKKSFKLSGFSFKKNKE) GEGGEAEGAAGASAEGGKDEASG -200 --EKEAA-EAESTEPASP--AEGEA--------SPKTEEGATPSSSStT SFKKSFKLSGFSFKKNKKE GEGAESEGGAAAAAEGGKEEAAA -166

CM-

-97

BCM- -AA-AAGGEGAAAPGEQAGGAGAEGAAGGEP---REAEAAEPEQPE-QPEQPAAE-EPQAEEQSEAAGEKAEEP---APGATAGDASSAAGPE--QEAPA -277 B- GAA-AAAGEAGAAPGEPTAAPGEEAAAGEEGAAGGDPQEAKPEEAAVAPEKPPARRGAKAVEEPSKAEEKAEEAGVSAAGAAGCEAPSAAGPGCPRAGGA -299 C- AAPEAAGGEEGKAAAEEASAAAAGSREAAKEEAG-DSQEAKSDEAA--PEKATGEEAP-AAEEQQQQQQEKAAEEAGAAATS-EAGSGE------QEAA -255

M- ATDEAA---ASAA-PAASPEPQPECSPEAPPAPTAE -309 B- PREEAAPPRASSACSAPSQEAQPECSPEAPPAEAAE -335 C- PAEEPAAAR------- QEAPSESSPEGP-AEPAE -281

FIG. 3. Alignment of the murine (M), bovine (B), and chicken (C) MARCKS amino acid sequence according to the method of Garmier et al. (25). The sequences are identified on the left, and the amino acids are numbered on the right. The Myr. box indicates the myristoylation consensus sequence (18), and the Phos. box indicates the phosphorylation domain.

2508

Biochemistry: Seykora et al. x

.A

H

P

Proc. Natl. Acad. Sci. USA 88 (1991)

B

13

- 23 - 6.6

0'

-44

C_

t.

28s --

I18S

- 2.0

4 -1.4

--

lBSG9DH w k

mur GPD H -

z ,0

E cn

y

25

-

6-) u

20

-

w u

15

-

ca -o

10

-

0

0

0 n a)

0

C

1

2

3

4

species of MARCKS mRNA in murine peritoneal macrophages (Fig. 4B). Analysis of Murine MARCKS Transfectants. Murine TK-L fibroblasts do not express the MARCKS mRNA or protein (10) (data not shown) and were, therefore, a recipient in transfection studies. Stable transfectants expressing the MARCKS protein were generated using a construct containing the complete coding region of the murine MARCKS gene and a neomycinresistance gene. The MARCKS protein expressed in clone TK-LM7 was characterized as follows. A 68-kDa myristoylated protein with a pl of 4.1 was immunoprecipitated from TK-LM7 cells by using an affinity-purified antibody to murine MARCKS (Fig. 5A). This protein comigrated precisely with immunoprecipitated murine peritoneal macrophage MARCKS labeled with [3H]myristic acid in a two-dimensional gel electrophoresis system (Fig. 5B), and S. aureus V8 protease digestion of the immunoprecipitated transfected protein yielded a myristoylated peptide map identical to that generated from authentic macrophage-derived MARCKS protein (data not shown). Treatment of TK-LM7 cells with phorbol esters resulted in a marked increase in the phosphorylation of the transfected MARCKS protein, confirming that it is a PKC substrate in the transfected cells (Fig. 5C). S. aureus V8 protease digestion of the immunoprecipitated phosphorylated protein yielded two characteristic phosphopeptides of 9 and 13 kDa, which were identical to those obtained from MARCKS that had been immunoprecipitated from 32P-labeled PMA-treated murine peritoneal macrophages (Fig. 5D).

