BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and...

BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by itsN-terminal myristoylation and palmitoylation, and interacts with a,but not b, subunit of Ca2+/calmodulin-dependent protein kinase II

Xin Wang,* Qing-Bao Tian,* Akira Okano,* Hiroyuki Sakagami,� Il Soo Moon,� Hisatake Kondo,�Shogo Endo§ and Tatsuo Suzuki*

*Department of Neuroplasticity, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Asahi,

Matsumoto, Japan

�Division of Histology, Department of Cell Biology, Graduate School of Medicine, Tohoku University, Sendai, Miyagi, Japan

�Department of Anatomy and Medical Institute of Dongguk University, College of Medicine, Dongguk University, Gyeongju, Korea

§Unit for Molecular Neurobiology of Learning and Memory, Initial Research Project, Okinawa Institute of Science and Technology

(OIST), JST, Gushikawa, Okinawa, Japan

Abstract

We cloned a rat BAALC 1-6-8 isoform cDNA (GenBank�Accession No. AB073318) that encoded a 22-kDa protein, and

identified endogenous BAALC 1-6-8 protein in the brain. The

gene was expressed widely in the frontal part of the brain, and

the protein was localized to the synaptic sites and was

increased in parallel with synaptogenesis. The protein inter-

acted with the a, but not b, subunit of Ca2+/calmodulin-

dependent protein kinase II (CaMKIIa). The interaction

occurred between the N-terminal 35-amino-acid region of

BAALC 1-6-8 protein and the C-terminal end of the regulatory

domain of CaMKIIa, which contains a isoform-specific

sequence. Thus, the interaction may be CaMKIIa-specific. We

also found that BAALC 1-6-8 protein, as well as CaMKIIa, was

localized to lipid rafts and that both myristoylation and palmi-

toylation of BAALC 1-6-8 N-terminal portion were required for

targeting of the protein into lipid rafts. These findings suggest

that BAALC 1-6-8 protein play a synaptic role at the postsy-

naptic lipid raft possibly through interaction with CaMKIIa.

Keywords: brain and acute leukemia cytoplasmic protein,

Ca2+/ calmodulin-dependent protein kinase II, myristoylation,

palmitoylation, postsynaptic density, postsynaptic lipid raft.

J. Neurochem. (2005) 92, 647–659.

BAALC (brain and acute leukemia, cytoplasmic) protein(Tanner et al. 2001) is a protein almost exclusivelyexpressed in neuroectoderm-derived tissues, hematopoieticprogenitor cells and acute leukemia, and is a marker ofhuman hematopoietic progenitor cells (Tanner et al. 2001;Baldus et al. 2003). The BAALC gene is localized onchromosome 8 in humans and has eight kinds ofalternatively spliced transcripts, all of which share N-terminal exon 1. An isoform consisting of exons 1, 6 and8, and one consisting of exons 1 and 8 (BAALC 1-6-8and BAALC 1-8, respectively) are expressed in the brain,whereas 1-2-6-8 and 1-5-6-8, as well as 1-6-8, isoformsare expressed in leukemic cells. BAALC is highlyconserved among mammals and zebra fish but absentfrom lower organisms. The expression of BAALC gene inacute leukemia is associated with an adverse prognosis ofthe disease.

Recently, we developed a method to identify a largenumber of mRNA species associated with the postsynapticdensity (PSD) fraction (Tian et al. 1999, 2003; Inaba et al.

Received May 27, 2004; revised manuscript received September 17,2004; accepted September 29, 2004.Address correspondence and reprint requests to Tatsuo Suzuki,

Department of Neuroplasticity, Institute on Aging and Adaptation,Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsu-moto 390–8621, Japan. E-mail: [email protected] used: CaMKII, Ca2+/calmodulin-dependent protein

kinase II; Dem, a molecule whose mRNA is localized in the dendrite;EGFP, enhanced green fluorescent protein; 1-6-8-EGFP, BAALC 1-6-8tagged with EGFP at its C-terminus; GST, glutathione S-transferase;ORF, open reading frame; PCR, polymerase chain reaction; PSD,postsynaptic density; RACE, rapid amplification of cDNA ends;RT–PCR, reverse transcriptase-based PCR; SPM, synaptic plasmamembrane; TGN, trans-Golgi network.

Journal of Neurochemistry, 2005, 92, 647–659 doi:10.1111/j.1471-4159.2004.02902.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 647–659 647

2004). During the identification of these dendriticallylocalizing mRNAs (Dems), we cloned a number of cDNAsincluding Dem A20-4. In the present study, we further clonedfull-length Dem A20-4 cDNA and characterized its geneproduct, because an initial database search of Dem A20-4detected no homologous protein. After the cloning of a full-length Dem A20-4 cDNA from a rat brain cDNA library(GenBank� Accession No. AB073318, registered on Octo-ber 24, 2001) we found that the encoded protein is anortholog of human BAALC 1-6-8 isoform protein (Gen-Bank� Accession No. NM024812). The BAALC 1-6-8clone of rat was registered in GenBank� (Accession No.NM144762) later on December 27, 2001 by the same groupthat found the human BAALC clone. Information aboutfunctional aspects of the BAALC protein in the brain has notbeen reported until now.

In this report we describe the properties of the BAALC1-6-8 protein in the rat brain, showing that the protein waslocalized to lipid rafts in the synaptic region and bound to theregulatory region of the a-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIa) through theN-terminal 35 amino acid residues.

Experimental procedures

Materials

pGEM-T easy vector was purchased from Promega Corporation

(Madison, WI, USA); ISOGEN was from Nippon gene KK. (Tokyo,

Japan); the DNA sequencing reagent kit was from Perkin-Elmer

Corporation (Urayasu, Japan); the 5¢/3¢-rapid amplification of cDNA

ends (RACE) kit and glycogen were from Boehringer Mannheim

Biochemica GmbH (Tokyo, Japan); DNase I, pQE-30 expression

vector and anti-tetraHis antibody (anti-His4) were from Qiagen

(Tokyo, Japan); Taq DNA polymerase and RNA polymerase chain

reaction (PCR) kit (AMV) Version 2.1 were from Takara Co. Ltd.

(Ohtsu, Japan); the RNA size marker kit was from Novagen

(Madison, WI, USA); the multi-tissue northern blot (7764–1) and

anti-flotillin-1 antibody (610821) were from BD Biosciences (Palo

Alto, CA, USA); pEGFP-N1 vector and ExpressHyb solution were

from Clontech (Cambridge, UK); pGEX-4T-1 was from Amersham

Biosciences (Tokyo, Japan); mouse monoclonal anti-CaMKII anti-

body (MAB3119; clone name, 6G9) was from Chemicon (Temec-

ulla, CA, USA); anti-CaMKIIb antibody was from Life Technologies

(Gaithersburg, MD, USA); protease inhibitor cocktail and anti-rabbit

IgG-agarose (A-1027) were from Sigma Chemical Company (St.

Louis, MO, USA); horseradish peroxidase-coupled goat anti-rabbit

immunoglobulin IgG (H & L) was from Calbiochem (San Diego,

CA, USA); horseradish peroxidase-coupled goat anti-mouse Ig (G

+A + M) was from Cappel (West Chester, PA, USA); anti-rabbit IgG

(H + L) labeled with Alexa Fluor 488 (A-11034) and anti-mouse IgG

(H + L) labeled with Alexa Fluor 488 (A-11029) or 568 (A-11004)

were from Molecular Probes (Eugene, OR, USA); SuperSignal,

SuperSignal ULTRA, and SuperSignal BLAZE, chemiluminescent

detection reagents for western blotting were from Pierce (Rockford,

IL, USA); 6-well tissue culture plates (No. 657–160) were

from Greiner Bio-One Co. Ltd. (Germany); and anti-His antibody

(anti-HisG), pcDNA4/HisMax, lipofectamine 2000, Neurobasal and

B27 supplement were from Invitrogen Life Technologies (Carlsbad,

CA, USA). All other chemicals were of reagent grade.

