JBC Papers in Press.

Published on August 5, 2002 as Manuscript M112277200

Shanks, et al.: Page 1

AKAP350 AT THE GOLGI APPARATUS: II. ASSOCIATION OF AKAP350 WITH A

NOVEL CLIC FAMILY MEMBER

Ryan A. Shanks*, M. Cecilia Larocca,* Mark Berryman**, John C. Edwards***,

Tetsuro Urushidani****, Jennifer Navarre* and James R. Goldenring*

*Departments of Medicine, Surgery and Cellular Biology and Anatomy

Institute of Molecular Medicine and Genetics
Medical College of Georgia and the
Augusta VA Medical Center
Augusta, Ga 30912

**Department of Biomedical Sciences

Ohio University College of Osteopathic Medicine
Athens, OH 45701

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***Department of Medicine
St. Louis University and the
St. Louis Veterans Affairs Medical Center
St. Louis, MO 63106

****Laboratory of Pharmacology and Toxicology

Graduate School of Pharmaceutical Sciences
The University of Tokyo
Tokyo, Japan 113-0033

Running title: AKAP350 and CLIC family interaction.

Address correspondence to present address:

James R. Goldenring M.D., Ph.D.
Epithelial Biology Program and Section of Surgical Sciences
Vanderbilt University School of Medicine
4160A MRB III
465 21st Avenue South
Nashville, TN 37232
TEL: 615-936-3726
FAX: 615-343-1591
E-mail: jim.goldenring@vanderbilt.edu

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
 Shanks, et al.: Page 2

ABSTRACT

AKAP350 can scaffold a number of protein kinases and phosphatases at the

centrosome and the Golgi apparatus. We performed a yeast two-hybrid screen of a rabbit

parietal cell library with a 3.2kb segment of AKAP350 (nucleotides 3611-6813). This

screen yielded a full-length clone of rabbit chloride intracellular channel 1 (CLIC1).

CLIC1 belongs to a family of proteins, which all contain a high degree of homology in

their carboxyl termini. All CLIC family members were able to bind a 133 amino acid

domain within AKAP350 through the last 120 amino acids in the conserved CLIC

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carboxyl termini. Antibodies developed against a bovine CLIC, p64, immunoprecipitated

AKAP350 from HCA-7 colonic adenocarcinoma cell extracts. Antibodies against CLIC

proteins recognized at least 5 CLIC species including a novel 46 kDa CLIC protein. We

isolated the human homologue of bovine p64, CLIC5B, from HCA-7 cell cDNA. A

splice variant of CLIC5, the predicted molecular weight of CLIC5B corresponds to the

molecular weight of the 46 kDa CLIC immunoreactive protein in HCA-7 cells.

Antibodies against CLIC5B co-localized with AKAP350 at the Golgi apparatus with

minor staining of the centrosomes. AKAP350 and CLIC5B association with Golgi

elements was lost following Brefeldin A treatment. Furthermore, GFP-CLIC5B(178-

410) targeted to the Golgi apparatus in HCA-7 cells. The results suggest that AKAP350

associates with CLIC proteins, and specifically, CLIC5B interacts with AKAP350 at the

Golgi apparatus in HCA-7 cells.

 Shanks, et al.: Page 3

INTRODUCTION

Cells contain a dynamic organization of signaling molecules and proteins required

for the transduction of intracellular signaling events. Thus, cellular organization is

crucial for intracellular signals to function correctly. The study of how the cell completes

this task has revealed molecular scaffolds, which allow spatial control of signaling events

(1,2). The study of signaling molecules scaffolded in specific locations within the cell

has led to an expanded understanding of the cellular functions that they control.

Scaffolding proteins are utilized in the cell to localize signaling proteins to

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specific areas facilitating precise cellular function. A-kinase anchoring proteins

(AKAPs) are one class of scaffolding protein utilized by the cell to anchor the regulatory

subunit of type II cAMP-dependent protein kinase A (3) to specific locations within the

cell (4). Besides the targeting of anchored A-kinase, another key function of AKAPs is

the recruitment of other signaling proteins (5). Often an AKAP will scaffold both kinases

and phosphatases, thereby coordinating specific signaling events (6). Thus, protein

scaffolds can assemble protein kinase and phosphatase systems in close proximity to their

intended substrates.

We have previously defined one AKAP, AKAP350, which is the product of a

multiply spliced gene family and is localized at the centrosome (7). This protein has also

been observed on the Golgi apparatus, where it was identified as the Centrosome and

Golgi apparatus localized PKN-associated protein (CG-NAP) (8). The sequence of CG-

NAP is identical to AKAP450 designated according to its 450kDa size (9), but we will

refer to AKAP350/CG-NAP/AKAP450 as AKAP350 based on the original identification

 Shanks, et al.: Page 4

of this protein by Keryer et al. as a 350kDa A-kinase anchoring protein at the centrosome

(10).

The function of a particular AKAP is defined by its localization and the proteins

bound to the scaffold (10). AKAP350 has putative binding sites for protein phosphatase

1, protein kinase N, protein phosphatase 2A (8), protein kinase

C e (11),phosphodiesterase 4D3 (29) as well as two A-kinase binding sites (7,8).

However, previous studies have not identified proteins other than kinases and

phosphatases interacting with AKAP350. We hypothesized that AKAP350 also bound to

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other signaling proteins based on the large size of the protein. Using yeast two-hybrid

screening we now show that the Chloride intracellular channel (CLIC) family of proteins

interact with a region of AKAP350 previously denoted as the pericentrin homology

region (PHR) due to its 41% similarity with the centrosomal protein pericentrin (7). In

addition, we identify a novel human CLIC family member, designated CLIC5B, which

represents the human homologue of bovine p64 (12). CLIC5B shares all but its first exon

with the previously published human CLIC5 protein (13), renamed here as CLIC5A.

