1 Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia
University College of Physicians and Surgeons, New York, NY 10032, USA
2 Department of Pathology, Columbia University College of Physicians and
Surgeons, New York, NY 10032, USA
3 Department of Anatomy and Cell Biology, Columbia University College of
Physicians and Surgeons, New York, NY 10032, USA
* Author for correspondence (e-mail: rkl2{at}columbia.edu)
Accepted 14 November 2002
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Summary |
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Key words: Gas2, GAR17, GAR22, Microfilament, Microtubule
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Introduction |
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The human Gas2-related gene on chromosome 22 (hGAR22) was
originally identified in a search for putative tumor suppressors
(Zucman-Rossi et al., 1996).
It is closely related to growth arrest specific gene 2 (Gas2) and is not a
member of the plakin family. The primary transcript of hGAR22
undergoes alternative splicing resulting in two mRNA splice-forms
(Zucman-Rossi et al., 1996
).
The sequences of the hGAR22 proteins contain both a putative calponin homology
(CH) actin-binding domain (ABD) and a putative microtubule-binding domain
(MTBD) of the recently described Gas2-related (GAR) family
(Sun et al., 2001
). Nothing is
known about the functions or biochemical properties of the protein products of
the hGAR22 gene.
Gas2 was identified in a screen for genes induced in murine fibroblasts by
growth arrest (Schneider et al.,
1988). Its protein product also contains a CH domain and a GAR
domain. Gas2 protein has been shown to associate with MFs in cultured cells,
presumably through its CH domain
(Brancolini et al., 1992
). Its
GAR domain colocalizes with MTs in transfected COS7 cells
(Sun et al., 2001
). Gas2 is
phosphorylated on serine and threonine during the G0 to G1 transition. This
phosphorylation event is thought to be specifically required for the formation
of membrane ruffles following serum stimulation of quiescent cells
(Brancolini and Schneider,
1994
). Gas2 is cleaved by caspases in cultured cells undergoing
apoptosis (Brancolini et al.,
1995
; Sgorbissa et al.,
1999
). The resulting N-terminal fragment contains the CH domain
and dramatically reorganizes the MF network of the cells, culminating in its
collapse around the nucleus. The caspase-mediated cleavage of Gas2 is
therefore hypothesized to be necessary for the morphological changes
characteristic of apoptotic cells. Consistent with a role of Gas2 in
apoptosis, Gas2 expression and cleavage are induced in hindlimb interdigital
tissues of mouse embryos between days 13.5 and 15.5, a developmental stage
characterized by extensive apoptosis in these tissues
(Lee et al., 1999
). Whether
Gas2 is an indispensable component of the apoptotic machinery in this or any
other in vivo system remains to be determined.
On the basis of the presence of a putative ABD and a putative MTBD in its sequence, hGAR22 could potentially mediate interaction between MFs and MTs in vivo. In this report, we describe the cytoskeleton-binding properties of the hGAR22 proteins as well as their individual domains. We characterize the regulation of GAR22 expression in a number of mammalian cell lines by growth arrest and the GAR22 mRNA and protein expression patterns in both human and mouse tissues. We also report the cDNA cloning of the mouse GAR22 (mGAR22) orthologue and a close human GAR22 homologue, the Gas2-related gene on chromosome 17 (hGAR17).
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Materials and Methods |
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All pFLAG-hGAR22 constructs used in this study are schematically depicted
in Fig. 1D. The original hGAR22
cDNA clone (GenBank/EMBL/DDBJ accession no. Y07846) was purchased from the
I.M.A.G.E. consortium. This clone was used as the template in the PCR
reactions performed to generate all the pFLAG-hGAR22 constructs. In order to
construct pFLAG-hGAR22 and pFLAG-hGAR22ß, the PCR products
obtained with sense primer 5'-ATGAATTCAATGGCAGACCCAGTGGCG-3' and
antisense primers 5'-ATTCTAGATTCACCTGGGTGGGGTCTCGGG-3' and
5'-ATTCTAGATTCACATCCAGGAATCTGG-3', respectively, were digested
with EcoRI and XbaI and ligated into pFLAG cut at these
sites. pFLAG-hCHD22 was similarly constructed using sense primer
5'-ATGAATTCAATGGCAGACCCAGTGGCG-3' and antisense primer
5'-ATTCTAGATTCACATGCGGGGGCCGCGGGC-3'. For the construction of
pFLAG-hGARD22, sense primer 5'-ATGAATTCAACACCCAGCGACCTGCGC-3' and
antisense primer 5'-ATTCTAGATTCACCTGGGTGGGGTCTCGGG-3' were used.
pFLAG-hCt22 was constructed using sense primer
5'-ATGAATTCACCCCGGGATCAGCTGCCC-3' and antisense primer
5'-ATTCTAGATTCACATCCAGGAATCTGG-3'. pFLAG-hGARt22 was constructed
by ligating the PCR product obtained with sense primer
5'-AACACCCAGCGACCTGCGCAAC-3' and antisense primer
5'-ATTCTAGATTCACATCCAGGAATCTGG-3' into pFLAG opened at the
EcoRV and XbaI sites. Prior to ligation, the product was
digested with XbaI and treated with T4 polynucleotide kinase to
introduce a phosphate at its 5' end.