Cloning of rat BAALC 1-6-8

Dem A20-4 double-stranded cDNA (110 bp in length) was obtained

from randomly amplified cDNAs prepared from mRNAs contained

in the PSD fraction (Tian et al. 1999). Antisense oligonucleotide A1(5¢-CACGGGGCTTGCTTTTGTTT-3¢, nt 2356–2337 of the final

sequence) was designed based on the sequence of the original

110-bp fragment of Dem A20-4. First, PCR was performed using a

cerebral cortex library of rat as the template, A1 and M13 reverse

primer of Lambda ZAP II vector as primers, and subsequently,

nested PCR produced a 1087-bp cDNA fragment. The Dem A20-4

sequence was extended by repeated 5¢-RACE. The last extended

sequence contained an in-frame ATG codon preceded by an

in-frame stop codon. Finally, we obtained a merged clone of

2369 bp. The nucleotide sequence was determined by the dideoxy

chain termination method with a DNA sequencer (model 370 A;

Perkin Elmer).

mRNA isolation, reverse transcriptase-based polymerase chain

reaction and northern blotting

Total RNA was isolated from the rat forebrain using ISOGEN

according to the manufacturer’s instructions. The RNA was

incubated with DNase I at 37�C for 30 min and treated with

ISOGEN again. RT–PCR essentially followed the protocol of the

RNA PCR kit (AMV) Version 2.1. One microgram of total RNA

was used for the RT–PCR, and cDNA was produced at 42�C for

30 min. The PCR reaction was carried out using 35 cycles of 30 s at

94�C, 30 s at 53�C and 2.5 min at 72�C. The PCR products were

separated by electrophoresis using a 1% agarose gel and visualized

by staining with ethidium bromide.

For northern blotting, a 536-bp cDNA that contained an open

reading frame (ORF) sequence was amplified by PCR using S2

and A7 primers, and was subcloned into pGEM-T easy vector.

The entire EcoRI fragment of this ORF was obtained from the

resultant plasmid, labeled with 32P using random hexamer primer,

and hybridized to a membrane blotted with 2 lg of mRNAs

prepared from the rat brain (Clontech, #7764-1) in ExpressHyb

solution at 57�C for 4 h. The blot was rinsed in 2 · saline sodium

citrate buffer containing 0.05% sodium dodecyl sulfate at room

temperature four times, washed with 0.1 · saline sodium citrate

buffer containing 0.1% sodium dodecyl sulfate at 62�C, and

exposed to an imaging plate (Fuji Xerox, Tokyo, Japan)

overnight.

Production of fusion or mutated constructs of BAALC 1-6-8

and Ca2+/calmodulin-dependent protein kinase II

and their expression

BAALC 1-6-8 cDNA encoding amino acid residues 3–145 was

amplified by PCR and then cloned into the pGEX-4T-1 vector to

produce glutathione S-transferase (GST) fusion protein. Following

transformation, Escherichia coli BL21 cells were grown in LB

medium with ampicillin (LBamp) at 25�C, and expression of

GST-BAALC 1-6-8 fusion protein was induced for 3 h in the

presence of 50 lM of isopropyl-1-thio-b-D-galactopyranoside. The

648 X. Wang et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 647–659

expressed proteins were solubilized by sonication and incubation

with Triton X-100 (final 1%) for 30 min at 4�C, and affinity-

purified using glutathione Sepharose 4B.

PCR mutagenesis was performed using wild-type BAALC 1-6-8

cDNA as a template. The wild-type and mutated BAALC 1-6-8

proteins were fused C-terminally with enhanced green fluorescent

protein (EGFP) by cloning in-frame into pEGFP-N1 vector and

expressed in Cos7 cells.

A polypeptide containing the full-length ORF of rat CaMKIIa(GenBank� Accession No. NM012920) was His-tagged at its N-

terminus by incorporation of the coding sequence in-frame into

pcDNA4/HisMax vector and expression in Cos7 cells.

The catalytic domain (aa 1–260), association domain (aa 321–

478), and regulatory domain-containing regions (aa 261–478) of rat

CaMKIIa were His-tagged at their N-termini by incorporation

in-frame into pQE-30 vector and expression in JM109 cells.

Nucleotide sequences of all constructs were checked by sequencing.

Production of antibody

A rabbit was immunized with GST-BAALC 1-6-8 fusion protein

coupled to Sepharose 4B. The antiserum, after removing anti-GST

antibody using GST coupled to Sepharose 4B, was affinity-purified

using GST-BAALC 1-6-8 fusion protein which was chemically

coupled to glutathione Sepharose 4B. The affinity-purified antibody

was used for all the immunological analyses in this study. Rabbit

polyclonal anti-CaMKII antibody (anti-CaML) was produced as

described (Suzuki et al. 1994).

In situ hybridization analysis

In situ hybridization was performed using Wistar rat brain (from 7-

week-old rats) as described previously (Sakagami et al. 1998; Inabaet al. 2004). An antisense oligonucleotide probe corresponding to

the nucleotides 5¢GTCGCTCAGAGGCATCCACGTTCAGT-GAGTGACACCCACTTCTTC3¢ of the cDNA for rat BAALC

1-6-8 (nt 834–790) was labeled with [a-35S]dATP by using terminal

deoxynucleotidyl transferase (Takara, Tokyo, Japan).

Cell culture and immunocytochemical examination

Cos7 cells were cultured in Dulbecco’s modified Eagle’s medium

supplemented with 10% fetal calf serum, 100 U/mL penicillin,

100 lg/mL streptomycin and 1 mM sodium pyruvate and main-

tained at 37�C in humidified air containing 5% CO2.

The hippocampus or cerebral cortex of embryonic day 18 rats

(Wistar) was mechanically dissociated and plated onto a glass slide

coated with polyethylenimine. Neurons were grown essentially as

described previously (Brewer et al. 1993) with Neurobasal medium

supplemented with B27.

Cultured cells were fixed with 4% paraformaldehyde and stained

with either anti-BAALC 1-6-8 (rabbit) or anti-CaMKII (6G9,

mouse) antibodies as described previously (Li et al. 2001).

Immunological signals were detected with anti-rabbit IgG (H + L)

labeled with Alexa Fluor 488 (green) or anti-mouse IgG (H + L)

labeled with Alexa Fluor 568 (red). They were examined and

photomicrographed with a Zeiss Axioskop microscope.

Pull-down assay

Pull-down assays were essentially performed as described previ-

ously (Li et al. 2001). Proteins of synaptic plasma membrane (SPM)

or PSD, or homogenate of JM109 cells in which domains of

CaMKIIa were expressed, was solubilized by boiling for 2 min in

sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample

buffer containing sodium dodecyl sulfate (final 1%) and 2-

mercaptoethanol (final 1.7%), mixed with 5 volumes of dilution

buffer (Li et al. 2001) containing 2% TX-100, and incubated for 2 h

at 4�C. This solubilization protocol is the only one at present that

enable both efficient solubilization of highly insoluble PSD proteins

and subsequent pull-down analysis or immunoprecipitation (Suen

et al. 1998; Li et al. 2001). Supernatant was obtained by centri-

fugation at 15 300 g for 15 min, and incubated at 4�C overnight in

the presence of GST protein fused with various regions of BAALC

1-6-8 chemically coupled to glutathione Sepharose 4B by dimeth-

ylpimelimidate (Brew et al. 1975). The gel was washed four times

by brief centrifugation and aspiration. The pull-down materials were

analyzed by western blotting.

Immunoprecipitation

Cos7 proteins were solubilized at 4�C for 2 h in 20 mM Tris-HCl

(pH 7.4) containing 150 mM NaCl, 1 mM EDTA, 1% TritonX-100,

and then clarified by centrifugation at 100 000 g for 30 min. The

solubilized proteins were incubated at 4�C for 3 h or overnight with

anti-BAALC 1-6-8 antibody or anti-CaML immobilized chemically

by dimethylpimelimidate with protein G plus/protein A-Agarose

(Sisson and Castor 1990). The gel was pelleted and washed three

times, and immunoprecipitated proteins were separated by sodium

dodecyl sulfate–polyacrylamide gel electrophoresis and western

blotted.