CLIC5B immunostaining colocalized with AKAP350 at the Golgi apparatus as well as, to

a lesser extent, the centrosome. The results suggest that AKAP350 may scaffold CLIC

proteins at discrete sites within the cell.

 Shanks, et al.: Page 5

MATERIALS AND METHODS

Materials - Oligonucleotides were synthesized by the Medical College of Georgia

Molecular Biology Core Facility. Cy3-conjugated species-specific secondary antibodies

were purchased from Jackson ImmunoResearch Labs (West Grove, PA). Prolong

Antifade, and Alexa 488-conjugated species-specific secondary antibodies were from

Molecular Probes, Inc. (Eugene, OR). [a-32P]dCTP was purchased from NEN Life

Science Products.

Yeast Two-Hybrid Screening- A rabbit parietal cell two-hybrid library in pAD-GAL4

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(14) was screened with 3.2 kb of AKAP350 sequence (nucleotides 3611-6813, GenBank

Accession AF083037) cloned into the binding domain vector pBDGal-4-Cam

(Stratagene) with the use of EcoRI and SalI sites. The Y190 yeast strain harboring the

HIS3 and b-galactosidase reporter genes were used to screen approximately 1 million

clones as previously described (14). The isolated clone was rescued into XL-1 Blue

bacteria, and plasmid DNA was prepared using Qiagen miniprep kits. All DNA

sequencing was performed using dye terminator chemistry automated sequencing in the

Molecular Biology Core Facility at the Medical College of Georgia. The!isolated clone

and the ‘bait’ pBDGal-4-Cam vector both gave negative results when analyzed by

themselves in yeast two-hybrid assays.

Yeast Two-Hybrid Binary Assays- Deletion constructs of the CLIC5B and the PHR

region of AKAP350 (nucleotides 3611-6813) were cloned into pADGal-4 and pBDGal-4-

Cam, respectively, using EcoRI and SalI sites, and tested for interaction in the yeast-two

hybrid assay as previously described (14). Full length human CLIC1, CLIC4, CLIC5A

and parchorin constructs (13,15) were cloned into pADGal-4, and tested for interaction
 Shanks, et al.: Page 6

with the AKAP350-PHR-pBDGal-4-Cam bait in the yeast two hybrid assay as previously

described (14). Positive results were recorded if b-galactosidase assay, was positive

within 3 hours. The pADGal-4 vectors produced negative results when analyzed by

alone.

Antibodies- The 14G2 monoclonal anti-AKAP350 antibody was developed as

previously described (7). The 121, 656 and B132 polyclonal anti-CLIC antibodies were

prepared as previously described (13,16). The specific polyclonal anti-CLIC5B antibody

was raised against the peptide containing amino acids 14 to 26 (QNERTYEVPDPEE) of

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the deduced amino acid sequence of CLIC5B (New England Peptides). The peptide was

synthesized in the Medical College of Georgia Molecular Biology Core Facility. The goat

polyclonal antibody was affinity purified using the CLIC5B peptide coupled to the

Aminolink system (Pierce), and eluted with 100 mM glycine pH 2.5.

Coimmunoprecipitation experiments- HCA-7 cells were incubated in lysis buffer (1%

NP-40, 50 mM Tris-HCl pH 7.4, 250 mM NaCl, 2mM EDTA, 5 mM benzamidine, 0.1

mM AEBSF, and protease inhibitors (Sigma)) for 5 minutes on ice and spun at 1000xg

for 15 minutes at 4oC. 300 mg of the HCA-7 lysate proteins were incubated with the 15

mL of 656 anti-CLIC antisera for 2 hours at room temperature. Dynabeads M-280 Sheep

anti-rabbit IgG were washed twice in phosphate buffered saline (PBS) with protease

inhibitors for 10 minutes at room temperature, and then blocked using 0.2% bovine

serum albumin and protease inhibitors (Sigma) for 1 hour at room temperature. The

lysate/antibody mixture was then incubated with the blocked Dynabeads M-280 Sheep

anti-rabbit IgG for 1 hour at room temperature (Dynal). The beads were washed with

phosphate buffered saline with protease inhibitors 3 times for 15 minutes. The beads
 Shanks, et al.: Page 7

were then boiled in the presence of 1% SDS and 1/5 volume of 2-mercaptoethanol. The

samples were separated on a 3-10% gradient SDS-PAGE gel and transferred to

nitrocellulose for western blot analysis.

Cellular fractionation- HCA-7 cells were grown to confluence and washed in PBS. The

cells were scraped in PBS and pelleted at 200 g, for 5 min at 4 C. The cells were then

homogenized in ice cold 250 mM sucrose, 10 mM HEPES pH 7.4, containing protease

inhibitors (Sigma) using a Potter Teflon on glass homogenizer. The homogenates were

centrifuged at 4 C sequentially at 1000 xg for 5 min (P0), 5000 xg for 15 min (P1), 17000

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xg for 20 min (P2), and 100000 xg for 60 min (P3). The pellets were resuspended in PBS

containing protease inhibitors and the high speed supernate (S3) was also saved.

Western Blot Analysis – HCA-7 cell homogenates and the different subcellular fractions

were resolved by SDS-PAGE using 3-10 % gradient gels for AKAP350 (14G2) and 10 %

gels for pan-CLIC (656) and anti-CLIC5B antibodies. For AKAP350 detection gels

were transferred 2 h at 750 mA to nitrocellulose, while for other antibodies gels were

transferred overnight at 100 mA to polyvinylidene difluoride (Immobilon P) membranes.