The pET vector system (Novagen) was used to create a prokaryotic expression
plasmid used to produce recombinant hGARD22 protein for immunization.
pET16b-hGARD22 was constructed by ligating the product of a PCR performed on
pFLAG-hGAR22 with sense primer
5'-CTCGACGAGCATATGAGGGAGATTCTG-3' and antisense primer
5'-CTCGGGATCCTGCATCTCACCTGGGTGG-3' into pET-16b opened at the
NdeI and BamHI sites. Prior to ligation, the PCR product was
digested with the same two enzymes.
All constructs were sequenced (DNA Sequencing Facility, Columbia University, New York) to ensure the absence of PCR-introduced errors.
cDNA cloning
mGAR22 cDNA was cloned from a mouse brain Marathon-ReadyTM cDNA
library (Clontech) by 5' RACE followed by full-length PCR. Two sets of
primers, one nested relative to the other, were used sequentially in each
5' RACE and full-length cDNA amplification. For 5' RACE, the first
set of primers consisted of Adaptor Primer 1 (Clontech) as the sense primer
(5'-CCATCCTAATACGACTCACTATAGGGC-3') and an antisense primer whose
sequence (5'-GAACTGTACCAGCCTCGGGGCAAGC-3') was based on that of a
partial mGAR22 EST clone (GenBank/EMBL/DDBJ accession no. AA718230). The
second nested set of primers consisted of the sense Adaptor Primer 2
(Clontech; 5'-ACTCACTATAGGGCTCGAGCGGC-3') and another EST
clone-based antisense primer (5'-AGCACCACACTCTTCTCGTTCTTG-3').
Products of the nested 5' RACE PCR were subcloned into pCR2.1-TOPO
(Invitrogen) and sequenced. These sequences were used to design the sense
primers used for the amplification of full-length mGAR22 cDNA
(5'-CGGGACTTCGTGCAGTGACTCCAC-3' first round primer;
5'-TGCAGTGACTCCACTGGCTCTGGGC-3' nested primer). The
antisense primers were based on a mouse polyA site sequence clone
(GenBank/EMBL/DDBJ accession no. M89786;
5'-TATAATGAAGGCTCAGTCCCCAAAA-3' first round primer;
5'-AAACAGGAGCTGGAGCAAGACTTTA-3' nested primer). Products
of the nested full-length PCR were subcloned into pCR2.1-TOPO and
sequenced.
Two sets of primers were used to clone full-length hGAR17 cDNA from a human Universal QuickCloneTM cDNA library (Clontech) by nested PCR. The first primer set consisted of sense primer 5'-CCACCTCCTGCCCTGCTGGGGTCCA-3' and antisense primer 5'-CATGGCCATCCTTTTTGCTTCCCTC-3'. The second, nested set consisted of sense primer 5'-GTCCAGCCATGTCCCAGCCTGCGGG-3' and antisense primer 5'-TTTTGCTTCCCTCCTACCCAACACG-3'. The products of the nested PCR were subcloned into pCR2.1-TOPO and sequenced.
Pfu polymerase (Stratagene) was used in all PCR reactions. Cycling parameters were set according to the library manufacturer's recommendations (Clontech). All sequencing was performed by the Columbia DNA Sequencing Facility (Columbia University, New York).
Antibody production
The C15 antibody was raised against a GAR-domain-containing fragment (AAs
213-337) encoded by pET16b-hGARD22 and containing a histidine tag at its
N-terminus. Recombinant protein was produced in BL21 bacteria and purified on
Ni2+ columns according to the pET system manual (Novagen).
Antibodies were raised in rabbits by Pocono Rabbit Farm & Laboratory, Inc.
(Canadensis, Pennsylvania). The C15 antibody was affinity purified on Affi-Gel
10 (Bio-Rad) conjugated to recombinant hGARD22 immunogen.
