Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 729-739
doi:10.1111/j.1745-7270.2008.00452.x
Proteome identification of binding-partners interacting with cell
polarity protein Par3 in Jurkat cells
Ying Zhou1,2#, Longhou Fang2#§, Dan Du2,3, Wenchao Zhou2,3, Xiujing Feng2,3, Jiwu Chen1, Zhe Zhang2, and Zhengjun Chen2*
1
School of Life Science,
East China Normal University, Shanghai 200062, China
2
State Key Laboratory of
Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200031, China
3
Graduate School of the
Chinese Academy of Sciences, Shanghai 200031, China
Received: April 3,
2008 Accepted: May 11,
2008
This work was
supported by grants from the National Natural Science Foundation of China (Nos.
30730055, 30700129 and 30623002) and the National Basic Research Program of
China (No. 2007CB914504)
#These authors
contributed equally to this work
§Current address:
Department of Medicine, University of California, San Diego, CA 92093, USA
*Corresponding
author: Tel, 86-21-54921081; Fax, 86-21-54921081; E-mail, [email protected]
The evolutionarily conserved cell polarity
protein Par3, a scaffold-like PDZ-containing protein, plays a critical role in
the establishment and maintenance of epithelial cell polarity. Although the
role of Par3 in establishing cell polarity in epithelial cells has been
intensively explored, the function of Par3 in hematopoietic cells remains
elusive. To address this issue, we generated GST-fusion proteins of Par3 PDZ
domains. By combining the GST-pull-down approach with liquid
chromatography-tandem mass spectrometry, we identified 10 potential novel
binding proteins of PDZ domains of Par3 in Jurkat cells (a T-cell line). The
interaction of Par3 with three proteinsnuclear transport protein importin-a4 and proteasome activators PA28b and PA28gwas confirmed using in vitro binding assay,
co-immunoprecipitation assay and immunofluorescence microscopy. Our results have
the potential to uncover novel functions of the cell polarity protein Par3 in
blood cells.
Keywords cell polarity; Par3; proteomics; importin-a4; PA28b; PA28g
Cell polarity is of crucial importance to the differentiation and functions
of most metazoan cells [1,2]. In the early embryogenesis of Caenorhabditis
elegans, Par3 localizes asymmetrically at the periphery of the one-cell
embryo and is essential for embryonic polarity. Loss-of-function mutation in Par3
gene results in mislocalizing cell fate determinants, incorrectly orientating
mitotic spindles and early embryonic lethality [3,4]. Bazooka, the Drosophila
orthologues of Par3, is also required for the generation of asymmetric
division of neuroblasts and oocyte differentiation [5,6]. In Xenopus,
the Par3 forms a complex with Par6/aPKC, which is asymmetrically localized to
the animal pole of oocytes [7]. In epithelial cells, Par3 interacts with
transmembrane JAM and/or nectin via its PDZ1 domain to facilitate membrane
localization [8,9]. As a scaffold protein, Par3 can also interact with
Par6/aPKC through its PDZ1 domain to establish tight junctions [10,11], while
its C-terminus interacts either with Tiam-1 to regulate the activity of Rac or
with LIM kinase 2 to regulate the phosphorylation of coffilin and, hence, the
organization of actin filament to promote the establishment of tight junctions
[12,13]. The Par3/Par6/aPKC complex also specifies the neuronal axons [14].