Blots were blocked with 5 % nonfat milk/Tris Buffered Saline (TBS), 0.5 % Tween-20,

and then probed with 14G2 (1:500), 656 (1:500) or anti-CLIC5B (1:50) in 3 % bovine

serum albumin/TBS-Tween for 1 h at room temperature. After washing three times with

TBS-Tween, blots were incubated with HRP-conjugated anti-mouse IgG (1:2500), anti-

rabbit IgG (1:20000) or anti-goat IgG (1:10000) in 1 % non-fat milk/TBS-Tween. Blots

were finally washed three times with TBS-Tween and three times with TBS, and

immunoreactivity was detected with chemiluminiscence (Supersignal, Pierce) and

subsequent exposure to BioMax film (Kodak)

 Shanks, et al.: Page 8

Cloning of CLIC5B - Total RNA were prepared from HCA-7 cells using RNASTAT-60

(Teltest). The Advantage RT for PCR kit (Clontech) was used to construct cDNA from 2

mg of HCA-7 total RNA using random hexamer primers. Gene specific primers were

constructed at the putative start site of CLIC5B and at the termination site for CLIC5A

(sense: GCGCGAATTCCCATGAATGACGAAGACTACAGCACCA, antisense:

GCGCGTCGACCTGGATCGGCTGAGGCGTTTGGCGACATCAG), and products

were amplified using Advantage Polymerase (Clontech) with 40 cycles: 95 C 15s, 60 C

10s, 68 120s. The resulting product was cloned into pTOPO (Invitrogen) and the insert

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was sequenced with flanking vector primers.

Northern blot analysis- A human multi-tissue northern blot was purchased from

Origene. The blot was sequentially probed with a 336 nucleotide probe corresponding to

the 5’ end of human CLIC5A (nucleotides 1-336, GenBank accession AF216941) or a

516 nucleotide probe corresponding to the 5’ end human CLIC5B (nucleotides 1-516

GenBank accession AY032602). Blots were probed overnight at 42 C and then washed

to high stringency (0.1X SSC, 65 C). Blots were exposed to X-ray film at -70 C for 24

hrs.

Immunocytochemistry - HCA-7 cells were grown on glass coverslips to confluence.

The cells were then incubated in the absence or presence of 10 mg/mL Brefeldin A (BFA)

for 1 hour at 37oC. Cells were washed in phosphate buffered saline and fixed with 4%

paraformaldehyde for 15 minutes at room temperature. Cells were blocked with 17%

donkey serum, 0.3% Triton-X100 in phosphate buffered saline and then incubated

simultaneously with 14G2 (mouse anti-AKAP350) (1:85), mouse anti-p58 (1:100),

and/or affinity-purified goat anti-CLIC5B (1:5) for 2 hours at room temperature. The
 Shanks, et al.: Page 9

cells were then simultaneously incubated with Alexa-488-conjugated anti-rabbit IgG and

Cy3-conjugated anti-mouse IgG for 60 minutes. Coverslips were inverted and mounted

with Prolong antifade. Cells were examined for triple labeling by confocal microscopy

(Molecular Dynamics, Sunnyvale, CA) using maximum intensity projections of 0.3 mm

optical sections in the apical region of the cell.

GFP-chimera expression- GFP-CLIC5B(178-410) was amplified utilizing primers

specific for exons 2-6 shared by CLIC5A and CLIC5B (sense:

GCGCGAATTCTTTGTGAAGGCTGGAATCGATGGAGA and anti-sense:

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GCGCGTCGACCTGGATCGGCTGAGGCGTTTGGCGACATCAG). EcoRI and SalI

sites were utilized to clone into EGFP-C2 (Clonetech). This construct was transfected

with Effectene (Qiagen) into HCA-7 cells, which were grown on glass coverslips

overnight. Some cells were treated with 10mg/ml Brefeldin A (BFA) for 1 hour at 370C

as detailed above. Cells were fixed and stained as described above.

 Shanks, et al.: Page 10

RESULTS

Identification of an interaction of the CLIC family of proteins with AKAP350-

AKAP350 interacts with several signaling proteins including protein phosphatase 1,

protein kinase N, protein phosphatase 2A (8), protein kinase Ce (11) and Type II A-

kinase (Fig 1A) (7,8). To investigate further the function of AKAP350 as a scaffolding

protein, we performed a yeast two-hybrid screen of a rabbit parietal cell library. We

screened the library with a 3.2 kb segment of AKAP350, previously termed the

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pericentrin homology region (PHR, 7) due to its weak homology (41% similarity) with

the centrosomal protein, pericentrin (Fig 1A). One positive clone was identified as a full-

length cDNA of the rabbit homologue of human CLIC1 (NCC27) (GenBank accession

number U93205). Subsequent yeast two-hybrid binary assays confirmed the interaction

of rabbit CLIC1 with the PHR of AKAP350. The rabbit CLIC1 sequence and deletion

constructs of the PHR of AKAP350 were used in yeast two-hybrid binary assays to map

the rabbit CLIC1 binding site in AKAP350. The CLIC binding region of AKAP350 was

limited to a 133 amino acid region between amino acids 1388 and 1515 of AKAP350

(Fig 1B).

Coimmunoprecipitation of AKAP350 and CLIC family members- To characterize the

CLIC/AKAP350 interaction, coimmunoprecipitation experiments were performed using a

rabbit polyclonal anti-CLIC antibody, 656. This antibody was chosen for its ability to

recognize a range of CLIC family members. The antibody was developed against a

region of the carboxyl terminus of the avian CLIC, p62, and is known to recognize avian
 Shanks, et al.: Page 11

p62, bovine p64 (16), human CLIC1 and CLIC4 (J. C. Edwards, unpublished results).

Figure 2A demonstrates that, in extracts of HCA-7 cells, the pan-CLIC 656 antibody

recognized major bands of 31, 32 and 46 kDa as well as a minor band at 85 kDa. Beads

with only anti-rabbit antibody or beads coated with the anti-pan-CLIC antibody, 656,

were incubated with 1% NP-40 extracts from HCA-7 human colon adenocarcinoma cells.