Sucrose density gradient flotation assay

For the sucrose density gradient flotation assay, proteins (500 lg) ofthe SPM fraction were suspended by repeated passage through a 26-G

needle in 1.5 mL of TNE buffer (20 mM Tris-HCl, pH 7.4, 150 mM

NaCl, 1 mM EDTA, 200 lM phenylmethylsulfonyl fluoride, 10 lg/mL pepstatin A, 10 lg/mL leupeptin and 10 lg/mL aprotinin)

containing 0.15%TritonX-100, and solubilized on a rocking platform

at 4�C for 30 min. Cos7 cells transfected with various plasmids

(30 lg) and grown for 48 h in 10-cm dishes were scraped into 2 mL

of TNE buffer containing 1% Triton X-100, and solubilized with brief

sonication. The sample was adjusted to 40% sucrose by the addition

of 80% sucrose in TNE buffer, placed at the bottom of a centrifuge

tube and overlaid successively with TNE buffer containing 30%

sucrose and then 5% sucrose, respectively (3.7 mL each). The sample

was then centrifuged at 256 000 g (max) for 30 h at 4�C. Aftercentrifugation, 11 fractions of 1 mL each were collected sequentially

from the top and the pellet was resuspended in 1 mL of TNE buffer.

Proteins in each fraction were precipitated in 10% trichloroacetic acid

solution and resuspended in 200 lL of TNE buffer. Equal volumes of

the fractions were separated and analyzed by western blotting.

Detection of lipid modification on BAALC 1-6-8 protein

Metabolic labeling of BAALC 1-6-8-EGFP with [3H]myristic acid

and [3H]palmitic acid followed essentially the method described

previously (DeSouza et al. 2002; Konno et al. 2002). pEGFP-N1vector inserted with BAALC cDNA (either wild-type or one of two

mutated types) (5 lg/3.5-cm ø dish) was transfected into Cos7 cells

using Lipofectamine 2000. Cells were pre-incubated for 30 min in

serum-free Dulbecco’s modified Eagle’s medium with 3 lg/mL of

BAALC 1-6-8, lipid rafts and CaMKIIa 649


cerulenin and 10 mg/mL of fatty-acid-free bovine serum albumin

48 h after transfection. [3H]Myristic acid or [3H]palmitic acid at 333

or 500 lCi/mL, respectively, was added to the culture medium and

maintained for 5 h at 37�C. Cells were rinsed once with phosphate-

buffered saline and scraped in 1 mL of cold phosphate-buffered

saline, centrifuged and suspended in 1 mL of cold radioimmuno-

precipitation analysis (RIPA) buffer containing 1% NP-40, 0.5%

sodium deoxycholate and 0.1% sodium dodecyl sulfate in

phosphate-buffered saline. Cell extracts were rocked at 4�C for

1 h, and the supernatant obtained after centrifugation at 15 000 g for20 min was immunoprecipitated. The immunoprecipitated materials

were analyzed by western blotting using anti-BAALC 1-6-8

antibody or fluorography using a BAS-TR2040 Imaging plate (Fuji

Film, Japan) and a FUJIFIX Bioimage Analyzer BAS1500.

Other methods

Western blotting was performed according to standard protocols

using the chemiluminescent substrate as described previously

(Murata et al. 2000; Suzuki et al. 2001). Subcellular fractions of

the forebrain, including PSD and postsynaptic lipid raft fractions,

were prepared from Wistar rats (6 weeks old, male) as described

previously (Suzuki et al. 1999; Murata et al. 2000). Immunohist-

ochemical examination at the light microscopic level using brains

from Wistar rats (6 weeks old, male) essentially followed the

protocol described previously (Murata et al. 2000). All preparationswere stored at )80�C until use. Animals were handled in accordance

with the National Institutes of Health Guide for Care and Use ofLaboratory Animals (NIH Publications no. 80–23).

Results

cDNA cloning of BAALC 1-6-8 and sequence analysis

Our initial database search of the 110-bp Dem A20-4 cDNAfailed to identify any homologous protein. Therefore, weelongated the Dem A20-4 cDNA fragment by repeated 5¢-RACE and finally obtained a full-length cDNA. RT–PCRusing primers S1 (5¢-TTAGGGGACCGATGTCGTG-3¢, nt175–193) and A2 (5¢-ACAAGGAGTCAAGGTGAGAG-3¢,nt 2291–2272), which produced a single band of thepredicted size, confirmed the presence of a transcriptcontaining the complete ORF of Dem A20-4 in vivo (notshown). Thus, we judged that the full-length Dem A20-4sequence we obtained represents a real transcript.

The full-length cDNA thus obtained was composed of2369 bp and contained an ORF that encoded a protein of 145amino acids. The calculated molecular mass and pI are15475 Da and 6.87, respectively. The nucleotide sequencesurrounding the predicted ATG start site was confirmed to bethe Kozak consensus for efficient translation, and included a5¢-GC-rich region (Kozak 1991). The 3¢ non-coding regionincluded a polyadenylation signal, AATAAA, and a singleATTTA sequence (Fig. 1), which has been implicated indestabilizing mRNAs (Shaw and Kamen 1986). A BLASTsearch of Dem A20-4 revealed that the Dem A20-4 proteinwas an ortholog of human BAALC 1-6-8 protein. A MOTIF

search using ProDom (http://motif.genome.ad.jp/motif-bin/)revealed that the N-terminus of BAALC 1-6-8 (amino acidresidues 1–52) constitutes a domain with unknown function(PD299808).

Analyses of the BAALC 1-6-8 transcript by reverse

transcriptase-based polymerase chain reaction and

northern blotting

To analyze the expression of the BAALC gene, RT–PCR andnorthern blotting were performed using the specific primers(S3 and A6) shown in Fig. 1. RT–PCR detected a singleband representing BAALC 1-6-8 nearly exclusively in theforebrain, although a very faint band with similar size wasalso detected in the lung (Fig. 2a). Hybridization to 2 lg ofmRNA prepared from the rat brain produced three bands of4.4, 3.0 and 2.5 kb (Fig. 2b). The bands of 3.0 and 2.5 kbwere dense and were also detected, but less densely, in heart.The 4.4-kb band was faint and was not detected in the tissuesother than the brain under the conditions used.

Distribution of BAALC 1-6-8 mRNA in adult rat brain

Expression of the BAALC gene was investigated by in situhybridization using the adult rat brain with an oligonucleotideprobe. In the adult rat brain, BAALC 1-6-8 mRNA waspredominantly expressed in most of forebrain structures,including the cerebral cortex, hippocampal formation, olfac-tory bulb, anterior olfactory nuclei, piriform cortex, tenia tectaand amygdaloid nuclei (Fig. 3). In the cerebral cortex, intenseexpression was detected throughout layers II–VI (Figs 3c andd). In the hippocampal formation, BAALC 1-6-8 mRNAwasexpressed intensely in the dentate granule cells and pyramidalcells throughout the hippocampal CA1–3 regions (Fig. 3d). Inthe olfactory bulb, the mRNAwas expressed intensely in theneuronal layers, including the glomerular, mitral cell andinternal granular layers (Fig. 3b). Regarding the forebrainstructures, no significant expression was detectable in thecaudate putamen or globus pallidus (Fig. 3c). The detailedregional expression of BAALC 1-6-8 is summarized inTable 1. In the control experiment, hybridization in thepresence of a 50-fold excess of unlabeled oligonucleotideprobe resulted in complete attenuation of the hybridizationsignal described above, suggesting the specificity of thepresent in situ hybridization analysis (data not shown).

Analyses of BAALC 1-6-8 protein by western blotting

Before investigation by western blotting, we checked theaffinity-purified anti-BAALC 1-6-8 antibody raised by usingGST-BAALC 1-6-8 fusion protein as an antigen. Theaffinity-purified antibody reacted with the 46-kDa GST-BAALC 1-6-8 fusion protein but not with GST protein(Fig. 4a). The antibody also reacted with BAALC 1-6-8protein expressed in Cos7 cells (not shown). Thus, theaffinity-purified antibody specifically recognized the BA-ALC 1-6-8 protein.

650 X. Wang et al.


The tissue distribution of the BAALC 1-6-8 protein wasexamined by using this affinity-purified antibody (Fig. 4b).The antibody reacted with a single band in the brainhomogenate with a size (22 kDa) similar to the predictedone, and this band was not detected in the presence of 50 lgof GST-BAALC 1-6-8 protein (not shown). Therefore, weconcluded that the 22-kDa band detected in the brain is theendogenous BAALC 1-6-8 protein. The 22-kDa BAALC1-6-8 protein was distributed specifically in the brain, amongthe tissues tested (Fig. 4b).