Although the function of Par3 has been extensively investigated in epithelial
cells and neuron cells, its role in other tissues, such as hematopoietic cells,
has barely been explored. While Par3 180K is believed to be involved in the
formation of tight junctions, Par3 150K is the major form detected in Jurkat cells
[15], strongly indicating that Par3 has different function in the hematopoietic
cells. Par3 protein contains three different PDZ domains. Hitherto, protein
partners for the PDZ domains of Par3 have been poorly understood, although some
interacting proteins have been revealed [8,10,16]. PDZ domains were originally
identified in the postsynaptic density protein PSD95, Drosophila septate
junction protein Dlg, and the epithelial tight junction protein ZO-1, hence
acronym PDZ. It is one of the well-known domains that mediates protein-protein
interactions with a variety of functions, including cell polarization,
migration, tumorigenesis, and metastasis. PDZ-containing polarity proteins,
include Par3, are differentially localized throughout polarized T cells and
regulate T-cell polarity and functions [17]. However, the molecular mechanism
by which Par3 participates in T-cell functions is not understood. To find novel
Par3- interacting proteins, we carried out GST-fusion protein pull-down assays
in Jurkat cells (a T-cell line) by using its PDZ domains as bait. In
combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS), 10
potential binding proteins of Par3 were identified. Three of them were further
confirmed to be associated with Par3. Our results should help to uncover novel
functions of Par3 in blood cells.
Materials and methods
Antibodies and plasmidsMonoclonal antibody against hemagglutinin A (HA) and proteinase
inhibitor cocktail tablets (for a broad spectrum of serine and cysteine proteases)
were purchased from Roche (Basel, Switzerland). Monoclonal antibody against
green fluorescent protein (GFP) was purchased from Santa Cruz Biotechnology
(Santa Cruz, USA). Monoclonal antibody against a-tubulin was from
Sigma-Aldrich (St. Louis, USA). Polyclonal antibody against Par3 was purchased
from Upstate (New York, USA). Antibody against GST was prepared by our own
laboratory.Par3 plasmid was a kind gift from Dr. Ian Macara (University of
Virginia, Charlattesville, USA). Par3 PDZ1 domain (251–385 aa) and Par3 PDZ2-3
(425–695 aa) domain were amplified and inserted in-frame into pGEX2T
(Amersham Pharmacia Biotech, Forster, USA) or pET-3E-His (kindly provided by
Dr. J. Ding of Institute of Biochemistry and Cell Biology, Shanghai, China). To
be expressed in mammalian cells, the Par3 PDZ1 cDNA was cloned in-frame into
pEGFPC1 by the forward primer 5‘-GGAATTCAGAACCTGTTGGACATGCTG-3‘
and the reverse primer 5‘-CGGGATCCGTCAGGGCTAAAACGGCTT-3‘.Par6A cDNA was cloned from normal human liver tissue by the forward
primer 5‘-CGGGATCCATGAACCGAAGTTTTCACAAG-3‘ and the reverse
primer 5‘-GCTCTAGAGAGCGTGACCGCGGGC-3‘. Par6C cDNA was cloned
from normal human liver tissue by the forward primer 5‘-CGGAATTCCACGAGGCACCTGCGCCTCG-3‘
and the reverse primer 5‘-GCTCTAGAGAGGCTGAAGCCACTACCATCTC-3‘
and then cloned into pEGFPC1 or pcDNA3VSV. PA28b and PA28g (kindly
provided by Dr. Sherwin Wilk of the Mount Sinai School of Medicine, New York
City, USA) were recloned into a mammalian expression vector, pEGFPC1, by the PA28b forward primer
5‘-CCGCTCGAGCTATGGCCAAGCCGTGTGG-3‘ and the PA28b reverse primer
5‘-AAAACTGCAGTCAGTACATAGATGGCTTTTC-3‘ ; and by the PA28g forward 5‘-CCCAAGCTTCGATGGCCTCGTTGCTGAAG-3‘
and the PA28g reverse primer 5‘-CCGGAATTCTCAGTACAGAGTCTCTGCA-3‘ respectively.GST-fusion proteins were produced in Escherichia coli BL21DE
and purified on Glutathione Sepharose 4B (Amersham Pharmacia Biotech, Forster
City, USA), His-tagged Par3 PDZ2-3 was produced in BL21DE3 and was purified on
TALON Metal Affinity Resin (Clontech, USA) according to the standard protocol
[18].Importin-a4 subunit complementary DNA (cDNA) was cloned from human placenta by
the forward primer CGGGATCCCGAGCCATGGCGGACAAC and the reverse primer CCGCTCGAGCTAAAACTGGAACCCTTCTG.
