From the Howard Hughes Medical Institute, Cardiovascular Division,
Children's Hospital, Harvard Medical School, Boston, Massachusetts
02115
Since its cloning and tentative identification as
a chloride channel, the function of the pICln protein has been debated. Although there is no consensus regarding the specific function of
pICln, it was suggested to play a role, directly or indirectly, in the
function of a swelling-induced chloride conductance. Previously, the
protein was shown to exist in several discrete protein complexes. To
determine the function of the protein, we have begun the systematic identification of all proteins to which it binds. Here we show that
four proteins firmly bind to pICln and identify the 72-kDa pICln-binding protein by affinity purification and peptide
microsequencing. The interaction between this protein and pICln was
verified several ways, including the extraction of several pICln clones
from a cDNA library using the 72-kDa protein as a bait in a yeast
two-hybrid screen. The protein is homologous to the yeast Skb1 protein.
Skb1 interacts with Shk1, a homolog of the
p21Cdc42/Rac-activated protein kinases (PAKs). The
known involvement of PAKs in cytoskeletal rearrangement suggests that
pICln may be linked to a system regulating cell morphology.
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INTRODUCTION |
Expression of the pICln cDNA in Xenopus laevis
oocytes was correlated with the appearance of a nucleotide-sensitive
chloride current (I = current, Cl = chloride, n = nucleotide-sensitive) (1-4). Although
pICln was tentatively identified as an integral component of the
chloride channel (1), several observations were inconsistent with the
channel hypothesis for pICln. First, pICln lacks predicted hydrophobic
membrane-spanning domains and structural homology to known channel
proteins (1). Second, in mammalian cells and Xenopus
oocytes, pICln was abundant and exhibited a predominantly cytoplasmic
and nuclear localization, whereas a small fraction (<5%) was
associated with the cytoskeleton (5). No pICln was detected in the
plasma membrane.
The chloride conductance associated with expression of pICln was
similar to an endogenous Xenopus oocyte chloride current elicited by hypotonic challenge (6). An anti-pICln antibody specifically ablated the swelling-induced chloride current in Xenopus oocytes (5), a finding supported by antisense
experiments in mammalian cells (7). For the reasons stated above, we
proposed that pICln was a cytosolic regulator of a swelling-induced
chloride channel rather than a channel itself (5). In contrast,
Paulmichl and co-workers (8) maintain that pICln is the
swelling-induced chloride channel itself. Recently, data were presented
suggesting that the chloride channel evoked by pICln expression has
properties different from the swelling-induced chloride current,
including a higher permeability to NO3
, stronger
outward rectification, and voltage-dependent nucleotide block (3). The molecular identification of the swelling-induced chloride channel has proven difficult, and several proteins including P-glycoprotein, pICln, ClC-2, and ClC-3 have been proposed to constitute this channel (9, 10). Although it seems unlikely that either
pICln or P-glycoprotein are themselves chloride channels, both ClC-2
and ClC-3 are well established members of a family of chloride channel
proteins. In contrast, pICln exhibits no significant homology to any
known mammalian protein and contains no domains that suggest a specific
function.
Although work from several laboratories supports a link between pICln
expression and activation of a chloride current, the nature of this
link is not clear. Currently there are no data to suggest that pICln
directly regulates a chloride channel. Indeed, pICln may act far
upstream from any plasma membrane-associated event and participate in
such diverse functions as transcriptional or translational regulation,
cytoskeletal rearrangement, or any one of several signal transduction
cascades. pICln was shown previously to exist in several discrete
complexes with other cytosolic proteins (5). We reasoned that the
identification of proteins interacting with pICln might reveal
functional connections to signaling pathways or known cellular
mechanisms. Here we report the identification of one such
pICln-interacting protein, a 72-kDa protein that appears to be the
human homolog of Skb1. Skb1 is a yeast protein that interacts with
Shk1, a homolog of the p21Cdc42/Rac-activated protein
kinases (PAKs).1 Although the
function of PAKs are only beginning to be understood, they appear to
affect cell morphology through interactions with the cytoskeleton
(11).
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EXPERIMENTAL PROCEDURES |
IBP72 Affinity Purification--
Rat pICln coding sequence was
subcloned into the pGEX-2T plasmid (Amersham Pharmacia Biotech). The
GST-pICln fusion protein was expressed in BL-21 bacteria and purified
over glutathione-Sepharose according to the manufacturer's protocols.