Because BAALC 1-6-8 mRNAwas derived from the PSDfraction-associated mRNAs, its products were expected to belocalized to postsynaptic areas. Thus, we examined thelocalization of BAALC 1-6-8 protein by western blottingusing an affinity-purified anti-BAALC 1-6-8 antibody andvarious subcellular fractions prepared from the rat forebrain.A single BAALC 1-6-8-immunoreactive 22-kDa band wasdetected in P2, synaptosome, SPM, and PSD fractions, as wellas brain homogenate, but not in the soluble fraction (Fig. 4c).BAALC 1-6-8 protein was highly concentrated in synapto-some and SPM fractions, although the amount of the proteinexpressed was very low (about 0.01% of the total SPMprotein) according to the quantification by western blotting.

The developmental profile of BAALC 1-6-8 protein wasexamined in rat forebrain homogenate (Fig. 4d). BAALC

1-6-8 protein was barely detected at postnatal day 1, slightlydetected at 1 week after birth, and increased dramaticallyduring 2 and 3 weeks after birth. The protein level wasslightly decreased at 6 weeks after birth. This developmentalprofile was in parallel with that of synaptogenesis in theforebrain (Kelly and Cotman 1981; Suzuki et al. 1997) andsimilar to those of several other PSD-enriched proteins, suchas PSD-95 (Cho et al. 1992), N-methyl-D-aspartic acid-typeglutamate receptors 1 and 2A (Zhong et al. 1995) andSAP97 (Muller et al. 1995). This suggests a role for BAALC1-6-8 protein in the synaptogenesis.

Distribution of BAALC 1-6-8 protein in the rat brain

The localization of the BAALC 1-6-8 protein was examinedimmunohistochemically in the rat cerebral cortex and CA1region of the hippocampus using anti-BAALC 1-6-8antibody (Fig. 5). In the neocortex, most pyramidal cells,as well as small cells, were positive for anti-BAALC 1-6-8antibody. Cells with a glia cell-like morphology were notstained. Nuclei appeared to be negative for the staining.Dendrites as well as somas of large pyramidal neurons inthe cortex were stained (Fig. 5a), whereas staining ofdendrites of the pyramidal neurons in the hippocampus wasnot clearly evident (Fig. 5b). The validity of immunohist-ochemical staining with affinity-purified anti-BAALC 1-6-8

Fig. 1 Nucleotide sequence and deduced

amino acid sequence of rat BAALC 1-6-8.

The original Dem A20-4 is a 110-bp

sequence at the 3¢ tail. Positions of various

primers (S1, S2, S3, A1, A2, A6 and A7)

and a probe for in situ hybridization (ISH)

are indicated by arrows. Polyadenylation

and mRNA instability signals (AATAAA and

ATTTA, respectively) are underlined. The

thick underline indicates exon 6, and the

regions 5¢ and 3¢ of it are exon 1 and exon

8, respectively. The double underline shows

the amino acid sequence of BAALC 1-8

isoform protein. The numbers of nucleotide

and amino acids are shown on both sides of

the sequence.



antibody was confirmed by the complete elimination ofstaining when tissues were treated without the primaryantibody (Fig. 5c).

Identification of BAALC 1-6-8-interacting protein

We tested interaction of BAALC 1-6-8 protein with CaMKIIby a pull-down assay using immobilized GST-BAALC 1-6-8fusion protein because we routinely screen for proteinsinteracting with CaMKII (Fig. 6). The results showed thatthe a, but not b, subunit of CaMKII was pulled down fromthe SPM and PSD proteins.

The interaction ofBAALC1-6-8 proteinwithCaMKIIawasconfirmed by co-immunoprecipitation using Cos7 cells thatco-expressed BAALC 1-6-8 and His-tagged CaMKIIa (His-CaMKIIa). The anti-BAALC 1-6-8 antibody co-immuno-precipitated His-CaMKIIa and, conversely, anti-CaMKIIantibody co-immunoprecipitated BAALC 1-6-8 (Fig. 7).

The association of BAALC 1-6-8 protein with CaMKIIawas also examined by cytochemical methods using culturedcortical neurons. BAALC 1-6-8 protein was localized inneurons, in both somas and dendrites, as also shown forCaMKIIa (Fig. 8a), and the two proteins were co-localized,

although only partly, in the cortical neuronal processes. Theco-localization was also examined using Cos7 cells co-expressing BAALC 1-6-8 tagged with EGFP at its C-terminus (1-6-8-EGFP) and His-CaMKIIa (Figs 8b and c).1-6-8-EGFP was distributed in cytoplasmic aggregates andthe plasma membrane. EGFP alone was distributed dif-fusely throughout the cytoplasm (Fig. 8b). His-CaMKIIawas distributed in the cytoplasmic region and plasmamembrane. These two proteins appeared to be co-localized,at least partly, both in the plasma membrane and cytoplas-mic region, as seen by fluorescent microscopic observation(Fig. 8b). This was confirmed by observation using confo-cal microscopy (Fig. 8c). The morphology of the 1-6-8-EGFP-positive aggregates in the cytoplasmic region sug-gested that the structure was the trans-Golgi network (TGN)(Humphrey et al. 1993). We tested this possibility byexamining the co-localization of 1-6-8-EGFP and TGN38.The results showed that 1-6-8-EGFP in the cytoplasmicregion showed overlapping distribution, although partly,with TGN38 (Fig. 8d). Localization of BAALC 1-6-8 in the

Fig. 3 Distribution of the mRNA for BAALC 1-6-8 in adult rat brain

examined by in situ hybridization analysis. (a) A dark-field emulsion

autoradiogram of a sagittal section, showing predominant expression

of BAALC 1-6-8 in the forebrain structures, including the cerebral

cortex (Cx), hippocampal formation (Hip), olfactory bulb (OB) and

anterior olfactory nuclei (AON). (b–d) Dark-field photographs of cor-

onal sections of the olfactory bulb (b), striatum (c) and hippocampal

formation (d). Acc, nucleus accumbens; Amy, amygdaloid nuclei;

CA1–3, fields of CA1–3 of Ammon’s horn; Cb, cerebellar cortex; cc,

corpus callosum; CP, caudate putamen; Cx, cerebral cortex; DG,

dentate gyrus; EPL, external plexiform layer; GL, glomerular layer; IC,

inferior colliculus; IGL, internal granular layer; Mi, mitral cell layer; MO,

medulla oblongata; OT, olfactory tubercle; Pi, piriform cortex; SC,

superior colliculus; Th, thalamus; TT, tenia tecta; VMH, ventromedial

hypothalamic nucleus; ZI, zona incerta. Scale bars: 100 lm in b; 1 mm

in a, c, d.

(a)

(b)

Fig. 2 Analyses of BAALC 1-6-8 mRNA expression. Expression of

BAALC 1-6-8 transcripts in various tissues of rat was examined by

RT–PCR (a) and northern blotting (b). Total RNAs prepared from the

rat forebrain were used for RT–PCR. S3 and A6 primers (see Fig. 1)

were used. A membrane blotted with size-fractionated poly(A)+ RNA

prepared from various rat tissues (2 lg of mRNA per lane, Clontech)

was used for northern blotting. G3PDH refers to glyceraldehyde 3-

phosphate dehydrogenase. FB, He, Br, Sp, Lu, Li, SM, Ki and Te refer

to forebrain, heart, brain, spleen, lung, liver, skeletal muscle, kidney

and testis, respectively.

652 X. Wang et al.


TGN is in good agreement with the report that TGNcontains a lipid raft-like membrane domain (Gkantiragaset al. 2001) (see also the section about raft localization ofBAALC 1-6-8 protein).

Determination of binding regions

The binding site for CaMKIIa in BAALC 1-6-8 protein wasidentified by pull-down assays using GST-fusion proteinswith various domains of the BAALC 1-6-8 protein. Theresults showed that the minimum region required for thebinding was amino acid residues 3–35 from the N-terminus(Fig. 9).