The bound protein was separated by SDS-PAGE and subjected to western blot analysis

with the monoclonal anti-AKAP350 antibody, 14G2. The anti-CLIC antibody 656,

specifically immunoprecipitated AKAP350 when compared to beads coated with only

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anti-rabbit antibody (Fig 2B). The western blot analysis with 656 confirmed that it did

not recognize AKAP350 (data not shown). These biochemical data confirmed the in situ

association of AKAP350 with a CLIC family member in HCA-7 cells.

CLIC5B: A novel CLIC family member in HCA-7 cells- As noted in Figure2a, the 656

pan-CLIC antibody recognized at least four discrete proteins in HCA-7 cells. The 31kDa

and 32kDa bands could represent CLIC1 and CLIC4, due to their apparent molecular

weight and the known ability of the 656 antibody to cross-react with CLIC1 and CLIC4

(J. C. Edwards unpublished results).

Previously, no CLIC protein had been described

migrating at 46kDa. Another pan-CLIC rabbit polyclonal antibody (121), which

recognizes the conserved CLIC carboxyl terminus of CLIC1 (13), also recognized a 46

kDa band in HCA-7 cells (data not shown). We employed several specific CLIC

antibodies to determine if they recognized this protein. Antibodies specific for CLIC1

and parchorin did not recognize this protein (data not shown). However, only a

polyclonal antibody affinity purified against CLIC5 (B132) did recognize a 46kDa band
 Shanks, et al.: Page 12

(data not shown). These results suggested that a CLIC5 immunoreactive protein might

represent the 46 kDa CLIC species in HCA-7 cells.

The high homology between bovine p64 and the more recently documented

CLIC5 led us to hypothesize that a CLIC5 splice variant might represent a human

homologue of bovine p64 (12,13). Human CLIC5 and bovine p64 show extensive

identity in their carboxyl termini, but have little similarity in their amino terminal

sequences. We performed a search with the 5’ coding sequence from bovine p64 against

genomic BAC sequences containing the human CLIC5 cDNA. This search revealed an

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alternate initiation site for CLIC5 that is present 65 kb upstream of the published start site

of human CLIC5. Utilizing a sense primer against this putative alternative start site and

an antisense primer against the 3’ end of the CLIC5 coding sequence we were able to

amplify a 1230 bp sequence from HCA-7 cell cDNA. This sequence encodes for a

protein with an apparent molecular mass of 46 kDa, corresponding to the molecular

weight of the CLIC-immunoreactive band detected by the 656 antibody (Fig 2A). We

named this protein CLIC5B (GenBank accession number AY032602) and have renamed

the previously published CLIC5 as CLIC5A (13). The only difference between these

proteins is found in their first exons. The first exon of CLIC5A encodes for its first 21

amino acids, while the first exon of CLIC5B encodes for its first 180 amino acids (Fig

4B). Since human CLIC5B shows 72% identity with bovine p64, CLIC5B most likely

represents the human homologue of bovine p64.

Northern analysis of CLIC5B- To determine the tissue distribution of these two

proteins, northern blot analysis of 12 different human tissues was performed. Due to the
 Shanks, et al.: Page 13

CLIC5 splicing, we designed a CLIC5A-specific probe which included a region of the 5’

untranslated sequence and the unique first exon of CLIC5A. The message size of

CLIC5A was consistent with the previously published size of 6.4 kb (13) (Fig 4A). We

noted the highest expression in lung, with a lower level of message in the heart, kidney

and skeletal muscle than previously reported with a probe designed against full length

CLIC5A (13). CLIC5A mRNA was also detected in colon and placenta upon longer

exposure of the blot to film (data not shown). As Berryman et al. described (13), the

CLIC5A specific probe also recognized less intense bands at 3.8 kb and 2.3 kb in some

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tissues (data not shown). A probe designed against the unique first exon of CLIC5B was

used to evaluate the tissue distribution of CLIC5B. CLIC5B expression was detected

only in the small intestine. The CLIC5B mRNA migrated at a slightly higher size (6.6kb)

than CLIC5A (Fig 4B).

CLIC5B interacts with AKAP350- The CLIC family of proteins is defined by their high

homology in their carboxyl termini which contain two conserved putative transmembrane

domains (17). To determine the specificity of AKAP350 binding for members of the

CLIC family, clones of human CLIC1, CLIC4, CLIC5A, CLIC5B and rabbit parchorin

(13,15) were paired with AKAP350 in yeast two-hybrid binary assays. Each of these

CLIC family members interacted with AKAP350 (Fig 5A). These results suggested that

the conserved carboxyl terminus in the CLIC family contained the binding site for

AKAP350 (Fig 5A).

Constructs, which contained the first and second halves of the conserved region of

CLIC5B, were used in yeast two-hybrid binary assays to determine the region responsible
 Shanks, et al.: Page 14

for binding to AKAP350. The binding region was contained in the final 120 amino acids

of CLIC5B. The entire region was necessary for the binding of AKAP350 to CLIC5B,

since any further truncations of this region were unable to bind to AKAP350 (Fig 5B).

This region contains extensive identity in all CLIC family members (Fig 5B).

CLIC5B staining colocalizes with AKAP350 in the Golgi apparatus in HCA-7 cells - In

the accompanying paper, we have characterized AKAP350A as a Golgi apparatus

specific splice variant of AKAP350 (Shanks et al. 2002). To explore the localization of

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AKAP350 and CLIC5B, we prepared a goat polyclonal antiserum against CLIC5B-

specific peptide sequence. Figure 6 demonstrates that antibodies against CLCI5B

detected a single 46 kDa band in fractions of HCA-7 cells with the highest concentrations

in heavy microsomes and the high speed supernate. All CLIC5B immunoreactivity was

abolished by incubation with 10 µM competing CLIC5B peptide (data not shown).

AKAP350 was also present in these fractions, but was also detected in the lighter

microsomal fractions.