5

Time (h)

FIG. 4. (A) Genomic Southern blot analysis. Murine high molecular weight DNA (15 ,g) was digested with BamHI (lane B), HindIlI (lane H), Pst I (lane P), and Xba I (lane X), fractionated, and probed. The numbers at the right represent DNA size markers in kb. (B) Northern blot analysis of total cellular RNA from murine peritoneal macrophages. Total cellular RNA was isolated from murine peritoneal macrophages that were exposed for 3 hr to LPS (100 ng/ml; lane L) or no stimulus (lane C). The total cellular RNA (15 Jg per lane) was fractionated, transferred to nitrocellulose, and hybridized as described above. 28S and 18S show the positions of these rRNA species on the nitrocellulose blot. murGPDH denotes the standardization of the blot by hybridizing the stripped blot with 32P-labeled probe generated from murine glyceraldehyde-3-phosphate dehydrogenase DNA. (C) A time course analysis of MARCKS mRNA induction in murine peritoneal macrophages by LPS. Murine peritoneal macrophages were exposed to LPS (100 ng/ml) for the indicated times or no stimulus. Total cellular RNA was isolated, processed, and hybridized. The relative amounts of MARCKS mRNA were determined by autoradiogram laser densitometry.

shown). All mouse tissues examined expressed three species of MARCKS mRNA of -2.1, -2.6, and -4.4 kb, as shown for macrophages in Fig. 4B. Differential polyadenylylation can account for the 2.1-kb and 2.6-kb mRNA species. This is inferred from the observation that clone MM3 utilized the most 5' polyadenylylation signal and the observation that clone MM1 utilized a polyadenylylation signal 614 nucleotides 3' to that used by clone MM3. Previous evidence from bovine cDNA clones suggests that the most plausible explanation for the 4.4-kb MARCKS mRNA species is the presence of a 1794-bp intron combined with the utilization of the most 3' polyadenylylation signal (10). LPS Induces MARCKS mRNA in Macrophages. Treatment of murine peritoneal macrophages with LPS at 100 ng/ml resulted in an 8-fold increase in MARCKS mRNA within 1 hr (Fig. 4C). After 3 hr of exposure to LPS, MARCKS mRNA had increased 29-fold relative to control cells (Fig. 4 B and C) and, after 5 hr of exposure to LPS, the levels of MARCKS mRNA were still 23-fold greater than the levels in control cells. At the time points examined, LPS induced all three

DISCUSSION MARCKS is a specific PKC substrate that is phosphorylated during macrophage and neutrophil activation, neurosecretion, A

B

h.

5-,

-7

::

C,-

ps r.

FIG. 5. (A) Two-dimensional electrophoresis of murine MARCKS protein immunoprecipitated from [3H]myristic acidlabeled TK-LM7 cells. The pH of the isoelectric-focusing dimension is designated at the top. The molecular weight standards of the SDS/PAGE dimension are shown by the numbers at the right. (B) As in A except using murine peritoneal macrophages that were exposed to LPS (100 ng/ml) for 3 hr. (C) Treatment of TK-LM7 cells with phorbol ester induces the phosphorylation of the transfected murine MARCKS protein. TK-LM7 cells were labeled with 32P and exposed to 100 nM phorbol 12-myristate 13-acetate (PMA) for 15 min. Labeled murine MARCKS protein was immunoprecipitated from control cell lysates (lane Con) and PMA-treated cell lysates (lane PMA) containing equal amounts of protein and then subjected to Laemmli SDS/PAGE. The number on the right indicates the apparent molecular mass (kDa) of the murine MARCKS protein. (D) Phosphopeptide maps after limited proteolysis with S. aureus V8 of murine MARCKS protein immunoprecipitated from TK-LM7 (lane S7) cells and murine peritoneal macrophages (lane M) previously labeled with 32P and exposed to 100 ,uM PMA for 15 min. The numbers on the right indicate the positions of molecular mass (kDa) standards.