In vitro binding assays
MF and MT-binding assays were performed as described previously
(Leung et al., 1999a). The STP3
T7 reticulocyte in vitro transcription/translation system (Novagen) was used
to produce [35S]methionine-labeled hGAR22 and hGAR17 proteins. All
proteins were prespun before incubation with MFs or MTs. The Non-Muscle Actin
Binding Protein Spin-Down Biochem Kit (Cytsokeleton, Inc.) was used for the
MF-binding assays. The Microtubule Associated Protein Spin-Down Assay Kit
(Cytoskeleton., Inc.) was used for the MT-binding assays. BioMax X-ray film
(Kodak) was used for autoradiography.
Northern blot analyses
Human or mouse Poly A+ RNA Multiple Tissue Northern
(MTNTM) blots (Clontech) were hybridized with
[35P]dCTP-labeled random-primed species-specific probes
corresponding to the +83/+413 region of hGAR22 or mGAR22 or to the +1147/+2385
region of hGAR17ß. Hybridization and washing parameters recommended by
the manufacturer were used. BioMax X-ray film (Kodak) was used for
autoradiography.
Cell culture, transient transfections and fluorescent microscopy
All cells were cultured at 37°C in an atmosphere containing 5%
CO2. COS7 cells were grown in DMEM (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (FBS). CAD cells [a gift from Dona
Chikaraishi, Duke University (Qi et al.,
1997)] were grown in DMEM-F12 (Life Technologies, Inc.)
supplemented with 8% FBS. NIH 3T3 cells were grown in DMEM with 10% calf
serum. GC4spc cells [a gift from Peter Burfeind, University of Gottingen,
Germany (Tascou et al., 2000
)]
were grown in DMEM with 10% FBS. In serum starvation experiments, CAD cells
were cultured in serum-free DMEM-F12; COS7, NIH 3T3 and GC4 cells were
cultured in their appropriate media with the serum content reduced to 0.1%. In
nocodazole treatment experiments, cells were treated with 10 µM nocodazole
(Sigma) in standard medium for 1.5 hours before being fixed and stained. For
transient transfection, cells were seeded on coverslips and transfected with
plasmid DNA using LipofectAMINE PLUS reagent (Life Technologies, Inc.). 24
hours after transfection, the cells were fixed with methanol for 5 minutes at
20°C or with 4% paraformaldehyde in PBS for 10 minutes at room
temperature, followed by extensive washing in PBS. The cells were then
extracted with 0.5% Triton X100 in PBS for 15 minutes at room temperature,
blocked with 5% normal goat serum for 30 minutes and incubated with primary
antibodies or phalloidin for 1 hour. After washing three times with PBS, the
cells were incubated with secondary antibodies for 1 hour, washed with PBS
again, and mounted onto slides in Aquamount (Lerner Laboratories). The
following reagents were used for cell staining: phalloidin-AlexaFluor-594,
phalloidin-AlexaFluor-350 (Molecular Probes), mouse monoclonal anti-FLAG M2
antibody (Sigma), rabbit polyclonal anti-tubulin antibody (Sigma),
AlexaFluor-594-conjugated goat-anti-rabbit antibody and
AlexaFluor-488-conjugated goat-anti-mouse antibody (Molecular Probes). A Nikon
Eclipse 800 fluorescent microscope and a SPOT digital camera were used to
capture images of the stained cells. All images were processed using Adobe
Photoshop 6.0.
Quantification of triple colocalization
COS7 cells were transiently transfected with pFLAG-hGAR17ß,
pFLAG-hGAR22ß, pFLAG-hGAR17 or pFLAG-hGAR22
and
triple-stained for actin, tubulin and the FLAG epitope. 30 cells transfected
with each construct were selected randomly and their pictures taken to
generate an RGB triple image. A cell was scored as positive if it contained at
least one triple-stained (white) filament equal or exceeding one fifth of the
length of the cell or at least two triple-stained filaments each equal or
exceeding one tenth of the length of the cell. The
2 criterion
with the Yates discontinuity correction factor was used to compare the
observed frequencies of triple colocalization
(Bailey, 1981
).
Immunohistochemistry
Cryostat sections of paraformaldehyde-fixed testes from 3-week- or
5-month-old C57BL/6 mice were mounted on glass slides, blocked with 5% normal
goat serum for 30 minutes at room temperature and incubated with the C15
antibody for 1 hour. The sections were then washed with PBS several times and
incubated with TRITC-conjugated goat-anti-rabbit antibody (Sigma) for 1 hour.
After extensive washing with PBS, the sections were sealed with coverslips and
Aquamount (Lerner Laboratories) and analyzed by fluorescent microscopy.