GST-pICln was immobilized using ActiGel ALD (Sterogene) at 2 mg of
protein/ml of gel. Bovine ventricular tissue was minced and homogenized
by Polytron (setting 7) for 3 × 30 s in MB buffer (10 mM Na-HEPES (pH 7.5), 20 mM KCl, 1 mM EGTA, 3 mM MgCl2, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 2 mg/ml each of aprotinin, leupeptin, and pepstatin). Following centrifugation at 100,000 × g, the
supernatant (2.6 g of protein) was loaded onto a 2 × 25 cm
DEAE-Sephacel (Amersham Pharmacia Biotech) column and washed with MB
containing 100 mM NaCl. pICln-containing complexes (as
detected by Western blotting) were eluted with MB + 400 mM
NaCl. The eluate was supplemented with Triton X-100 (1% final
concentration) and rotated overnight with 100 µl of GST-pICln resin.
After washing beads with MB, 400 mM NaCl, 1% Triton X-100,
bound proteins were solubilized in SDS loading buffer, separated by
SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene
fluoride film, and visualized with Coomassie staining. The 72-kDa
protein band was excised, digested with trypsin and cyanogen bromide,
and microsequenced (Mayo Foundation).
Constructs, Northern Blot, Yeast Two-hybrid Analysis, and Cell
Transfection--
IBP72 coding sequence was subcloned into pGEX-2T,
and the GST-IBP72 fusion protein was produced and purified as described above. The full-length IBP72 clone was subcloned into pCDNA 3.1(
) (Invitrogen) and translated in vitro using the
TNT system (Promega). pICln deletions were generated by
polymerase chain reaction (PCR) subcloning of truncated versions of
human pICln coding sequence into pCDNA3.1(+) or (
); all
constructs were "tagged" with an amino-terminal FLAG epitope
(DYKDDDDK). 10 µg of each construct was used for calcium phosphate
transfection of 50-70% confluent HEK293 cells plated in 10-cm culture
dishes. 48 h after transfection, proteins were in vivo
labeled for 5-12 h using 50 µCi/ml [35S]methionine
(Amersham). IBP72 coding sequence was subcloned into pBTM-116KN vector
(18) using restriction sites introduced by PCR. The resultant construct
was verified by DNA sequencing and used as the bait for two-hybrid
screening of a human heart Matchmaker library
(CLONTECH) in the yeast strain L40 (18).
Full-length, FLAG-tagged pICln coding sequence was cloned into pGAD424
(CLONTECH). Yeast his3 expression was
assayed by growth on dropout plates lacking histidine, tryptophan, and
leucine and supplemented with 5 mM 3-aminotriazole.
-Galactosidase activity was measured using o-nitrophenyl-
-D-galactopyranoside (Sigma) as
substrate. The multi-tissue human Northern blot
(CLONTECH) was probed according to the
manufacturer's specifications with a [32P]dCTP
random-labeled fragment (Stratagene) consisting of the entire human
IBP72 human coding sequence.
Cell Lysis, Immunoprecipitation, and Immunoblotting--
Total
cell lysate for immunoprecipitation was obtained as 100,000 × g supernatant after cell lysis in buffered solution
containing 1% Triton X-100 and 350 mM NaCl. Soluble
proteins were isolated by Dounce homogenization followed by pelleting
of microsomal fractions at 40,000 rpm in an SW-55 rotor for 30 min at
4 °C. The supernatant was supplemented with Triton X-100 to a final
concentration of 1% and with NaCl to 350 mM. For
immunoprecipitation, samples were precleaned with 40 µl of Protein
A/G-Sepharose (Amersham Pharmacia Biotech) for 1.5 h at 4 °C.
pICln was immunoprecipitated using the aFP antibody as described (5).
GST-pICln (1 µg) was used for precipitation of in vitro
translated IBP72. Precipitation of the deletion constructs and
interacting proteins was achieved using Protein G-Sepharose and
anti-FLAG M2 monoclonal antibody (Kodak). The rabbit anti-IBP72
antibody was generated against the GST-IBP72 fusion protein and
affinity-purified.
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RESULTS |
pICln was immunoprecipitated from
[35S]methionine-labeled Madin-Darby canine kidney (MDCK)
total cell lysates using a polyclonal antibody generated to a GST-pICln
fusion protein. Several proteins consistently co-immunoprecipitated
with pICln (pICln-binding proteins (IBP)) with electrophoretic
mobilities corresponding to molecular masses of 72, 43, 29, and 17 kDa
(Fig. 1, lane 1). Since the
same set of associated proteins was co-immunoprecipitated from the water-soluble cell fraction, we concluded that the IBPs are not membrane-associated proteins. Their association with pICln was judged
to be specific because the same set of proteins was
co-immunoprecipitated with a different anti-pICln antibody (data not
shown).