Next, we searched for the binding sites for BAALC 1-6-8protein in CaMKIIa. Five fragments of CaMKIIa: catalyticdomain at the N-terminus (aa 1–260), the association domainin the C-terminal region (aa 321–478), and the fragments

Table 1 Summary of the expression of BAALC 1-6-8-mRNA in adult

brain

Brain structure BAALC 1-6-8 mRNA

Olfactory bulb

Glomerular layer +++

Mitral cell layer +++

Internal granular layer +++

Anterior olfactory nuclei +++

Olfactory tubercle +/–

Tenia tecta +++

Neocortex +++

Piriform cortex +++

Hippocampal formation

Pyramidal cell layers CA1–3 +++

Dentate granule cell layer +++

Basal ganglia

Caudate putamen –

Globus pallidus –

Nucleus accumbens + � +/–

Septum +

Amygdaloid nuclei +++

Epithalamus

Medial habenular nucleus +

Lateral habenular nucleus –

Thalamic nuclei ++

Zona incerta +

Hypothalamus + � ++

Midbrain

Central gray +

Superior colliculus +

Inferior colliculus +

Substantia nigra pars compacta +

Pons

Pontine nuclei +

Dorsal tegmental nucleus +

Locus coeruleus +

Lateral parabrachial nucleus +

Medulla oblongata +

Nucleus of solitary tract +

Cerebellum

Molecular layer –

Purkinje cell layer –

Granule cell layer + � +/–

Deep cerebellar nuclei –

Spinal cord

Ventral horn + � +/–

Dorsal horn +

Expression levels of BAALC 1-6-8 mRNA were evaluated in emulsion

autoradiogram as follows: –, background; +, low; ++, moderate; +++,

high.

(a) (b)

(c) (d)

Fig. 4 Western blot analyses of BAALC 1-6-8 protein. (a) Verifica-

tion of the specificity of the anti-BAALC 1-6-8 antibody. Purified GST-

BAALC 1-6-8 fusion protein (G-1-6-8), which was used as an immu-

nogen for antibody production, and GST protein alone (1 lg each)

were western blotted with anti-BAALC 1-6-8 antibody. CBB and WB

refer to Coomassie Brilliant Blue R-250 staining and western blotting,

respectively. (b) Tissue distribution of BAALC 1-6-8 protein. Proteins

from various tissues (100 lg each) were separated by sodium dodecyl

sulfate–polyacrylamide gel electrophoresis and western blotted using

anti-BAALC 1-6-8 antibody. He, FB, Sp, Lu, Li, Mu, Ki, Te, Th, St, SI

and Pa refer to heart, forebrain, spleen, lung, liver, muscle, kidney,

testis, thymus, stomach, small intestine and pancreas, respectively.

(c) Subcellular distribution of BAALC 1-6-8 protein in the rat brain.

Proteins of various subcellular fractions prepared from rat forebrain

(50 lg each) were separated by electrophoresis on a 12% polyacryl-

amide gel and western blotted using anti-BAALC 1-6-8 antibody (1/200

dilution, 4�C overnight). P1 contains nuclei and cell debris, and P2 is a

crude mitochondrial fraction. H, S and Syn refer to forebrain homo-

genate, soluble and synaptosome, respectively. (d) Developmental

changes in BAALC 1-6-8 protein in the forebrain. Proteins (100 lg)

prepared from rats at various ages (1 day through 6 weeks after birth)

were western blotted. Molecular masses are indicated in kDa on the

left.



containing the regulatory domain in the middle (aa 261–478,aa 1–309 and aa 310–478) (Tobimatsu and Fujisawa 1989),each tagged with His at its N-terminus, were expressed inJM109 cells and pulled down by the GST-aa3–35 fragmentof BAALC 1-6-8 protein. We used a fragment that containedthe regulatory domain (aa 1–309, aa 261–320 and aa

310–478), instead of the regulatory domain alone, becausethe regulatory domain alone could not be expressed well forunknown reasons. The results showed that N-terminalresidues 3–35 of BAALC 1-6-8 protein pulled down onlythe regulatory domain-containing fragments (aa 261–478 andaa 310–478) (Fig. 10), which suggests that BAALC 1-6-8protein interacts with the regulatory region of CaMKIIa,especially C-terminal end of the regulatory region (aa 310–320). Interaction with the aa 310–320 region was muchstronger than that with aa 261–320 region, which might bedue to inhibition by the aa 261–309 region. Furthermore, theregion aa 310–320 contains a subunit-specific sequence(Fig. 11a). The peptide with a subunit-specific sequence(SGGKSGG) inhibited the interaction between the BAALC1-6-8 protein and the CaMKIIa (Fig. 11b). Therefore, asubunit-specific sequence may be important for the bindingto BAALC 1-6-8 protein and the interaction may be specificto the a isoform.

Lipid raft localization of BAALC 1-6-8 protein and its

targeting signal

The intracellular localization of the BAALC 1-6-8 proteinwas further investigated by a sucrose density gradientflotation assay of Triton X-100-treated SPM protein(Fig. 12). BAALC 1-6-8 protein was distributed in lipid raftfractions (fractions 5 and 6), soluble fractions (fractions8–11) and the PSD-containing pellet. There might be twopopulations of BAALC 1-6-8 protein: those in lipid raft andin non-raft. Alternatively, the BAALC protein detected in thenon-raft fractions might also reside in lipid raft in vivo butsolubilized with excess Triton X-100, because distributionanalysis used depends on solublization conditions such asconcentration of detergent. Therefore, part of BAALC 1-6-8protein recovered in the soluble fraction may also be derivedfrom lipid raft. Similarly, CaMKIIa was distributed in thelipid raft fractions in addition to the PSD-containing pellet.

Fig. 5 Localization of BAALC 1-6-8 protein in rat brain at the light

microscopic level. Rat brain slices were stained with (a and b) or

without (c) affinity-purified anti-BAALC 1-6-8 antibody. (a, c) Cerebral

cortex. (b) CA1 region of hippocampus. O, Py and R refer to stratum

oriens, stratum pyramidale and stratum radiatum, respectively. Bar,

50 lm.

Fig. 6 Pull-down of CaMKIIa with BAALC 1-6-8 protein. Proteins

(15 lg) of SPM or PSD were solubilized by boiling for 2 min in the

presence of sodium dodecyl sulfate and reducing agent, then mixed

with 5 volumes of Triton X-100-containing buffer, and incubated with

either GST-BAALC 1-6-8 fusion protein or GST immobilized on

glutathione Sepharose 4B (see Methods). The pulled-down proteins

were western blotted with anti-CaMKII (anti-CaML) or anti-CaMKIIb

antibodies. Ten and 7.5 lg of SPM and PSD proteins were loaded

onto the gels, respectively. Molecular weights are shown on the left of

the gel. WB refers to western blotting. Anti-CaML recognizes mainly

CaMKIIa. a and b indicate the positions of CaMKIIa and CaMKIIb,

respectively.

Fig. 7 Co-immunoprecipitation of BAALC 1-6-8 with CaMKIIa. BAALC

1-6-8 and His-CaMKIIa (abbreviated as 1-6-8 and H-CaMKIIa,

respectively) were co-expressed in Cos7 cells. Cos7 proteins were

solubilized in a TNE buffer, clarified by centrifugation at 100 000 g for

30 min, and used for immunoprecipitation with anti-BAALC 1-6-8

antibody or anti-CaML immobilized chemically by dimethylpimelimi-

date to protein G plus/protein A-Agarose. Co-immunoprecipitated

proteins were detected with anti-BAALC 1-6-8 or anti-HisG antibodies.

The immunoprecipitated and control materials were processed sim-

ultaneously under the same conditions. IP refers to immunoprecipi-

tation.

654 X. Wang et al.


The positions of lipid raft fractions were confirmed by thepresence of flotillin-1 and GM1 ganglioside cholera toxin-binding subunit, both of which are lipid raft markers (Suzuki2002; Hering et al. 2003).

Many proteins have specific signals for targeting tospecific sites in the cell. BAALC 1-6-8 protein has candidatesites for N-terminal myristoylation and palmitoylation(Fig. 13a), both of which are potential membrane-targetingsignals (Alland et al. 1994; Shenoy-Scaria et al. 1994;Milligan et al. 1995; Robbins et al. 1995). We confirmed,in the first place, the myristoylation and palmitoylation of theBAALC 1-6-8 protein at the N-terminal region using wild-type and two kinds of mutated proteins, G2A and C3S. Theresults showed that Gly2 and Cys3 were myristoylated andpalmitoylated, respectively (Fig. 13b). The G2A mutantlacked the myristoylation, and C3A mutant lacked palmi-toylation (Fig. 13b). Both myristoylation and palmitoylationmay not occur in the Gly2 mutant, because N-terminalmyristoylation is a prerequisite for the Cys3 palmitoylation.