Immunoprecipitation, western blotting and phosphatase treatment
To prepare cell lysates for immunoprecipitation, cells were washed wish
PBS, scraped into 1 ml of lysis buffer (PBS, pH 7.4; 1% Triton X100, protease
inhibitors), vortexed briefly, incubated on ice for 10 minutes, and spun at
maximum speed in a tabletop centrifuge. The supernatant was collected and
rotated at 4°C for 1 hour with 20 µl of the C15 antibody. 40 µl of
Sepharose-Protein-A beads (Sigma) was then added and the sample rotated at
4°C for another hour. The beads were washed four times with lysis buffer,
boiled in SDS-containing denaturing buffer, and the resulting sample run on an
SDS-PAGE gel. The proteins were transferred onto ImmobilonTM-P
membrane (Millipore), probed with the C15 antibody followed by incubation with
HRP-conjugated goat-anti-rabbit antibody. The HRP staining was visualized
using the ECL system (Amersham/Pharmacia). Total cell lysates were prepared by
boiling cells in denaturing buffer. Mouse tissue extracts were prepared by
homogenizing dissected tissues in 50 mM Tris-HCl, pH 6.8, 1% SDS on ice,
centrifuging the extract to pellet the debris and boiling the supernatant in
denaturing buffer. In phosphatase treatment experiments, GAR22 protein was
immunoprecipitated with the C15 antibody from CAD or COS7 cells serum starved
for 48 hours, incubated with protein phosphatase 1 or T-cell phosphatase (New
England Biolabs) for 30 minutes at 30°C in buffer supplied by the
manufacturer and western-blotted and stained as described above.
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Results |
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Both hGAR17 isoforms contain a CH domain. hGAR17ß also contains a GAR
domain and an unstructured C-terminal sequence that we have designated C-tail
17 (Ct17). The N-terminal portions of hGAR17ß and hGAR22ß display a
high degree of sequence similarity. Over the first 337 amino acids, the
proteins are 56% identical, with the CH domains exhibiting
62% and
the GAR domains
75% sequence identity. The remaining C-terminal sequences
are relatively poorly conserved (
30% identity). The hGAR17 sequences are
available from GenBank/EMBL/DDBJ under accession Nos. AF508784 (hGAR17
)
and AF508785 (hGAR17ß).
Cytoskeleton-binding properties of hGAR17 and hGAR22 isoforms
Alternative splicing gives rise to two hGAR22 mRNA splice-forms
(Zucman-Rossi et al., 1996)
(Fig. 1C). The longer mRNA
encodes the hGAR22
protein of 337 amino acids with a deduced molecular
mass of 36.3 kDa. The shorter mRNA encodes hGAR22ß protein of 681 amino
acids with a deduced molecular mass of 72.6 kDa. The first 337 amino acids of
hGAR22ß are identical to those of hGAR22
and contain a putative
ABD of the CH family followed by a putative MTBD of the GAR family. The 334
additional amino acids present at the C-terminus of hGAR22ß do not
contain any known domains or extended secondary structures. We refer to this
portion of hGAR22ß as the C-tail 22 (Ct22).
In order to study the cytoskeleton-binding properties of the hGAR17 and
hGAR22 isoforms, we subcloned their cDNAs into the eukaryotic expression
vector pFLAG, a derivative of pcDNA3 described previously
(Leung et al., 1999b)
(Fig. 1B,D). The resulting
constructs were transiently transfected into NIH 3T3 or COS7 cells. The former
cell line showed better stress fibers and was used to study actin
colocalization; the latter cell line had a better microtubule network and was
used to study MT colocalization. To visualize the transfected proteins
(FLAG-tagged at the NH2 terminus), we used a monoclonal anti-FLAG
antibody. Both
isoforms localized predominantly in the cytoplasm,
exhibiting strong colocalization with actin filaments, particularly stress
fibers (Fig. 2C,F). Neither
hGAR17
nor hGAR22
colocalized with MTs, even though the GAR22
protein contains a GAR domain (Fig.
2I,L). Both ß isoforms colocalized with actin filaments
(Fig. 3C,F). They also showed
some colocalization with MTs, which was more evident in some cells than others
(Fig. 3I,L). Some of the
protein was also distributed in the cytoplasm in a diffuse pattern.