Since the patterns of AKAP350 and CLIC5B immunoreactivity were overlapping

but not identical, CLIC5B antibodies were used to stain HCA-7 cells. Figure 7A

demonstrates that, although considerable cytosolic staining was observed, CLIC5B

immunoreactivity localized to the Golgi with extensive colocalization with the p58 Golgi

marker. A pan-CLIC antibody (121) also demonstrated strong staining of the Golgi

apparatus (data not shown). Treatment of cells with Brefeldin A led to disruption of the

the Golgi and vesiculation of p58 staining with dispersal of CLIC5B staining into a

predominantly cytosolic distribution (Fig 7A). We also observed colocalization of

 Shanks, et al.: Page 15

CLIC5B with AKAP350 staining with the 14G2 monoclonal antibody (Fig 7B).

Brefeldin A treatment dispersed the Golgi staining by both CLIC5B and AKAP350

antibodies. However, in Brefeldin-treated cells, it was also apparent that a small amount

of CLIC5B staining was also present at centrosomes whose AKAP350 staining was

unaltered by brefeldin treatment (Fig 7B). These results demonstrate that CLIC5B

localizes predominantly to the Golgi apparatus, with a lesser association with the

centrosome.

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GFP-CLIC5B(178-410) targets to the Golgi apparatus- To clarify the localization of

CLIC5B with the Golgi apparatus, a GFP-chimera (GFP-CLIC5B(178-410)) was

constructed utilizing amino acids 178-410 of CLIC5B. This region represents the shared

exons 2-6 of CLIC5A and CLIC5B (Fig. 4). The HCA-7 cells were then stained with the

Golgi apparatus marker p58 (Fig 8). HCA-7 cells expressing GFP-CLIC5B (178-410)

displayed colocalization with the Golgi apparatus. Staining with the anti-AKAP350

14G2 antibody was unchanged in cells expressing this GFP-chimera (data not shown).

Treatment of cells with brefeldin A caused dispersal of GFP-CLIC5B (178-410)

confirming its localization to the Golgi apparatus (data not shown). These data suggest

that the region coding for exons 2-6 is sufficient for targeting of CLIC5B to the Golgi

apparatus.
 Shanks, et al.: Page 16

DISCUSSION

AKAPs scaffold signaling proteins to specific locations required for specialized

functions throughout cells. The signaling proteins associated with an AKAP often define

the functions of these scaffolds (5). In this report, we have identified the interaction of

AKAP350 with CLIC family members, and specifically the colocalization of CLIC5B

and AKAP350 at the Golgi apparatus of HCA-7 cells. For the first time, these results

demonstrate the interaction at the Golgi apparatus of AKAP350 with a effector protein

with no known enzymatic activity. In addition, these studies establish a mechanism for

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the scaffolding of CLIC proteins at discrete locations within the cell.

The CLIC protein family consists of seven members, which all have several

common attributes. The family consists of parchorin, bovine p64 and CLIC1-CLIC5.

Each protein in this family has a high amino acid homology at the carboxyl terminal end

of the protein. This homologous region consists of approximately 200 amino acids and

contains a putative hydrophobic membrane-spanning domain. Although expression

patterns and localization differ for each of the family members, all of these CLIC proteins

associate with membranous organelles.

The data presented here suggest that the CLIC5 gene encodes for at least two gene

products. Northern blot analysis for CLIC5A and CLIC5B also demonstrated different

mRNA expression patterns for these proteins. Multiple message sizes have been reported

for CLIC1, CLIC4, CLIC5A and p64 (12,13,22,24) suggesting that each of the members

of this family of proteins contains several multiply spliced species. Due to the high

homology among CLIC family members in their carboxyl termini, amino terminal region

splicing events could provide distinguishing characteristics enabling a single gene to

 Shanks, et al.: Page 17

encode for proteins with a diverse range of functions. Berryman and Bretscher utilized

the full length CLIC5A cDNA as a probe to perform northern analysis of a multi-tissue

blot (13). To distinguish CLIC5A from CLIC5B, we utilized probes specific for the

unique first exons of CLIC5A and CLIC5B. The CLIC5B probe recognized a single 6.6

kb message detectable only in the small intestine. The CLIC5B message was slightly

longer than the CLIC5A message of 6.4 kb observed in several tisues (13). Interestingly,

the CLIC5A-specific probe still recognized other shorter message sizes of 3.8 kb and 2.3

kb (data not shown), which correlate with the reported mRNA sizes observed with the

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probe designed against full length CLIC5A (13). These smaller mRNAs could represent

shorter message sizes created from splicing events downstream of the first exon. The

bovine homologue of CLIC5B (p64) also showed similar multiple message sizes by

northern analysis (12). The differences observed between each of these studies warrants

further investigation to determine the functional diversity of CLIC5/p64 splice variants.

Previous studies have demonstrated the association of CLIC family members with

the cytoskeleton (CLIC5A), nucleus (CLIC3, CLIC1), Golgi apparatus (bovine p64),

mitochondria (CLIC4), and the sub-apical membrane compartment (CLIC1, CLIC4,

CLIC5A, and parchorin) (12,13,15,21,22,25). Targeting domains in CLICs responsible

for their localization have not been elucidated. The 46 kDa CLIC5B protein appears to

be a major immunoreactive CLIC in HCA-7 cells. Immunocytochemical studies

demonstrated the predominant localization of CLIC5B immunoreactivity with the Golgi

apparatus in HCA-7 cells. CLIC5B, formed through an alternate initiation site of the

CLIC5A gene, most likely represents the human homologue of bovine p64. CLIC5B and

p64 share 72% identity, and p64 was originally isolated from kidney microsomes
 Shanks, et al.: Page 18

containing Golgi apparatus markers (12). Interestingly, the region conserved in CLIC5A

and CLIC5B (exons2-6) localized to the Golgi apparatus when overexpressed as a GFP-

chimera protein in HCA-7 cells. Therefore, the ability to localize to the Golgi apparatus

is at least partially maintained in the conserved region of CLIC5A and CLIC5B.