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Fig. 1.
Purification of the 72-kDa protein
interacting with pICln. Lane 1, pICln was immunoprecipitated
from the cytosolic fraction of MDCK cells labeled in vivo
with [35S]methionine. Four proteins interact specifically
with pICln. Lane 2, large scale isolation of IBP72 from
bovine heart using immobilized GST-pICln. Lane 3, proteins
bound to immobilized GST.
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IBP72 was purified by affinity to pICln. Cytosolic extracts from bovine
heart were used as the source of IBP72 since initial experiments
indicated that it was relatively abundant in this tissue. IBP72 was
enriched significantly in eluates from the GST-pICln resin (Fig. 1,
lane 2). Immobilized GST did not bind this protein, indicating that the 72 kDa protein interacted with GST-pICln
specifically (Fig. 1, lane 3). The purified 72-kDa protein
was digested with trypsin and cyanogen bromide, and five different
peptides were sequenced. The peptide sequences obtained from the 72-kDa
protein were used to screen the expressed sequence tag (EST) data base. Several overlapping clones were identified that predicted a single open
reading frame (ORF) of 1911 base pairs and whose translation contained
sequences identical with the 72-kDa protein-derived peptides.
Subsequently, two EST clones containing an identical 2.4-kb insert were
identified (GenBankTM accession numbers R13970 and
AA099674) that spanned the ORF. Using 5'-rapid amplification of
cDNA ends with a human fetal brain library, an additional 60 bases
of 5'-untranslated sequence were identified.
The ORF predicts a protein of 637 amino acids with a molecular mass of
72.6 kDa (Fig. 2A). Consistent
with this prediction, in vitro translation of the cDNA
yielded a protein with an apparent molecular mass of 72 kDa (Fig.
2B). Only two residues are not conserved between the bovine
and human proteins within the sequence specified by the five fragments.
A 2.4-kb transcript for IBP72 was identified in a wide range of human
tissues including skeletal muscle, brain, heart, placenta, kidney,
pancreas, lung, and liver (Fig. 2C). Although the cloned
72-kDa protein has no significant homology with other cloned mammalian
proteins and contains no consensus structural motifs, it does exhibit
moderate homology to putative proteins encoded in the
Caenorhabditis elegans and Saccharomyces
cerevisiae genomes, as well as significant homology to the
skb1 gene product (12) from Schizosaccharomyces
pombe (52% homology; Fig. 2A). Recently, a human
cDNA identical with our IBP72 was cloned by homology to Skb1 and
submitted to GenBankTM (accession number AF015913).

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Fig. 2.
Primary structure and alignment, in
vitro translation, and tissue expression of the cloned IBP72.
A, amino acid sequence of IBP72 and alignment with Skb1.
Microsequenced peptides are underlined, and amino acid
residues not conserved between the bovine peptides and predicted human
protein sequence are labeled with an asterisk. B, in
vitro translation of IBP72 produced a single band with an apparent
molecular mass of 72 kDa, consistent with the value predicted from the
ORF translation. C, Northern blot containing mRNA from
several human tissues demonstrates the ubiquitous expression pattern of
IBP72 mRNA, a single species of 2.4 kb.
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The cloned 72-kDa protein appears to be IBP72 as indicated by several
approaches. First, the in vitro translated protein exhibited specific binding to the GST-pICln fusion protein but not to GST alone
(Fig. 3, left panel). Second,
an affinity-purified polyclonal antibody raised against the recombinant
72-kDa protein recognized IBP72 co-immunoprecipitated with pICln (Fig.
3, right panel). Third, a LexA-IBP72 fusion protein
specifically interacts with a GAL4 activation domain-ICln fusion
protein in the yeast two-hybrid system (Table
I). Moreover, when a human heart cDNA
library was screened in the yeast two-hybrid system using the
LexA-IBP72 fusion protein as bait, six independent ICln clones were
obtained. These results argue strongly that our cloned protein is
IBP72.

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Fig. 3.
The cloned IBP72 is identical to the native
protein interacting with pICln. Lanes 1 and 2, GST-pICln fusion protein (lane 1) but not GST alone
(lane 2) binds in vitro translated,
[35S]methionine-labeled IBP72. Lane 3, the
antibody raised to recombinant IBP72 recognized IBP72
co-immunoprecipitating with pICln; lane 4 shows no
recognition by the anti-IBP72 antibody of a 72-kDa protein in control
immunoprecipitates with an unrelated antibody. The pICln antibody does
not immunoprecipitate in vitro translated IBP72. The
lower band in lanes 3 and 4 corresponds to the heavy chain of the primary antibodies recognized by
the anti-rabbit secondary antibody.