It was then inserted in-frame into pcDNA3-N-HA (HA tag in the N-terminus,
kindly provided by Dr. G. Pei of the Institute of Biochemistry and Cell
Biology, Shanghai, China).
Cell culture and transfectionHuman embryonic kidney (HEK) 293T, or 293 cells and HeLa cells (ATCC),
were maintained in Dulbeccos modified Eagles medium (Gibco, Carlsbad, USA)
supplemented with 10% newborn calf serum (Gibco). Jurkat cells were cultured in
RPMI 1640 medium (Gibco) supplemented with 10% newborn calf serum. For
exogenous expression of proteins in cells, we used calcium-phosphate-mediated
transfection method [19].
In vitro binding assayTen million cells were lysed with 1 ml
radio immunoprecipitation assay (RIPA) buffer [50 mM Tris-Cl with pH 7.5, 1%
Triton X-100, 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM NaF, 100 mmol phenylmethylsulphonyl
fluoride and cocktail (Roche, Basel, Switzerland)]. Then the lysate was
incubated with purified GST-fusion proteins conjugated to glutathione 4B beads;
for Coomassie Brilliant Blue staining, 3 mg purified proteins were
used, while for Western blotting, 0.3 mg GST-fusion proteins were used. The beads
were washed with HNTG buffer (20 mM HEPES with pH7.5, 150 mM NaCl, 0.1%
Trition-X-100 and 10% glycerol), unless otherwise mentioned, and eluted with
SDS sample buffer. Proteins were separated by Sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed using
Coomassie Brilliant Blue R250 staining.
Western blotCells were lysed with RIPA buffer. Lysates were quantified using the
Bradford quantification method [20]. Proteins were fractionated by
one-dimensional SDS-PAGE and analyzed by immunoblotting with proper antibodies.
Antibodies were detected by enhanced chemiluminescence (Pierce, Rockford, USA).
ImmunoprecipitationHEK293 cells were washed three times with 0.01 M phosphate-buffered
saline (pH 7.4) 24 h after transfection. Cells were lysed in RIPA buffer on
ice. After 15 min, the lysates were sonicated for 2?2 s at 100 W and
centrifuged at 13,000 rpm for 5 min at 4 ?C. The pellets were then discarded. For immunoprecipitation, rabbit anti-PDZ1 polyclonal antibodies were
used. Lysates were mixed with primary antibodies and Protein A-Sepharose beads at
4°C overnight on a head-over-tail rotator. The beads were washed three times
with HNTG buffer before loading buffer was added. Then the samples were
subjected to SDS-PAGE.
Protein identification by one dimensional-LC-MS/MSAll bands were analyzed using a LCQ Deca XP system (Thermo Finnigan,
San Jose, USA). Protein identification by one dimensional-LC-MS/MS was
performed as described previously [21]. Data from LC-MS were identified by
TurboSequest software (Thermo Finnigan), which used mass spectrometry (MS) and
MS/MS spectra of peptide ions to search against the Swiss-Prot non-redundant
database (species: Homo sapiens). The protein identification criteria
that we used were based on Delta Cn (g 0.1) and Xcorr (one charge g 1.9, two
charges g 2.2 and three charges g 3.75).
Immunofluorescent microscopyCells grown on glass cover slips (Fisher Biotech, Fairlawn, USA)
were washed several times with ice-cold phosphate-buffered saline (PBS), fixed
with 3.7% formaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 for
3–5
min. The cells were then blocked in Tris-buffered saline solution with Tween
(TBST) containing 1% bovine serum albumin for 1 h at room temperature. Primary
antibody incubations were performed at 4 ?C overnight in TBST containing 1%
bovine serum albumin. After being washed three times in TBST, samples were
incubated for 1 h with the secondary antibodies, Alexa Fluor 546 goat
anti-rabbit or Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes
Invitrogen, Carlsbad, USA). Cover slips were mounted using PermaFluor Aqueous
Mounting Medium (Immunotech, Marseille Cedex, France). Samples were analyzed
with a laser scanning confocal microscope (Leica, Wetzlar, Germany).