Previous studies have localized CLIC5A predominantly to the supapical compartment

(13). The first exon of CLIC5A may therefore code for a motif required for Golgi

apparatus export or targeting to the apical membrane domain. Although AKAP350 can

bind to CLIC5B and could sequester it to the Golgi apparatus, we have shown that

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AKAP350 can potentially bind to all CLIC family members. Therefore, interactions with

AKAP350 may not be sufficient for organelle specific targeting of CLIC family

members.

The association of AKAP350 and CLIC5B was affected by structural changes in

the Golgi apparatus. In brefeldin A treated HCA-7 cells, both CLIC5B and AKAP350

staining were dispersed in the cytosol. These data suggest that the structural integrity of

the Golgi apparatus is necessary for the association of CLIC5B and AKAP350 with the

Golgi cisternae. These results support a model for AKAP350 scaffolding of CLIC5B at

the assembled trans-Golgi cisternae. In addition, western analyses demosntrate a large

amount of both CLIC5B and AKAP350 protein in the supernate fraction after

homogenization. These data suggest a dynamic association of CLIC5B with AKAP350,

and provide a mechanism for the modulation of CLIC function at the Golgi apparatus.

Although the function of CLIC5B at the Golgi apparatus is unclear, previous

studies have described CLIC family members as independent channels. Harrop, et al (26)

have recently described a 23 amino acid sequence in carboxyl terminus of CLIC1 as a

 Shanks, et al.: Page 19

putative transmembrane domain (26). Indeed, this region is highly conserved among all

the CLIC family members. A mounting volume of evidence describes the CLIC proteins

as potential integral membrane proteins that can exist as cytosolic and/or membrane

proteins based on a significant conformational change in the amino terminus (26,27).

This new conformation would expose the putative transmembrane domain. The large

conformational change required in CLIC1 for the exposure of this domain could require

signaling through kinases and phosphatases. Since AKAP350 contains putative binding

sites for protein phosphatase 1, protein kinase N, protein phosphatase 2A (8), protein

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kinase Ce (11), phosphodiesterase 4D3 (28) and A-kinase (7,8), it is conceivable that

these enzymes could play a role in the function of CLIC5B as well as other CLIC family

members. Edwards and colleagues have suggested phosphorylation and

dephosphorylation of CLIC5B’s bovine homologue, p64, has an effect on its ability to

control chloride movement (29). Further investigations into CLIC5B’s function as a

chloride anion channel will have to be completed before it is determined that this CLIC

protein mediates chloride movement at the Golgi apparatus.

Signaling events will likely control the movement and function of the CLIC

family of proteins. In this report, we describe the interaction of all CLIC family members

with a conserved region found in all AKAP350 splice variants (7). Therefore, it is

possible that other CLIC family members will be scaffolded by AKAP350 in other

intracellular locations in particular cell types. Although we have not established that

AKAP350 is present wherever CLIC family members are localized, a more

comprehensive study of other cell types could lead to the discovery of other

AKAP350/CLIC interactions. This interaction is intriguing because of the diversity of

 Shanks, et al.: Page 20

localized signaling molecules that AKAP350 can potentially target to control the function

of CLICs. Many of the CLIC family members share phosphorylation motifs for enzymes

scaffolded by AKAP350. For example CLIC4, p64, CLIC5A and CLIC5B all share a

conserved A-kinase phosphorylation site in their extreme carboxyl termini (30,31).

Thus, cAMP-dependent phosphorylation of CLIC proteins by A-kinase could regulate

their distribution and function.

In summary, we have described the interaction of CLIC family members with

AKAP350. In HCA-7 cells, CLIC5B, the human homologue of bovine p64, colocalizes

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predominantly with AKAP350 at the Golgi apparatus with lesser localization at the

centrosome. This report represents the first demonstration of AKAP350 with a non-

enzymatic effector protein at the Golgi apparatus. These studies indicate that AKAP350

not only scaffolds enzymatic signaling molecules, but also conveys organelle-specific

interactions with non-kinase/phosphatase effector molecules. The relationships between

the signaling proteins scaffolded to AKAP350 and CLIC proteins will provide further

insights into the function of the CLIC family of proteins and AKAP350 in specific

subcellular compartments.
 Shanks, et al.: Page 21

ACKNOWLEDGEMENTS

We would like to thank Dr. Lynne A. Lapierre for her assistance with the yeast

two-hybrid work, and her intellectual input into this manuscript. Furthermore, we would

like to thank Dr. Hank Schmidt for his assistance with several of the techniques utilized

here. This work was supported by the following grants: to JRG from NIH NIDDK

(DK48370 and DK43405) and a Veterans Administration Merit Award, to JCE from NIH

RO1 AR44838 and a Veterans Administration Merit Award.

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 Shanks, et al.: Page 22

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 Shanks, et al.: Page 25

FIGURE LEGENDS

Fig. 1: CLIC interaction with AKAP350: A: Diagram of the full length human gastric

clone splice variant of AKAP350 including the 4 leucine zipper regions (LZ), and the

binding sites for RII, PKN, PP1, and PKCe. Also shown is the Pericentrin Homology

Region (PHR). B: Constructs of the PHR used to map the CLIC binding region (shown

in grey). Rabbit CLIC1 binding to the PHR constructs in yeast two-hybrid assays is

indicated at the right.

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Fig. 2: Anti-CLIC antibodies immunoprecipitate AKAP350: A. Western blot

analysis of anti-CLIC staining in HCA-7 cells. 25 µg of HCA-7 lysate protein were

resolved on 10% SDS-PAGE gels and western blots were probed with polyclonal ant-

CLIC (656) antibodies. The positions of molecular mass standards are indicated at left.