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In order to ascertain that the CH and GAR domains of the GAR proteins are functional ABDs and MTBDs, respectively, we created pFLAG constructs encoding amino acids 1-200 of hGAR22 (pFLAG-hCHD22, for CH domain 22), amino acids 201-337 of hGAR22 (pFLAG-hGARD22, for GAR domain 22) or amino acids 204-281of hGAR17ß (pFLAG-hGARD17; Fig. 1B,D). The resulting constructs were transfected into NIH 3T3 or COS7 cells and the expressed proteins visualized by antibody staining. hCHD22 protein colocalized with actin filaments (Fig. 4C), suggesting that the CH domain contained within it is likely to be an ABD. Both hGARD17 and hGARD22 proteins exhibited colocalization with MTs, although some of the proteins were also diffusely distributed in the cytoplasm (Fig. 4F,I). These observations indicate that the GAR domains of hGAR17 and hGAR22 are likely to be MTBDs. We also studied the intracellular localization of proteins corresponding to the C-tail portions of hGAR17ß (amino acids 282-880, encoded by pFLAG-hCt17) and hGAR22ß (amino acids 338-681, encoded by pFLAG-hCt22) as well as of proteins encompassing the GAR domain and the C-tail of the two ß isoforms (hGAR17ß amino acids 204-880, encoded by pFLAG-hGARt17; hGAR22ß amino acids 201-681, encoded by pFLAG-hGARt22; Fig. 1B,D). Both hCt17 and hCt22 proteins colocalized with MTs in a significant fraction of transfected cells; these proteins also tended to accumulate in the nucleus, possibly due to an overall positive charge (Fig. 5C,F). hGARt17 and hGARt22 both colocalized with and apparently bundled MTs in the majority of transfected cells (Fig. 5I,L). These data suggest that the C-tail sequences of the two ß isoforms possess an intrinsic MT-binding capacity. This added capacity is probably responsible for the pronounced MT-bundling ability of the GARt fragments. The GARD, Ct and GARt constructs of both hGAR17 and hGAR22 did not exhibit any significant degree of colocalization with MFs in transfected cells. Conversely, hCHD22 did not colocalize with MTs (data not shown).
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To assess the MT-stabilizing potential of the various truncated hGAR17 and hGAR22 proteins, we examined their ability to protect MTs from depolymerization by nocodazole in transfected COS7 cells. Consistent with their MT-bundling capacity, only the GARt fragments were able to protect MTs from depolymerization; the preserved MTs also appeared bundled. No other transfected hGAR17 or hGAR22 constructs used in this study conferred on MTs any protection from nocodazole (data not shown).
hGAR17 and hGAR22 proteins bind MFs and MTs in vitro
To verify our findings concerning the cytoskeleton-binding properties of
the hGAR17 and hGAR22 proteins in transfected cells, we conducted a series of
in vitro binding assays using the same constructs that were used for transient
cell transfection. In these assays, radioactively labeled hGAR17 or hGAR22
proteins produced using a reticulocyte lysate in vitro
transcription/translation system were incubated with preassembled MFs or MTs,
the reactions spun at high speed and the pellet and supernatant fractions run
on an SDS-PAGE gel. The labeled proteins were visualized by autoradiography.
Any filament-bound protein would be expected to cosediment with the filaments
and be present in the pellet fraction, whereas non-bound protein would remain
in the supernatant.
As shown in Fig. 6, both
hGAR17 and hGAR22
bound MFs in vitro, consistent with their
colocalization with actin filaments in transfected cells. Neither
isoform cosedimented with MTs, indicating that the GAR domain of hGAR22
is likely to be masked. The ß isoforms bound both MFs and MTs, consistent
with their localization in transiently transfected cells. hCHD22 protein
cosedimented with MFs but not with MTs. Combined with the finding that
hGAR17
binds only MFs in vitro, these data confirm that the CH domains
of hGAR17 and hGAR22 are ABDs. hGARD17 and hGARD22 proteins cosedimented only
with MTs, indicating that the GAR domains of hGAR17 and hGAR22 can bind MTs.
Similar results have been shown for the GAR domains of MACF, BPAG1a and Gas 2
(Sun et al., 2001
). In
addition, like the C-terminal domains of MACF and BPAG1a, the Ct domains of
hGAR17ß and hGAR22ß also bind MTs. Therefore, these two domains may
bind cooperatively to MTs.
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The cytoskeleton-binding properties and domain organization of the hGAR17
isoforms are remarkably similar to those of the hGAR22 isoforms. The only
notable exception is the absence of a GAR domain in hGAR17. However,
the absence of the GAR domain in hGAR17
does not result in
cytoskeleton-binding characteristics that are significantly different from
those of hGAR22
.
Molecular cloning of mGAR22 cDNA
At present, the mouse is the mammalian model of choice for biomedical
researchers, mainly because of its amenability to genetic modification. We
therefore were interested in cloning the mouse orthologue of hGAR22.