Results
Expression of Par3 150K and 100K in Jurkat cellsMost normal tissues, epithelial cells [10], and tumor cells (data
not shown) contain primarily Par3 180K, 150K and 100K. The role of Par3 180K is
to establish and maintain tight junctions in epithelial cells. However, there
are no tight junctions in Jurkat cells as they are hematopoietic cells.
Therefore, the expression pattern of Par3 in Jurkat cells has yet to be
determined. SDS-PAGE was used to separate 40 µg lysates from Jurkat cells or
Madin-Darby canine kidney (MDCK) cells; they were then analyzed using
immunoblotting with antibody against Par3. As shown in Fig. 1, unlike
MDCK cells, which expressed large amount of all the three isoforms of Par3,
Jurkat cells contained mainly Par3 150K, though there were trace amounts of
Par3 180K and Par3 100K (Fig. 1). No signal was detected in the IgG
control lanes, and the specific signals of Par3 could be blocked using the
purified His-PDZ2-3 protein, which covered 82.9% of the Par3 antigen,
suggesting the signals of Par3 in the two cell lines are specific. The different
expression pattern of Par3 isoforms in Jurkat cells may be indicative of its
different role or different interacting partner(s) in the cells.
Identification of proteins from Jurkat cells interacting with Par3
PDZ domainsWe next investigated the possible Par3 PDZ domains-interacting
proteins from Jurkat cells. Jurkat cell lysates were first pre-cleared by
Glutathione Sepharose 4B beads (GST beads) in order to remove non-specific GST
beads-binding proteins. Then the pre-cleared cell lysates were incubated with
the recombinant GST-PDZ1 or GST-PDZ2-3 domains of Par3 immobilized on GST beads
[Fig. 2(A)]. As shown in Fig. 2(B), four bands, respectively
sized 86, 60, 30 and 18 kDa, were specifically pulled down by GST-PDZ1 fusion
protein of Par3, and an 80 kDa band and a 70 kDa band by Par3 GST-PDZ2-3 fusion
protein. Subsequently, all these bands were selected for LC-MS/MS analysis. In
total, 10 different proteins were identified, including proteasome activators
PA28b and PA28g, nuclear transport protein, importin-a4, ribosomal proteins and
heat shock proteins. Using bioinformatics analysis, the identified proteins
were implicated in different cellular functions, such as protein metabolism,
energy metabolism and protein transportation (Table 1). The peptides
identified by the LC-MS/MS were shown in supplementary data.
Confirmation of the interaction between Par3 and importin-a4, PA28b and PA28g by co-overexpression in HEK293
cellsTo confirm the interaction between Par3 and the above identified proteins
from Jurkat cells, three important functional proteins, importin-a4, PA28b and PA28g, were selected
for further study. After molecularly cloning the cDNA of the selected genes,
HA-tagged importin-a4, GFP-tagged PA28b and GFP-tagged PA28g were constructed. Again, in vitro protein pull-down assays
were performed by incubating GST-fused Par3 PDZ1 or PDZ2-3 domains with
different lysates from HEK293 cells transiently expressing the constructed
proteins. As expected, overexpressed importin-a4, PA28b and PA28g could
specifically bind to Par3 PDZ1 domain, but not to Par3 PDZ2-3 domains or GST
alone (Fig. 3). These findings were consistent with the LC-MS/MS
findings.We next tested whether Par3 is associated with the three proteins in
vivo. To clarify this issue, full-length Par3 and PDZ1 domain of Par3
tagged by c-myc were co-transfected with HA-importin-a4, GFP-PA28g or GFP-PA28b in HEK293
cells. We found that Par3 PDZ1 was able to co-precipitate with GFP-PA28? [Fig.
4(A)], but not with HA-importin-a4 and GFP-PA28b (data not shown).
Similarly, the full-length Par3 could interact with PA28? [Fig. 4(B)],
but not with importin-a4 and PA28b (data not shown).