Next, we investigated the effect of the lipid modificationon the targeting of BAALC 1-6-8 protein to lipid rafts. Wild-type and two types of mutant proteins C-terminally taggedwith EGFP were expressed in Cos7 cells, and the sucrosegradient flotation assay or fluorescent microscopic observa-tion was carried out. The wild-type BAALC 1-6-8 protein(1-6-8-EGFP) was distributed to the lipid raft fractions (Fr. 4and 5 Fig. 13c), whereas neither type of mutant BAALC 1-6-8 protein (G2A-EGFP and C3S-EGFP) was targeted to thelipid raft fractions (Fig. 13c). Both mutant proteins were

localized in the Cos7 cells differently from the wild-typeprotein, being diffusely distributed in the cells (Fig. 13d).These data suggest that both myristoylation and palmitoyla-tion at the N-terminus of the BAALC 1-6-8 protein areimportant for targeting to the lipid raft.

Discussion

We cloned a full-length cDNA encoding rat BAALC 1-6-8protein, demonstrated that this protein is endogenous to thebrain and characterized various properties of the protein. Theprotein was highly selectively expressed in the brain,detected in the synaptic fractions, and localized in bothneuronal somas and dendrites. The BAALC gene wasexpressed widely in the frontal part of the brain, especiallyin the neocortex, hippocampus and olfactory bulb, and theexpression was increased in parallel with synaptogenesis. Wefound that the protein interacted with the a subunit ofCaMKII. The interaction was between the N-terminal33-amino-acid region of the BAALC 1-6-8 protein andC-terminal portion of the regulatory domain of CaMKIIa,which was a isoform-specific region (Fig. 11a). This result,together with the absence of interaction between BAALC1-6-8 protein and CaMKIIb in the PSD fraction (Fig. 6),suggests that the interaction with BAALC 1-6-8 protein isspecific to CaMKIIa. Furthermore, the N-terminal portion ofthe BAALC 1-6-8 protein was both myristoylated andpalmitoylated and the dual modification was required fortargeting of the protein into lipid rafts.

(a) (b)

(c)

(d)

Fig. 8 Subcellular localization of BAALC 1-

6-8 protein and CaMKIIa. Co-localization of

BAALC 1-6-8 protein with CaMKIIa was

examined in cultured cortical neurons

(E18P21) (a) in Cos7 cells co-expressing 1-

6-8-EGFP and His-CaMKIIa (b, c), and in

Cos7 cells expressing 1-6-8-EGFP (d).

Cells were double-stained with anti-BAALC

1-6-8 and CaMKIIa (6G9) antibody, which

were labeled with Alexa Fluor 488 (green)

and Alexa Fluor 568 (red), respectively, in

(a). Cells were stained with CaMKIIa (6G9)

antibody and then labeled with Alexa 568 in

(b). Two examples are shown and cells

expressing EGFP alone are also shown on

the right. Cells were stained with anti-

TGN38 antibody and then labeled with

Alexa 568 in (d). Cells were observed using

fluorescent microscopy (a, b and d) or

confocal microscopy (c). Bars, 20 lm.



The importance and involvement of the N-terminal portionof BAALC 1-6-8 in some functional role have beensuggested because this region is shared by all BAALCisoform proteins (Tanner et al. 2001). However, the func-tional properties of this portion have not been reported untilnow. We found that this region bound to CaMKIIa but not toCaMKIIb. Importantly, the site of interaction with BAALC1-6-8 protein was found to be C-terminal end of theregulatory region of the kinase (aa 310–320), which islocated near a calmodulin-binding site (Schulman and Lou1989) and autophosphorylation sites (Thr286, Ser305 andSer306), and contains another autophosphorylation site(Ser314) (Patton et al. 1990; Hanson and Schulman 1992).Therefore, a regulatory role of BAALC 1-6-8 protein toCaMKII activity could be suggested and the protein may actspecifically on a isoform. The N-terminal region of BAALCprotein may play a role in regulating cell proliferation inacute leukemia via an unknown mechanism involving theregulation of CaMKIIa. Similarly, in the normal brain,

BAALC, via this N-terminal region, may play roles insynaptogenesis during brain development and synapticplasticity in the adult brain, since the protein increaseddrastically during the synaptogenesis period (Fig. 4d).BAALC 1-6-8 protein does not appear to be a CaMKIIsubstrate, as the protein does not contain a consensus motif forCaMKII phosphorylation and our preliminary experimentshowed only slight phosphorylation, if any, of BAALC 1-6-8protein by the CaMKII endogenous to SPM (data not shown).

Another important finding is that the BAALC 1-6-8protein is targeted to the lipid raft, a cholesterol- andsphingolipid-enriched microdomain (Masserini et al. 1999;Simons and Ikonen 1997; Smart et al. 1999), in thepostsynaptic area (Figs 12 and 13). This finding indicatesthat, BAALC protein is distributed not only in the cytosoliccompartment but also in lipid rafts. The presence ofpostsynaptic lipid rafts has been proposed (Suzuki et al.2001; Suzuki 2002). Postsynaptic lipid rafts are believed tobe involved in membrane trafficking, signal processing, orregulation of the actin cytoskeleton (Hering et al. 2003) atpostsynaptic sites (Suzuki et al. 2001; Suzuki 2002).Disruption of lipid rafts leads to depletion of excitatory andinhibitory synapses, loss of dendritic spines, and instabilityof surface AMPA receptors (Hering et al. 2003). Thepostsynaptic lipid raft is proposed to be a major site for

Fig. 9 Identification of the site of interaction with CaMKIIa in BAALC

1-6-8 protein. PSD proteins (15 lg) were pulled down, as described in

the legend to Fig. 6, by GST proteins fused with various portions of

BAALC 1-6-8 protein, and CaMKII was detected by anti-CaML. a and b

indicate the positions of CaMKIIa and b, respectively. The regions of

BAALC 1-6-8 protein used for the pull-down and the binding of CaM-

KIIa are summarized in the lower part of the figure.

Fig. 10 Identification of the region of interaction with BAALC 1-6-8

protein in CaMKIIa. Five different CaMKIIa regions, each of which was

tagged with His (H-CaMKIIa), were expressed in JM109 cells and

pulled down, as described in the legend to Fig. 6, with GST protein

fused with aa 3–35 of BAALC 1-6-8 protein. The pulled down proteins

were western blotted with anti-His4 antibody. The regions of CaMKIIa

used for the pull-down and the binding of residues 3–35 of BAALC 1-6-

8 protein are summarized in the lower part of the figure.

656 X. Wang et al.


postsynaptic signal processing as well as PSD, and possessesa variety of proteins. BAALC 1-6-8 protein turned out to beone of them. We simultaneously confirmed the localization ofCaMKII in the postsynaptic lipid raft in vivo (Fig. 12), whichhas been suggested before (Suzuki et al. 2001). CaMKII hasbeen thought to be localized in two types of postsynaptic

(a)

(b)

Fig. 11 Inhibition of the interaction between BAALC 1-6-8 protein and

CaMKIIa by a peptide with CaMKIIa-specific sequence. (a) Amino acid

sequences of various CaMKII isoforms. Only variable regions are

shown. Amino acid numbers above the sequence are those of a

subunit. Lines are inserted to align the sequences. a subunit-specific

sequence is underlined. (b) Inhibition of pull-down by a peptide with

CaMKIIa-specific sequence. His-tagged CaMKIIa region (aa. 310–

478) was pulled down with GST protein fused with aa 3–35 of BAALC

1-6-8 protein, as described in the legend to Fig. 10, in the presence or

absence of peptide (SGGKSGG, 1 mg/lane).

Fig. 12 Localization of BAALC 1-6-8 protein in the postsynaptic lipid

raft. SPM proteins were subjected to sucrose density gradient cen-

trifugation after treatment with TX-100 (see Methods). Equal volumes

of each fraction obtained after the centrifugation were subjected to

immunoblotting using anti-BAALC 1-6-8, anti-CaML or anti-flotillin-1

antibody. Cholera toxin-binding subunit (CTBS) was detected by dot

blot. Flotillin-1 and CTBS are markers for lipid raft.