Arrowheads indicate the positions of 4 CLIC immunoreactive species. B. Anti-

AKAP350 (14G2) western blot analysis of an immunoprecipitation experiment

performed with beads coated with either the anti-CLIC antibody (656) or just anti-rabbit

IgG (Beads alone). Lanes represent the HCA-7 NP-40 protein lysate starting material (25

mg of protein = 1/12 volume of starting material) (Lysate), protein that did not bind to the

beads (1/12 volume = Void), and protein bound to the beads (all bound protein from 300

mg total starting material) (Eluate).

Fig. 3: CLIC5B sequence: A: Nucleotide sequence and deduced amino acid sequence

of CLIC5B, amplified from an HCA-7 cDNA. The highlighted sequence and

corresponding amino acids represent the novel first exon of CLIC5B created by an
 Shanks, et al.: Page 26

alternate start site upstream of the start site of CLIC5A. B: Diagram of the CLIC5 gene

in chromosome 6 (GenBank reference 13643945) showing the alternate start site of

CLIC5B upstream of CLIC5A. Nucleotide numbers which correspond to the following

exons are: CLIC5B exon 1 (195221-194681), CLIC5A exon 1 (130521-130458), exon 2

(54373-44262), exon 3 (48510-48380), exon 4 (40780-40674), exon 5 (13537-13355),

and exon 6 (2384-2218). The bar represents 10 kb and the double line breaks represent

20 kb.

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Fig. 4: Northern blot analysis of CLIC5A and CLIC5B expression in human tissues:

A: Northern blot analysis of a human multi-tissue blot with a probe created from 5’

untranslated and exon 1 of CLIC5A. B: Northern blot analysis of the same human multi-

tissue blot reprobed with a probe representing the first exon of CLIC5B. Lanes

containing mRNA from whole organs are denoted as: 1-brain, 2-colon, 3-heart, 4-kidney,

5-liver, 6-lung, 7-skeletal muscle, 8-placenta, 9-small intestine, 10-spleen, 11-stomach,

12-testis.

Fig. 5: AKAP350 interaction with CLIC: A: Diagram representing parchorin, CLIC1,

CLIC4, CLIC5A and CLIC5B which all interact with AKAP350 in yeast two-hybrid

assays (indicated at the right). Constructs of the conserved region (gray) in CLIC5B

were used to define where AKAP350 binds. The last 120 amino acids (290-410) were

able to interact with AKAP350, but deletion constructs of this region were unable to bind.

B: Carboxyl terminal AKAP350 binding domain of CLIC family proteins: Protein

alignment of the last 120 amino acids of CLIC5B, which bind to AKAP350, compared to
 Shanks, et al.: Page 27

parchorin, CLIC1, CLIC4, CLIC5A and p64 bovine. The highlighted regions represent

homology when compared to CLIC5B. A putative transmembrane domain is

underscored with X’s.

Fig. 6: CLIC5B immunoreactivity in fractions from HCA-7 cells: HCA-7 were

fractionated into homogenate (H), 5000xg microsomes (P1), 17,000xg microsomes (P2),

100,000xg microsomes (P3) and a high speed supernate (S3). From each fraction, 25 µg

of protein for CLIC5B detection or 100 µg of protein for AKAP350 detection were

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resolved on SDS-PAGE gels and western blots were performed for CLIC5B and

AKAP350.

Fig. 7: Immunocytochemical localization of AKAP350 and CLIC5B in HCA-7 cells:

A: Confocal microscopy of HCA-7 cells stained with the anti-CLIC5B antibody (first

column) and the Golgi marker anti-p58 antibody (second column) The first row depicts

single confocal fluorescence optical sections from untreated control HCA-7 cells and the

second row shows the staining of brefeldin A treated cells (+BFA). B: Confocal

microscopy of HCA-7 cells stained with the anti-CLIC5B antibody (first column) and the

anti-AKAP350 (14G2) antibody (second column). The first row represents control HCA-

7 cells and the second row represents BFA treated cells (+BFA). Note the focal co-

labeling of centrosomes by both AKAP30 and CLIC5B antibodies in brefeldin A-treated

cells.
 Shanks, et al.: Page 28

Fig. 8: Targeting of GFP-CLIC5B in HCA-7 cells: HCA-7 cells were transfected with

GFP-CLIC5B (amino acids 178-410). These cells were then dual stained with the Golgi

apparatus marker, p58. Note the colocalization of p58 with the GFP-CLIC5B chimeric

protein in transfected cells.

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FIGURE 1
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Shanks, et al.: Page 30

FIGURE 2
 Shanks, et al.: Page 31

FIGURE 3

A.
1 ATG AAT GAC GAA GAC TAC AGC ACC ATC TAT GAC ACA ATC CAA AAT GAG AGG ACG TAT GAG
1 M N D E D Y S T I Y D T I Q N E R T Y E
61 GTT CCA GAC CAG CCA GAA GAA AAT GAA AGT CCC CAT TAT GAT GAT GTC CAT GAG TAC TTA
21 V P D Q P E E N E S P H Y D D V H E Y L
121 AGG CCA GAA AAT GAT TTA TAT GCC ACT CAG CTG AAT ACC CAT GAG TAT GAT TTT GTG TCA
41 R P E N D L Y A T Q L N T H E Y D F V S
181 GTC TAT ACC ATT AAG GGT GAA GAG ACC AGC TTG GCC TCT GTC CAG TCA GAA GAC AGA GGC
61 V Y T I K G E E T S L A S V Q S E D R G
241 TAC CTC CTG CCT GAT GAG ATA TAC TCT GAA CTC CAG GAG GCT CAT CCA GGT GAG CCC CAG
81 Y L L P D E I Y S E L Q E A H P G E P Q
301 GAG GAC AGG GGC ATC TCA ATG GAA GGG TTA TAT TCA TCA ACC CAG GAC CAG CAA CTC TGC
101 E D R G I S M E G L Y S S T Q D Q Q L C
361 GCA GCA GAA CTC CAG GAG AAT GGG AGT GTG ATG AAG GAA GAT CTG CCT TCT CCT TCA AGC
121 A A E L Q E N G S V M K E D L P S P S S
421 TTC ACC ATT CAG CAC AGT AAG GCC TTC TCT ACC ACC AAG TAT TCC TGC TAT TCT GAT GCT
141 F T I Q H S K A F S T T K Y S C Y S D A