To clone mGAR22 cDNA, we used 5' RACE followed by PCR amplification of
full-length cDNA. We obtained two mGAR22 sequences, apparent products of
alternative splicing. One of them contains an open reading frame of 1011 bp
and encodes a putative protein of 337 amino acids with a deduced molecular
weight of 36.5 kDa. The sequence of this protein is 89% identical to that
of hGAR22
. This protein represents the mGAR22
isoform. The other
amplified cDNA sequence contains an open reading frame of 2034 bp and encodes
a putative protein product of 678 amino acids with a deduced molecular mass of
72.4 kDa. This protein is
87% identical to hGAR22ß and represents
the mGAR22ß isoform. As in the case of hGAR22, the first 337 amino acids
of mGAR22ß are identical to the sequence of mGAR22
. When
transiently expressed in COS7 cells, mGAR22
and mGAR22ß exhibited
localization patterns practically indistinguishable from those of the
corresponding human isoforms (data not shown). The sequences of the mGAR22
cDNAs are available from GenBank/EMBL/DDBJ under accession Nos. AF508323
(mGAR22
) and AF508324 (mGAR22ß).
Endogenous expression of GAR17 and GAR22 in tissues and cell
lines
Northern blot analysis of human tissues demonstrated that hGAR17 is
selectively expressed in skeletal muscle
(Fig. 7A). Since hGAR17
and ß splice-form transcripts differ by only 47 bp
(Fig. 1A), they cannot be
distinguished on a northern blot. However, we have shown by RT-PCR on human
muscle mRNA that the ß transcript is the predominant hGAR17 mRNA species.
In the absence of an anti-GAR17 antibody, we have not yet been able to detect
endogenous hGAR17 proteins.
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We performed northern blot analyses of GAR22 mRNA expression in both human
and mouse tissues. Species-specific probes corresponding to the sequence of
GAR22 exon 2 were hybridized with human and mouse multiple-tissue northern
blots. In humans, GAR22 mRNA was present in all tissues studied, albeit at
greatly varying levels (Fig.
7B). The highest levels of hGAR22 mRNA were detected in the brain,
kidney, heart and liver. In all human tissues, the transcripts of hGAR22ß
(expected size 2.6 kb) were much more abundant than the transcripts of
hGAR22
(
3.1 kb). In mice, the highest levels of GAR22 mRNA were
detected in the testes, heart and liver. Substantial amounts were also present
in the kidney, brain and lung (Fig.
7C). The shorter transcripts of mGAR22ß appeared to be the
predominant mGAR22 transcript as well.
To study the expression profiles of GAR22 proteins, we used a rabbit
polyclonal antibody, designated C15, that was raised against the hGARD22
region of hGAR22 (Fig. 1D) and
affinity purified on recombinant immunogen protein. On western blots of mouse
tissues, this antibody recognized a band with an apparent molecular mass of
75 kDa that comigrated with mGAR22ß protein ectopically expressed in
COS7 cells. The band was present in testis and, to a lesser degree, in the
brain (Fig. 8A). The identity
of this band as mGAR22ß protein was confirmed by mass spectrometry (data
not shown). No bands corresponding to mGAR22
were recognized in any of
the tissues studied. Although mGAR22 mRNA was expressed in multiple tissues,
the testis and brain appear to be the only mouse tissues that express
GAR22ß at levels detectable by western blotting. Post-transcriptional
inhibition of gene expression or enhanced protein degradation might account
for the apparent lack of GAR22 proteins in other GAR22 mRNA-containing
tissues. Immunostaining of mouse testicular sections with the C15 antibody
showed that mGAR22ß is predominantly expressed in the cytoplasm of germ
cells at all stages of differentiation. In spermatozoa, the protein was
concentrated in the head (Fig.
9).
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Gas2, a close homologue of GAR22, has previously been shown to be
upregulated in growth-arrested cultured cells
(Brancolini et al., 1992). We
therefore studied the effects of growth arrest on GAR22 protein expression in
a variety of cultured cell lines of different origin. CAD, NIH 3T3 and GC4 are
murine cell lines derived from CNS neurons, fibroblasts and spermatocytes,
respectively; COS7 is a simian epithelial cell line. The GAR22 protein was
immunoprecipitated from cells that were either actively proliferating or
arrested by contact inhibition or serum starvation (CAD cells) for 48 hours,
and the precipitated protein detected by western blotting. Both
immunoprecipitation and immunodetection were performed with the C15 antibody.
In all four cell lines, expression of GAR22ß protein was induced by
growth arrest (Fig. 8B). No
GAR22
was detected in any of the cell lines under any conditions.
Growth arrest brought about by serum starvation of the three non-neuronal
lines resulted in similar GAR22ß induction (data not shown). In CAD
cells, GAR22ß levels reached a maximum by day 5 of serum starvation
(Fig. 8C). In COS7 cells,
GAR22ß protein levels plateaued by 24 hours of serum starvation (data not
shown). As revealed by phosphatase treatment of GAR22ß immunoprecipitated
from growth-arrested CAD or COS7 cells, the induced protein is likely to be
phosphorylated on serine and threonine, but only to a much lesser degree, if
at all, on tyrosine (Fig. 8D).