(d)

(c)

(b)

(a)

Fig. 13 Lipid modification of BAALC 1-6-8 protein and its effect on the

cellular localization of the protein. (a) N-terminal amino acid sequence

of BAALC 1-6-8 protein. The N-terminal myristoylation consensus

sequence (MGxxxS/T) containing the palmitoylation site (Cys3) is

underlined. (b) Detection of N-terminal myristoylation (C14) and

palmitoylation (C16) of BAALC 1-6-8 protein fused C-terminally with

GFP (1-6-8-EGFP). Wild-type (WT) and mutant (G2A and C3S) 1-6-8-

EGFP proteins were expressed in the presence of 3H-labeled myris-

tylate or palmitate in Cos7 cells, and were immunoprecipitated with

anti-BAALC 1-6-8 antibody (anti-1-6-8). WT and two kinds of mutant

proteins were immunoprecipitated as shown in the leftmost picture.

The middle and rightmost pictures are fluorograms showing 3H-myr-

istoylated and 3H-palmitoylated bands, respectively. IP and WB refer

to immunoprecipitation and western blotting, respectively. (c) Cellular

localization of WT and two types of mutant 1-6-8-EGFP (G2A and

C3S) shown in the sucrose density gradient flotation assay and fol-

lowing detection with anti-BAALC 1-6-8 antibody. Lipid raft fractions

are fractions 4 and 5, as indicated by flotillin-1 distribution. (d) Cellular

localization of the WT and two types of mutant BAALC 1-6-8 protein

fused C-terminally with EGFP expressed in Cos7 cells.



sites: one is anchored to the PSD and SPM, and the other isdistributed in the postsynaptic cytoplasm, where the preciselocalization sites are not known at present, but are recoveredin the soluble fraction after centrifugation. CaMKII in thetwo sites (soluble and insoluble form) is interchangeable byits translocation in both physiological (Shen and Meyer1999; Shen et al. 2000; Gleason et al. 2003) and patholo-gical (Suzuki et al. 1994) conditions. The third site ofCaMKIIa targeting in the postsynaptic region was found tobe the postsynaptic lipid raft, which could be recovered in theTriton X100-insoluble fraction (Fig. 12). The mechanism ofCaMKII targeting to lipid raft and the role of BAALC 1-6-8protein in the lipid rafts are not known at present. It isunlikely that the BAALC protein works as an anchor forCaMKIIa into lipid raft, as CaMKIIa transfected in the Cos7cells was readily localized to the lipid raft even in theabsence of BAALC 1-6-8 protein (data not shown).

The mechanism of targeting of BAALC 1-6-8 protein tothe lipid raft involves N-terminal myristoylation and palmi-toylation. The exact amino acids that are to be modified bythese lipids are predicted to be Gly2 and Cys3 (myristoy-lation and palmitoylation sites, respectively), because there isat the NH2-terminus a two-signal motif (Met-Gly-Cys-) forboth myristoylation and palmitoylation (Fig. 13a), which iswell known in Ga family members and a group of Src-relatedprotein tyrosine kinases (Resh 1994). These sites were shownto be real sites for lipid modification in a labeling experimentusing G2A- and C3S-mutant BAALC 1-6-8 proteins(Fig. 13b). These modifications determined the targeting ofthe protein to specific intracellullar sites, including lipid rafts(Figs 13c and d), and dual modification was required. Theseresults are in good agreement with the following model.Myristoylation of proteins typically occurs at the N-terminusin a co-translational reaction via an amide bond (Wilcoxet al. 1987), whereas palmitoylation occurs post-translation-ally at Cys residue(s) via a thioester bond (Mumby 1997).This ‘two-signal model’ with tandem amide-linked myristateand thioester-linked palmitate is thought to be a targetingsignal to the lipid raft and caveolae (Alland et al. 1994;Shenoy-Scaria et al. 1994; Milligan et al. 1995; Robbinset al. 1995). Furthermore, palmitoylation is a reversible post-translational modification that is dynamically regulated byspecific physiological stimuli, including synaptic activity(Milligan et al. 1995; Ross 1995; Mumby 1997; El-Husseiniet al. 2002). Thus, targeting of BAALC 1-6-8 protein topostsynaptic lipid rafts may be regulated by the palmitoy-lation and de-palmitoylation at Cys3.

The findings reported here suggest that the N-terminalportion of BAALC 1-6-8 protein is functionally important intwo respects: (i) specific interaction with CaMKIIa andpossible control of the actions of CaMKIIa, and (ii)regulation of the intracellular localization of the protein.Interestingly, another isoform, BAALC 1–8, which consistsof only the N-terminal 54-amino acid portion (Fig. 1), is

expressed in the brains of humans, mice and rats (Tanneret al. 2001). A 33-amino-acid peptide highly homologous tothe BAALC N-terminal sequence has also been identified inzebra fish (GenBank� Accession No. CAD87801). The 1-8isoform, a portion of 1-6-8, may regulate the functions of1-6-8 by competing to bind CaMKIIa, and this regulationthrough alternative splicing could be triggered by certainstimuli, such as synaptic activity, as suggested in the case ofHomer (Xiao et al. 2000). Elucidation of the functions of the1-6-8 C-terminal portion awaits future experiments.

In conclusion, the findings in this study suggest theBAALC 1-6-8 protein plays a synaptic role at the postsy-naptic lipid raft by interacting with a subunit of CaMKII.

Acknowledgements

We greatly appreciate Dr N. Usuda, Fujita Health Science

University School of Medicine, Toyoake, Japan, for help with

immunohistochemistry. This research was supported in part by a

Grant-in-Aid for Scientific Research and a Grant-in-Aid for the

National Project of Protein Structure and Functional Analysis from

the Ministry of Education, Culture, Sports, Science & Technology of

Japan, Toyota Physical and Chemical Research Institute, Uehara

Memorial Foundation, and the Naito Foundation.

References

Alland L., Peseckis S. M., Atherton R. E., Berthiaume L. and Resh M. D.(1994) Dual myristylation and palmitylation of Src family memberp59fyn affects subcellular localization. J. Biol. Chem. 269, 16 701–16 705.

Baldus C. D., Tanner S. M., Kusewitt D. F., Liyanarachchi S., Choi C.,Caligiuri M. A., Bloomfield C. D. and de la Chapelle A. (2003)BAALC, a novel marker of human hematopoietic progenitor cells.Exp. Hematol. 31, 1051–1056.

Brew K., Shaper J. H., Olsen K. W., Trayer I. P. and Hill R. L. (1975)Cross-linking of the components of lactose synthetase withdimethylpimelimidate. J. Biol. Chem. 250, 1434–1444.

Brewer G. J., Torricelli J. R., Evege E. K. and Price P. J. (1993) Opti-mized survival of hippocampal neurons in B27-supplementedNeurobasal, a new serum-free medium combination. J. Neurosci.Res. 35, 567–576.

Cho K. O., Hunt C. A. and Kennedy M. B. (1992) The rat brainpostsynaptic density fraction contains a homolog of the Drosophiladiscs-large tumor suppressor protein. Neuron 9, 929–942.

DeSouza S., Fu J., States B. A. and Ziff E. B. (2002) Differentialpalmitoylation directs the AMPA receptor-binding protein ABP tospines or to intracellular clusters. J. Neurosci. 22, 3493–3503.

El-Husseini, Ael-D., Schnel E., Dakoji S. et al. (2002) Synaptic strengthregulated by palmitate cycling on PSD-95. Cell 108, 849–863.

Gkantiragas I., Brugger B., Stuven E., Kaloyanova D., Li X. Y., Lohr K.,Lottspeich F., Wieland and Helms J. B. (2001) Sphingomyelin-enriched microdomains at the Golgi complex. Mol. Biol. Cell 12,1819–1833.

Gleason M. R., Higashijima S., Dallman J., Liu K., Mandel G. andFetcho J. R. (2003) Translocation of CaM kinase II to synaptic sitesin vivo. Nat. Neurosci. 6, 217–218.

Hanson P. I. and Schulman H. (1992) Inhibitory autophosphorylation ofmultifunctional Ca2+/calmodulin-dependent protein kinase

658 X. Wang et al.


analyzed by site-directed mutagenesis. J. Biol. Chem. 267, 17 216–17 224.