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Table I
Interaction of IBP72 with pICln in the yeast two-hybrid system
Yeast-harboring plasmids expressing the indicated proteins were assayed
for growth on selective plates containing 5 mM
3-aminotriazole (AT) and for -galactosidase activity.
-Galactosidase activities are given in -galactosidase units (19)
and represent the average ± S.D. of three independent
measurements. GAD is the Gal4 activation domain (amino acids 768-881).
GAD-pICln-DN103 is a pICln clone that lacks the amino-terminal 103 amino acids.
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To identify the domain(s) of pICln critical for interaction with IBP72,
we generated several epitope-tagged (FLAG) human pICln deletion
constructs and examined their ability to bind native IBP72. The
full-length FLAG-pICln protein and endogenous pICln interacted with the
same set of proteins in human embryonic kidney (HEK293) cells (data not
shown). All deletion constructs were expressed at levels equivalent to
or higher than the full-length FLAG-pICln protein, as assessed by
immunocytochemical analysis and immunoprecipitation of
[35S]methionine-labeled proteins (data not shown). Based
on this approach, we conclude that the extreme carboxyl terminus of
human pICln, specifically the last 37 amino acids, is critical for
interaction with IBP72 (Fig. 4).
Consistent with this result, one of the pICln clones identified by
yeast two-hybrid selection has the amino-terminal 103 residues deleted
but retains full ability to interact with IBP72 (Table I).

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Fig. 4.
IBP72 interacts with the carboxyl terminus of
pICln. Sequential amino- and carboxyl-terminal truncations of the
human pICln cDNA were tagged with the FLAG epitope and transfected
into HEK293 cells. After [35S]methionine in
vivo cell labeling, cytosolic extracts were immunoprecipitated
with the anti-FLAG M2 antibody, and the presence of IBP72 was assessed
by autoradiography of SDS-electrophoresed proteins.
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DISCUSSION |
In an effort to identify the functional role of pICln, we are
characterizing IBPs. Four major IBPs consistently co-purify with pICln
in several tissues. We cloned the human cDNA for IBP72 based on
microsequence data obtained from affinity-purified bovine IBP72. The
interaction between the cloned human IBP72 and pICln was confirmed by
several lines of evidence, including the extraction of pICln from a
cDNA library using the full-length coding sequence for IBP72 as the
bait in a yeast two-hybrid screen. IBP72 is ubiquitously expressed and
has no identified mammalian homologs or recognizable structural motifs
that would suggest a specific function. Currently we are cloning the
other IBPs and will use similar approaches to verify the specificity of
their interaction with pICln.
Although IBP72 has no known human homologs, sequence similarity
suggests that IBP72 represents a human homolog of the Skb1 protein from
S. pombe (12). Skb1 was identified by a yeast two-hybrid screen using the S. pombe protein kinase Shk1 as bait. The
Shk1 kinase is linked to Ras- and Cdc42-dependent signaling
cascades regulating cell viability, morphology, and mitogen-activated
protein kinase-mediated pheromone responses (13). S. pombe
lacking Skb1 are less elongated than wild-type yeast, emphasizing the
role of this protein in the regulation of cell morphology. Shk1 is a
homolog of the mammalian PAK, of which there are three cloned isoforms
(14). PAK kinases are activated by GTP-bound forms of the small
GTP-binding proteins Rho, Rac, and Cdc42 and have been implicated in
control of cytoskeletal rearrangement and cell morphology (11, 14).
One consistent conclusion from previous studies of pICln function is
that its overexpression induces, either directly or indirectly, the
appearance of a chloride conductance (1, 2, 4). Given the biochemical
characteristics of pICln, we favor the hypothesis that pICln is not a
channel itself but rather part of a pathway either closely or remotely
connected to a chloride current, possibly through cytoskeletal
rearrangement. Indeed, actin co-immunoprecipitated with pICln, and a
fraction of pICln associated with insoluble cytoskeletal elements (5).
The protein identified in this report, IBP72, may provide a link
between pICln and cytoskeletal rearrangement. Regulation of
swelling-induced chloride channels is likely to involve cytoskeletal
rearrangement (15, 16). Also, recent evidence links
p21Rho-dependent cytoskeletal reorganization to
a swelling-induced chloride conductance (17). Whether pICln and IBP72
are linked to a swelling-induced chloride current (5-7), a
volume-insensitive chloride conductance (2, 3), or both will be
determined only by understanding all elements of the pathway.