In none of the cell lines studied could GAR22 protein be detected by direct
western blotting, even in arrested cells. Given that the C15 antibody readily
recognizes both GAR22 isoforms in lysates of transfected cells (first lane in
Fig. 8A,B and data not shown),
this observation suggests that the absolute amounts of endogenous GAR22
protein expressed in cultured cell lines under any conditions are low.
hGAR22ß and hGAR17ß are likely to crosslink MFs and MTs in
transfected cells
hGAR22ß and hGAR17ß proteins contain both an ABD and an MTBD and
can associate with either MFs or MTs in transfected cells
(Fig. 3). These findings
suggest that these proteins can physically crosslink MFs and MTs. To explore
this possibility, we transfected pFLAG-hGAR22ß and pFLAG-hGAR17ß
into COS7 cells and stained the cells with a polyclonal anti-tubulin antibody,
a monoclonal anti-FLAG antibody, and fluorescently labeled phalloidin.
Fluorescently labeled secondary antibodies were used to visualize the FLAG and
tubulin stainings. Pictures of the cells were taken at wavelengths suitable
for all three labels and superimposed to produce an RGB image. In such an
image, areas of triple-color overlay, which would correspond to triple
colocalization and probable crosslinking, are represented in white.
Significantly more cells transfected with pFLAG-hGAR22ß
(Fig. 10) exhibited triple
colocalization than did cells transfected with pFLAG-hGAR22, the hGAR22
isoform that does not interact with MTs either in vitro or in vivo. Similar
results were obtained with pFLAG-hGAR17ß and pFLAG-hGAR17
(data
not shown). To quantify the levels of triple colocalization elicited by the
pFLAG-hGAR17 or pFLAG-hGAR22 constructs, 30 cells transfected with each
construct were selected randomly and their pictures taken. A cell was scored
as positive if it contained at least one triple-stained (white) filament equal
or exceeding one fifth of the length of the cell or at least two
triple-stained filaments each equal or exceeding one tenth of the length of
the cell. The
2 criterion was used to statistically compare
the observed frequencies of triple colocalization
(Bailey, 1981
). The frequencies
of triple colocalization in cells transfected with the ß isoforms of both
hGAR17 and hGAR22 were found to be significantly (P<0.005) greater
than those in cells transfected with their respective
isoforms. These
findings indicate that both hGAR22ß and hGAR17ß are likely to be
able to crosslink MFs and MTs in vivo.
|
We did not observe any differences between the cytoskeleton-binding profiles of hGAR22 and hGAR17 proteins with or without a FLAG epitope tag (data not shown). It is therefore highly unlikely that the proposed crosslinking ability of the ß isoforms results from the presence of a FLAG tag at their N-terminus.
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Discussion |
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Both hGAR22 and hGAR22ß contain a functional ABD of the CH
family and associate with actin filaments in transfected cells. They also bind
MFs in spin-down binding assays, indicating that the association with actin
filaments observed in vivo is likely to be direct. This association is
mediated by the CH domain, which is present in both isoforms. Both
hGAR22
and hGAR22ß also contain the previously characterized MTBD
of the GAR family (Sun et al.,
2001
). The GAR domain of hGAR22
appears to be masked, since
this isoform does not associate with MTs either in transfected cells or in
vitro. By contrast, the longer hGAR22ß protein associates with MTs in
transfected cells as well as in spin-down assays. hGAR22ß contains a
second MT-binding motif in its C-terminal tail. Various protein structure
prediction programs failed to identify any known types of domains or motifs in
this portion of hGAR22ß (amino acids 338-681), and this region is
predicted to be mostly unstructured. Our preliminary data indicate that the
sequence responsible for the MT-binding capacity of this region is located
between amino acids 338 and 489. Although both the GAR domain and the
C-terminal tail of hGAR22ß associate with MTs, the GAR domain binds more
efficiently than C-terminal tail (especially in vivo). A protein combining the
GAR domain and the C-terminal tail not only binds MTs but also appears to
bundle them in the majority of transfected cells. Furthermore, this protein
also stabilizes MTs and protects them from depolymerization by nocodazole.
These findings indicate that the GAR domain and the C-tail may bind MTs
cooperatively, resulting in a higher avidity of interaction. This enhanced
MT-binding capacity appears to be negated by the presence of the N-terminal CH
domain in the full-length isoform. Protease cleavage of hGAR22ß under
specific physiological circumstances could potentially release a
GAR-domain-containing C-terminal fragment that would be able to stabilize MTs.