Biochemistry: Seykora et al. and growth factor-dependent mitogenesis (2-8). Although the precise function of MARCKS remains to be elucidated in these diverse systems, it is known to be a calmodulin-binding protein (12, 13) that in macrophages appears to have a role in reversibly linking the actin cytoskeleton to the plasma membrane (ref. 14; M. Thelen et al., unpublished data; J. Hartwig et al., unpublished data). We report the molecular characterization of the cDNA encoding the murine macrophage MARCKS protein. The fulllength gene encodes a 309-amino acid protein that is alanine-rich and has a calculated molecular mass of 29.661 kDa and a theoretical pI of 4.1. The calculated pI value is identical to that obtained by isoelectric focusing of MARCKS purified from mouse brain and that of the protein immunoprecipitated from murine macrophages (16). On the other hand, the calculated molecular mass of 29.7 kDa is at variance with the apparent molecular mass of 68 kDa obtained from SDS/PAGE analysis (7). However, transfection of the complete coding region produces a protein that migrates with an apparent molecular mass of 68 kDa on SDS/PAGE (Fig. 5). The anomolous migration of MARCKS in SDS/PAGE, which results in the discrepancy between the actual and apparent molecular mass of the protein, is attributable to its large Stoke's radius and to its rod-shaped dimensions (ref. 3; J. Hartwig etal., unpublished data). It has also been found that the cDNAs for bovine brain and chicken brain MARCKS encode proteins of 31.9 and 28.7 kDa, respectively, that migrate on SDS/PAGE with apparent molecular masses of 87 and 67 kDa (10, 11). The coding region of the murine MARCKS gene exhibits a highly repetitive structure that is manifested in the protein as short amino acid motifs that are repeated numerous times throughout the molecule (Fig. 2). For example the element Pro-Ala-Ala4Ala) is repeated seven times in the molecule and the element (Ala)-Ala-Ala-Pro is repeated three times (Fig. 1). These repetitive motifs are reminiscent of highly structured linear molecules such as collagen (26) and analysis of the protein's secondary structure by the method of Garnier et al. (25) suggests that 76% of the molecule is an extended helical structure. This is consistent with our rotary shadowing data that define MARCKS as a rod-shaped molecule with the dimensions of 33 nm x 2.5 nm (J. Hartwig et al., unpublished data). MARCKS is a calmodulin and actin binding protein whose attachment to the substrate-adherent plasma membrane is regulated by PKC (refs. 13 and 14; M. Thelen et al., unpublished data; J. Hartwig et al., unpublished data). As such it represents an ideal candidate molecule through which PKC might regulate the reversible association of the actin cytoskeleton with the substrate adherent plasma membrane. Comparison of the primary sequences of the murine, bovine, and chicken MARCKS reveals that the N-terminal and the phosphorylation domains are highly conserved whereas the remainder of protein is divergent. It is therefore reasonable to predict that these two conserved domains are important for the basic function of the MARCKS molecule. This appears to be the case. The N-terminal domain contains a myristoylation consensus sequence (18) that is necessary but not sufficient for membrane attachment of the protein (ref. 27; M. Thelen et al., unpublished data). The conserved domain spanning amino acids 128-180 of murine MARCKS contains all the known phosphorylation sites of the protein (Ser-152, -156, and -163) as well as the calmodulin (12) and actin binding sites (J. Hartwig et al., unpublished data). The predicted secondary structure of the nonconserved regions of MARCKS is primarily helical, which is further reinforced by the rod-shaped dimensions of the protein. It is therefore likely that the functional domain organization of MARCKS consists of a membrane-binding N-terminal region that is separated from the calmodulin and actin binding phosphorylation domain by an extended helical region. Southern blot analysis of murine genomic DNA reveals that the MARCKS gene is most likely present at a single copy per haploid

Proc. Natl. Acad. Sci. USA 88 (1991)