Co-localization of Par3 with importin-a4, PA28g or PA28b detected by
immunofluorescence To further study the interaction between Par3 and these candidate
proteins, their subcellular distributions were investigated using
immunofluorescent microscopy. HA-importin-a4, GFP-PA28g and GFP-PA28b were
respectively overexpressed in HeLa cells together with Par3. Overexpressed Par3
was, in most cases, localized in the cytosol and on the membrane, and had
relatively strong signals. Nuclear localization of overexpressed Par3 was
occasionally observed with weak signals (Fig. 5). Importin-a usually
localized at perinuclear regions. Confocal microscopy showed the perinuclear
co-localization of HA-importin-a4 and Par3, which suggested that these two proteins could interact
in cells. When PA28b, a cytoplasmic protein, co-transfected with Par3, a clear GFP-PA28b signal on the
membrane that codistributed with Par3 was detected. PA28g was reported as
a nuclear protein, and indeed, GFP-PA28g localized exclusively in
the nucleus, while most Par3 appeared in both the cytosol and nucleus.
Strikingly, a proportion of the cells exhibited a strong Par3 signal
co-localized with PA28g in the nucleus (Fig. 5, bottom row). Collectively, the
immunofluorescence data provided additional evidence for possible subcellular
co-localizations between Par3 and the three selected proteins.
Discussion
There are many techniques to systematically investigate
protein-protein interactions and protein networks. Among these approaches,
GST-pull down in combination with MS offers a fast and efficient way of
studying potential molecular interactions. Using this combination of
techniques, we identified several novel Par3 PDZ domain-interacting proteins in
Jurkat cell (a T-cell line), including importin-a4, PA28b and PA28g. The
interactions between these candidate proteins with Par3 were further confirmed
using several different approaches. Par3 is known as an essential player in polarity establishment and
maintenance. Interestingly, most identified Par3-interacting proteins in the
present work are associated with protein metabolism, such as heat shock
proteins (HSP) and proteasome activators PA28b and PA28g; the rest are
related to transport (e.g. importin-a4) or unknown functions. This may indicate
novel functions of Par3 in hematopoietic cells.
Importin-a Importin-a plays a critical role in nuclear localization signals (NLS) –
mediated nuclear importation process of selective protein [22–24]. In
eukaryotic cells, the selective transport of karyophilic proteins to the
nucleus is mediated by short amino acid sequences, which are commonly referred
to as NLS and are characteristically rich in basic amino acids [25–27]. We found
that Par3 from different sources (from Caenorhabditis elegans to human)
contained canonical NLS predicted by the PSORT program (http://psort.hgc.jp/)
(Table 2). Par3, although localized mainly in the tight junction of
Caco-2 or MDCK cells, also showed a clear nuclear signal before cells became
polarized [15]. It is not yet understood how Par3 translocates to the nucleus.
Our data showed that importin-a co-localized with Par3 in perinuclear structures, indicating that
Par3 might directly bind to importin-a to be transported to the nucleus. The
predicted NLS localizes in the C-terminus of human Par3 (Table 2), and
overexpressed Par3 C-terminus containing the predicted NLS localizes in the
nuclei of HeLa/MDCK cells (data not shown). It is possible that Par3
translocates to the nucleus by one or several NLS. However, Par3 does not
necessarily need to have a NLS of its own, as Par3 PDZ1 domain might interact
with nuclear proteins associated with importin-a; in this way, it can be translocated to the nucleus
through a piggyback mechanism [28,29]. Whether Par3 can govern its own
nuclear entry or requires an associated partner remains to be determined.
PA28Our results suggested that Par3 PDZ1 domain interacted with
proteasome activators PA28b and PA28g. In cells, proteasome 26S consists of a 20S central component and a
19S regulatory subunit [30,31]. There are two proteasome activators in higher
eukaryotes, called PA28 and PA200 [3234]. Both promote the degradation of
peptides and some poorly folded small proteins. PA28 heptamers form
donut-shaped rings that bind to the ends of the 20S proteasome and activate
peptide hydrolysis by opening the sealed ? subunit gate [35]. The PA28 family consists of a, b and g. PA28a and PA28b form 3a/4b-heteroheptamers
that are found mostly in the cytoplasm [36,37]. In addition to the cytoplasmic
signal, our data revealed that PA28b co-localized with Par3 on the membrane (Fig.