However, we have found no evidence for such a cleavage (see below). Using a
combined RACE and RT-PCR approach, we have also cloned two cDNA species of
mGAR22. The mGAR22 transcripts encode proteins that are extremely
similar to hGAR22
and hGAR22ß (89% and 86% amino acid identity for
the
and ß isoforms, respectively). In transfected cells, the
cytoskeleton-binding properties of mGAR22
and mGAR22ß appear to be
identical to those of their human counterparts.
A database search indicated that human chromosome 17 contains a
GAR22-like gene. We cloned two hGAR17 cDNA species by nested PCR
using a human multiple-tissue cDNA library as the template. These cDNAs encode
two protein isoforms, hGAR17 and hGAR17ß, that share significant
sequence similarity with the hGAR22 proteins. The smaller hGAR17 isoform,
hGAR17
, contains a CH domain and binds MFs in vitro and in vivo. Its
characteristics are generally analogous to those of hGAR22
. The larger
hGAR17 isoform, hGAR17ß, contains both a CH domain and a GAR domain.
These domains are functional, as hGAR17ß colocalizes with MFs and MTs in
transfected cells and cosediments with both types of filaments in spin-down
assays. The overall structure and properties of hGAR17ß are similar to
those of hGAR22ß. Northern blot analysis indicates that hGAR17 mRNA is
expressed selectively in skeletal muscle. We do not yet have any information
regarding the hGAR17 protein expression pattern.
A number of proteins have been identified that can crosslink different
components of the cytoskeleton. Many of these proteins belong to the plakin
protein family (Svitkina et al.,
1996; Wiche, 1998
;
Leung et al., 2002
); some of
them have been shown to simultaneously interact with MFs and MTs in
transfected cells. Our laboratory has previously demonstrated that two members
of the plakin family, microtubule actin crosslinking factor, MACF, and BPAG1a,
can bridge MFs and MTs (Leung et al.,
1999a
; Leung et al.,
2001
). Certain non-plakin cytoskeleton-associated proteins, such
as MAP1 and MAP2, can do so in vitro
(Pedrotti et al., 1994
;
Pedrotti and Islam, 1997
;
Ozer and Halpain, 2000
). We
have found that the larger isoforms of hGAR22 and hGAR17, hGAR22ß and
hGAR17ß, increase the extent of coalignment of MFs and MTs in transfected
COS7 cells, most probably by crosslinking the two types of filaments. Thus,
the physiological functions of these proteins may involve mediation of
specific MF-MT interactions.
Gas2 protein is induced in cultured cells upon growth arrest
(Brancolini et al., 1992). It
has also been implicated in the apoptosis-associated rearrangement of the
cytoskeleton both in cultured cells
(Brancolini et al., 1995
) and
possibly in developing mammalian tissues
(Lee et al., 1999
). Caspase 3
is the main enzyme responsible for Gas2 cleavage in apoptotic cells
(Sgorbissa et al., 1999
), and
the N-terminal CH-domain-containing product of the cleavage affects the
changes in the cytoskeleton observed in cells undergoing apoptosis. We were
unable to detect hGAR22 protein cleavage in a variety of cell lines treated
with several apoptotic stimuli (data not shown). The expression of either
full-length hGAR22 isoforms or the CH domain-containing N-terminus in
transfected COS7 cells does not result in any notable changes in cell
morphology. Although hGAR22 protein expression is also induced by growth
arrest in cultured cells, the absolute levels of expression appear to be low.
Thus, we have been unable to visualize endogenous GAR22 protein in several
types of arrested cells by indirect immunofluorescent staining with a number
of anti-GAR22 antibodies (data not shown). The levels of expression in mouse
testis and brain, the only tissues with detectable amounts of endogenous GAR22
protein, are also low. Notably, it is only the larger GAR22ß isoform that
can be detected in both cultured cells and mouse tissues. Consistent with the
GAR22ß transcript being the predominant splice-form in both human and
mouse tissues, GAR22
protein is undetectable in mouse tissues as well
as in cultured cells. Gas2 protein is phosphorylated on serine and threonine
during the G0 to G1 transition, which is thought to be a mechanism of rapid
Gas2 inactivation following serum stimulation of arrested cells
(Brancolini and Schneider,
1994
). By contrast, we have found that endogenous GAR22ß is
heavily phosphorylated on serine and/or threonine in quiescent, G0-arrested
cells. Most of the phosphorylation probably occurs on the C-tail sequence,
which contains multiple putative PKA, PKC and casein kinase II target sites.
On the basis of the differences between Gas2 and GAR22 mentioned above, we
believe that the in vivo function of GAR22 is different from that of Gas2.
Further studies will be required to determine the physiological roles of GAR22
and GAR17 proteins.
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