Hering H., Lin C.-C. and Sheng M. (2003) Lipid rafts in the maintenanceof synapses, dendritic spines, surface AMPA receptor stability.J. Neurosci. 23, 3262–3271.

Humphrey J. S., Peters P. J., Yuan L. C., Juan S. and Bonifacino J. S.(1993) Localization of TGN38 to the trans-Golgi Network:Involvement of a Cytoplasmic Tyrosine-containing Sequence.J. Cell Biol 120, 1123–1135.

Inaba Y., Tian Q. B., Okano A. et al. (2004) Brain-specific novelguanine nucleotide exchange factor candidate for Arf, synA-rfGEFc, is localized to postsynaptic density. J. Neurochem. 89,1347–1357.

Kelly P. T. and Cotman C. W. (1981) Developmental changes in mor-phology and molecular composition of isolated synaptic junctionalstructures. Brain Res. 206, 251–257.

Konno D., Ko J-A, Usui S. et al.(2002) The postsynaptic density anddendritic raft localization of PSD-Zip70, which contains an N-myristoylation sequence and leucin-zipper motifs. J. Cell Sci. 115,4695–4706.

Kozak M. (1991) Structural features in eukaryotic mRNAs that modulatethe initiation of translation. J. Biol. Chem. 266, 19 867–19 870.

Li W., Okano A., Tian Q. B., Nakayama K., Furihata T., Nawa H. andSuzuki T. (2001) Characterization of a novel synGAP isoform,synGAP-?. J. Biol. Chem. 276, 21 417–21 424.

Masserini M., Palestini P. and Pitto M. (1999) Glycolipid-enrichedcaveolae and caveolae-like domains in the nervous system.J. Neurochem. 73, 1–11.

Milligan G., Parenti M. and Magee A. I. (1995) The dynamic role ofpalmitoylation in signal transduction. Trends Biochem. Sci. 20,181–187.

Muller B. M., Kistner U., Veh R. W., Cases-Langhoff C., Becker B.,Gundelfinger E. D. and Garner C. C. (1995) Molecular charac-terization and spatial distribution of SAP97, a novel presynapticprotein homologous to SAP90 and the Drosophila discs-largetumor suppressor protein. J. Neurosci. 15, 2354–2366.

Mumby S. M. (1997) Reversible palmitoylation of signaling proteins.Curr. Opin. Cell Biol. 9, 148–154.

Murata S., Usuda N., Okano A., Kobayashi S. and Suzuki T. (2000)Occurrence of a transcription factor, signal transducer and activa-tors of transcription 3 (Stat3), in the postsynaptic density of the ratbrain. Mol. Brain Res. 78, 80–90.

Patton B. L., Miller S. G. and Kennedy M. B. (1990) Activation of typeII calcium/calmodulin-dependent protein kinase by Ca2+/calmod-ulin is inhibited by autophosphorylation of threonine within thecalmodulin-binding domain. J. Biol. Chem. 265, 11 204–11 212.

Resh M. D. (1994) Myristylation and palmitylation of Src familymembers: the fats of the matter. Cell 76, 411–413.

Robbins S. M., Quintrell N. A. and Bishop J. M. (1995) Myristoylationand differential palmitoylation of the HCK protein-tyrosine kinasesgovern their attachment to membranes and association withcaveolae. Mol. Cell. Biol. 15, 3507–3515.

Ross E. M. (1995) Palmitoylation in G-protein signaling pathways. Curr.Biol. 5, 107–109.

Sakagami H., Saito S., Kitani T., Okuno S., Fujisawa H. and Kondo H.(1998) Localization of Ca2+/calmodulin-dependent protein kinasekinases in the adult rat brain. Mol. Brain Res. 54, 311–315.

Schulman H. and Lou L. L. (1989) Multifunctional Ca2+/calmodulin-dependent protein kinase: domain structure and regulation. TrendsBiochem. Sci. 14, 62–66.

Shaw G. and Kamen R. (1986) A conserved AU sequence from the 3untranslated region of GM-CSF mRNA mediates selective mRNAdegradation. Cell 46, 659–667.

Shen K. and Meyer T. (1999) Dynamic control of CaMKII translocationand localization in hippocampal neurons by NMDA receptor sti-mulation. Science 284, 162–166.

Shen K., Teruel M. N., Connor J. H., Shenolikar S. and Meyer T. (2000)Molecular memory by reversible translocation of calcium/calmod-ulin-dependent protein kinase II. Nat. Neurosci. 3, 881–886.

Shenoy-Scaria A. M., Dietzen D. J., Kwong J., Link D. C. and Lublin D.M. (1994) Cysteine3 of Src family protein tyrosine kinase deter-mines palmitoylation and localization in caveolae. J. Cell Biol.126, 353–363.

Simons K. and Ikonen E. (1997) Functional rafts in cell membranes.Nature 387, 569–572.

Sisson T. H. and Castor C. W. (1990) An improved method for immo-bilizing IgG antibodies on protein A-agarose. J. Immunol. Meth.127, 215–220.

Smart E. J., Graf G. A., McNiven M. A., Sessa W. C., Engelman J. A.,Scherer P. E., Okamoto T. and Lisanti M. P. (1999) Caveolins,liquid-ordered domains, and signal transduction. Mol. Cell. Biol.19, 7289–7304.

Suen P. C., Wu K., Xu J. L., Lin S. Y., Levine E. S. and Black I. B.(1998) NMDA receptor subunits in the postsynaptic density of ratbrain: expression and phosphorylation by endogenous proteinkinases. Mol. Brain Res. 59, 215–228.

Suzuki T. (2002) Lipid rafts at postsynaptic sites: distribution, functionand linkage to postsynaptic density. Neurosci. Res. 44, 1–9.

Suzuki T., Okumura-Noji K., Tanaka R. and Tada T. (1994) Rapidtranslocation of cytosolic Ca2+/calmodulin-dependent protein kin-ase II into postsynaptic density after decapitation. J. Neurochem.63, 1529–1537.

Suzuki T., Mitake S., Okumura-Noji K., Shimizu H., Tada T.and Fujii T. (1997) Postsynaptic mechanisms. Localization ofa-internexin in the postsynaptic density of the rat brain. BrainRes. 765, 74–80.

Suzuki T., Usuda N., Murata S., Nakazawa A., Ohtsuka K. and TakagiH. (1999) Presence of molecular chaperones, heat shock cognate(Hsc) 70 and heat shock proteins (Hsp) 40, in the postsynapticstructures of rat brain. Brain Res. 816, 99–110.

Suzuki T., Ito J., Takagi H., Saitoh F., Nawa H. and Shimizu H. (2001)Biochemical evidence for localization of AMPA-type glu-tamate receptor subunits in the dendritic raft. Mol. Brain Res. 89,20–28.

Tanner S. M., Austin J. L., Leone G. et al. (2001) BAALC, the humanmember of a novel mammalian neuroectoderm gene lineage, isimplicated in hematopoiesis and acute leukemia. Proc. Natl Acad.Sci. USA 98, 13 901–13 906.

Tian Q. B., Nakayama K., Okano A. and Suzuki T. (1999) Identificationof mRNAs localizing in the postsynaptic region. Mol. Brain Res.72, 147–157.

Tian Q. B., Okano A., Nakayama K., Miyazawa S., Endo S. and SuzukiT. (2003) A novel ubiquitin-specific protease, synUSP, is localizedat the post-synaptic density and post-synaptic lipid raft. J. Neuro-chem. 87, 665–675.

Tobimatsu T. and Fujisawa H. (1989) Tissue-specific expression of fourtypes of rat calmodulin-dependent protein kinase II mRNAs.J. Biol. Chem. 264, 17 907–17 912.

Wilcox C., Hu J. S. and Olson E. N. (1987) Acylation of proteins withmyristic acid occurs cotranslationally. Science 238, 1275–1278.

Xiao B., Tu J. C. and Worley P. F. (2000) Homer: a link between neuralactivity and glutamate receptor function. Curr. Opin. Neurobiol.10, 370–374.

Zhong J., Carrozza D. P., Williams K., Pritchett D. B. and Molinoff P. B.(1995) Expression of mRNAs encoding subunits of the NMDAreceptor in developing rat brain. J. Neurochem. 64, 531–539.



BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and...

Documents

Transcript of BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and...