2509

genome and has a simple gene structure. However, under conditions of low stringency, multiple bands on Southern blot analysis are detected (data not shown), suggesting the possibility of other sequences that bear a moderate degree of homology to the MARCKS gene and implying the possibility of a family of related proteins. The steady-state level of MARCKS mRNA in murine macrophages is increased >20-fold by prior exposure of the cells to LPS. This is consistent with our previous observation which demonstrated that the MARCKS protein is strongly induced by LPS in murine macrophages and in human neutrophils (7, 8). In addition, MARCKS is also induced by tumor necrosis factor a in human neutrophils, where it constitutes 90o of all protein synthesized in response to this cytokine (8). Both LPS and tumor necrosis factor a are important immunomodulatory molecules that prime phagocytes for markedly increased PKC-dependent responses without themselves activating the kinase (for review, see ref. 28). Our data suggest that these immunomodulators potentiate PKC-dependent signaling pathways by inducing the specific PKC substrate MARCKS. We thank Karen Keenan and Amalia Pavlovec for excellent technical assistance. We thank A. Nairn for helpful discussions. J.T.S. is a recipient of the Merck M.D.-Ph.D. Fellowship. A.A. is an Established Investigator of the American Heart Association. This work was supported in part by National Institutes of Health Grant Al 25032 (A.A.) and National Institutes of Health grants awarded to J.V.R. 1. Farago, A. & Nishizuka, Y. (1990) FEBS Lett. 268, 350-354. 2. Wu, W. S., Walaas, S. I., Nairn, A. C. & Greengard, P. (1982) Proc. Natl. Acad. Sci. USA 79, 5249-5253. 3. Albert, K. A., Nairn, A. C. & Greengard, P. (1987) Proc. Natl. Acad. Sci. USA 84, 7046-7050. 4. Kligman, D. & Patel, J. (1986) J. Neurochem. 47, 298-303. 5. Rozengurt, E., Rodriguez-Pena, M. & Smith, K. A. (1983) Proc. Natl. Acad. Sci. USA 80, 7244-7248. 6. Blackshear, P. J., Wen, L., Glynn, B. P. & Witters, L. A.-(1986)J. Biol. Chem. 261, 1459-1469. 7. Aderem, A. A., Albert, K. A., Keum, M. M., Wang, J. K. T., Greengard, P. & Cohn, Z. A. (1988) Nature (London) 332, 362-364. 8. Thelen, M., Rosen, A., Nairn, A. C. & Aderem, A. (1990) Proc. Natl. Acad. Sci. USA 87, 5603-5607. 9. Patel, J. & Kligman, D. (1987) J. Biol. Chem. 262, 16686-16691. 10. Stumpo, D. J., Graff, J. M., Albert, K. A., Greengard, P. & Blackshear, P. J. (1989) Proc. Natl. Acad. Sci. USA 86, 4012-4016. 11. Graff, J. M., Stumpo, D. J. & Blackshear, P. J. (1989) Mol. Endocrinol. 3, 1903-1906. 12. Graff, J. M., Young, T. M., Johnson, J. D. & Blackshear, P. J. (1989) J. Biol. Chem. 264, 21818-21823. 13. McIlroy, B. K., Walters, J. D., Blackshear, P. J. & Johnson, J. D. (1991) J. Biol. Chem. 266, 4959-4966. 14. Rosen, A., Keenan, K. F., Thelen, M., Nairn, A. C. & Aderem, A. A. (1990) J. Exp. Med. 172, 1211-1215. 15. Aderem, A. A., Cohen, D. S., Wright, S. D. & Cohn, Z. A. (1986) J. Exp. Med. 164, 165-179. 16. Rosen, A., Nairn, A. C., Greengard, P., Cohn, Z. A. & Aderem, A. A. (1989) J. Biol. Chem. 264, 9118-9121. 17. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 18. Towler, D. A., Gordon, J. I., Adams, S. P. & Glaser, L. (1988) Annu. Rev. Biochem. 57, 69-99. 19. Fawcett, T. W. & Bartlett, S. G. (1990) BioTechniques 9, 46-48. 20. Stumpo, D. J., Graff, J. M., Albert, K. A., Greengard, P. & Blackshear, P. J. (1989) Nucleic Acids Res. 17, 3987-3988. 21. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 22. Lehrach, H., Diamond, D., Wozney, J. M. & Boedtker, H. (1977) Biochemistry 16, 4743-4751. 23. Aderem, A. A., Keum, M. M., Pure, E. & Cohn, Z. A. (1986) Proc. Natl. Acad. Sci. USA 83, 5817-5821. 24. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448. 25. Garnier, J., Osguthorpe, D. J. & Robson, B. (1978) J. Mol. Biol. 120, 97-120. 26. Prockop, D. J. (1990) J. Biol. Chem. 265, 15349-15352. 27. Graff, J. M., Gordon, J. I. & Blackshear, P. J. (1989) Science 246, 503-506. 28. Aderem, A. (1991) in Current Topics in Microbiology and Immunology, eds. Russel, S. W. & Gordon, S. (Springer, New York), in press.