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481 GAA GGT TTG GAA GAA AAG GAG GGA GCT CAC ATG AAC CCT GAG ATT TAC CTC TTT GTG AAG
161 E G L E E K E G A H M N P E I Y L F V K
541 GCT GGA ATC GAT GGA GAA AGC ATC GGC AAC TGT CCT TTC TCT CAG CGC CTC TTC ATG ATC
181 A G I D G E S I G N C P F S Q R L F M I
601 TTT TGG CTG AAA GGA GTC GTG TTC AAT GTC ACC ACT GTG GAT CTG AAA AGA AAG CCA GCT
201 F W L K G V V F N V T T V D L K R K P A
661 GAC CTG CAC AAC TTA GCC CCC GGC ACG CAC CCG CCC TTC TTG ACC TTC AAC GGG GAC GTG
221 D L H N L A P G T H P P F L T F N G D V
721 AAG ACA GAC GTC AAT AAG ATC GAG GAG TTC CTG GAG GAG ACC TTG ACC CCT GAA AAG TAC
241 K T D V N K I E E F L E E T L T P E K Y
781 CCC AAA CTG GCT GCA AAA CAC CGG GAA TCC AAC ACA GCG GGC ATC GAC ATC TTT TCC AAG
261 P K L A A K H R E S N T A G I D I F S K
841 TTT TCT GCC TAC ATC AAA AAT ACC AAG CAG CAG AAC AAT GCT GCT CTT GAA AGA GGC CTA
281 F S A Y I K N T K Q Q N N A A L E R G L
901 ACC AAG GCT CTA AAG AAA TTG GAT GAC TAC CTG AAC ACC CCT CTA CCA GAG GAG ATT GAC
301 T K A L K K L D D Y L N T P L P E E I D
961 GCC AAC ACT TGT GGG GAA GAC AAG GGG TCC CGG CGC AAG TTC CTG GAT GGG GAT GAG CTG
321 A N T C G E D K G S R R K F L D G D E L
1021 ACC CTG GCT GAC TGC AAT CTG TTG CCC AAG CTC CAT GTG GTC AAG ATT GTG GCC AAG AAA
341 T L A D C N L L P K L H V V K I V A K K
1081 TAC CGC AAC TAT GAT ATC CCG GCT GAG ATG ACA GGC CTG TGG CGG TAC CTC AAG AAC GCC
361 Y R N Y D I P A E M T G L W R Y L K N A
1141 TAT GCC CGT GAT GAG TTC ACC AAC ACC TGT GCA GCT GAC AGT GAG ATC GAG TTG GCC TAC
381 Y A R D E F T N T C A A D S E I E L A Y
1201 GCT GAT GTC GCC AAA CGC CTC AGC CGA TCC
401 A D V A K R L S R S

B.
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Shanks, et al.: Page 32
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Shanks, et al.: Page 33

FIGURE 4
 Shanks, et al.: Page 34

FIGURE 5

A.

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B.
520 DANEIYEKSLLKALKKLDAYLNSPLPDEVDAYS-TEDVAVSGRKFLDGDDLTLADCNLLPKLHIIKIV PARCHORIN
125 ALNDNLEKGLLKALKVLDNYLTSPLPEEVDE-TSAEDEGVSQRKFLDGNELTLADCNLLPKLHIVQVV CLIC1
135 EANEALERGLLKTLQKLDEYLNSPLPDEIDENS-MEDIKFSTRRFLDGDEMTLADCNLLPKLHIVKVV CLIC4
132 QNNAALERGLTKALKKLDDYLNTPLPEEIDANTCGED-KGSRRKFLDGDELTLADCNLLPKLHVVKIV CLIC5A
317 QSNAALERGLTKALKKLDDYLNTPLPEEIDADTRGDDEKGSRRKFLDGDELTLADCNLLPKLHVVKIV BOVINE P64
291 QNNAALERGLTKALKKLDDYLNTPLPEEIDANTCGED-KGSRRKFLDGDELTLADCNLLPKLHVVKIV CLIC5B
XXXXXXXXXXXXXXXX
587 AKKYRDFEFPPEMTGIWRYLNNAYARDEFINTCPADQEIEHAYSDVAKRMK PARCHORIN
192 CKKYRGFTIPEAFRGVHRYLSNAYAREEFASTCPDDEEIELAYEQVAKALK CLIC1
202 AKKYRNFDIPKGMTGIWRYLTNAYSRDEFTNTCPSDKEVEIAYSDVAKRLTK CLIC4
199 AKKYRNYDIPAEMTGLWRYLKNAYARDEFTNTCAADSEIELAYADVAKRLSRS CLIC5A
385 AKKYRNYDFPAEMTGLWRYLKNAYARDEFTNTCAADSEIELAYADVAKRLSRS BOVINE P64
358 AKKYRNYDIPAEMTGLWRYLKNAYARDEFTNTCAADSEIELAYADVAKRLSRS CLIC5B
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FIGURE 6
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Shanks, et al.: Page 36
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Shanks, et al.: Page 37

FIGURE 8
 AKAP350 at the Golgi apparatus: II. Association of AKAP350 with a novel CLIC
family member
Ryan A. Shanks, M. Cecilia Larocca, Mark Berryman, John C. Edwards, Tetsuro
Urushidani, Jennifer Navarre and James R. Goldenring
J. Biol. Chem. published online August 5, 2002

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