5). The significance of this translocation of PA28b is not clear. It has been
reported that cell polarity or tight junction protein stability is regulated by
proteasome degradation. During development, tissue repair and tumor metastasis,
both cell-cell dissociation and cell migration occur and appear to be
intimately linked, and what happened during epithelial scattering is a good
example for that. Interestingly, proteasome inhibitors prevented the
redistribution of proteins of the tight junction and the adherens junction
during cell scattering. Moreover, proteasome inhibition partly preserved cell
polarity during scattering. Thus, a proteasome-dependent step during scattering
appears to be involved in the loss of polarity protein [38]. The Par3-PA28b interaction may
be involved in proteasome-dependent degradation of tight junction components
during cell scattering. Yet, as a tight junction protein itself, Par3 can be a
substrate of PA28b as well.PA28g (originally named Ki antigen) forms homoheptamers that localize in
the nucleus. Consistent with this, overexpressed Par3 and PA28g co-localized in
the nuclei of HeLa cells in our study. Our previous results showed that Par3
may play a role in DNA repair via binding to Ku70/Ku80 [39]. Whether nuclear
co-localized PA28 is involved in Par3-Ku70/Ku80-regulated DNA repair is an
interesting question. One relevant observation was that a certain proportion of
cells exhibited strong nuclear signal of Par3 co-localized with PA28g when both were
overexpressed (Fig. 5, bottom row). This phenomenon did not appear when
Par3 was co-transfected with importin-a4 or PA28b. It is accepted that Par3,
as a tight junction protein, plays different roles by forming different
complexes in its variant subcellular locations. For example, Par3 was reported
to localize in centrosome during cell mitosis, indicating the involvement of
Par3 in cell proliferation [40]. Par3 was also shown to localize at the leading
edge of lamellipodia in migration cells [41]. All these data indicate that to
function properly, Par3 may localize differently in cells at different stages
and/or under distinct stimuli to interact with the applicable local binding
partners.
Other candidate proteinsSeveral HSPs were pulled down by Par3 PDZ domains. HSP 70 from E.
coli can bind to ribosome-bound nascent polypeptides [42]. Here, both HSP
and ribosome were found to associate with Par3, and the interaction might be
involved in the protection of tight junctions in epithelial cells during heat
stress.It should be noted that we unequivocally detected peptides
corresponding to the P isoform of phosphofructokinase (PFK), which likely
constitute the intense band observed in around 80 kDa [Fig. 2(B)] (Table 1).
This probably reflects this enzymes great abundance within the pool of
proteins that associate with Par3 PDZ1. In fact, enzymes, such as GADPH,
aldolase, pyruvate kinase, PFK and lactate dehydrogenase, have been observed to
assemble at microtubules. Their interactions are advantageous for glycolysis
[43,44]. Interestingly, Par3 associates with microtubule-based molecular motors
dynein LC8 [45]. Par3 might form a complex with PFK-dynein so that they
function together.It has been reported that T lymphocytes expressed mainly Par6C and a
low level of Par6B [46]. However, we failed to identify Par6 in the present
analysis, though the GST-Par3 PDZ1 fusion protein was proved to bind Par6 in
other cell lines, such as HEK293 (data not shown). In our assay, the 45 kDa
band of Par6C may be too close to the GST-PDZ1 band, and thus be masked [Fig.
2(B)]; the amount of the proteins around 55-60 kDa (the size of Par6B)
pulled down by GST-PDZ1 was too low for MS analysis. Though people often refer
to a Par3/Par6/aPKC complex with the implication that these three proteins are
constitutively linked to one another, this complex can in fact be both
functionally and spatially separate [47,48]. We previously identified Ku70/Ku80
as Par3-interacting factors in HeLa and A431 cells[39]. In Jurkat cells, using GST-Pars
PDZ2-3, we pulled down protein bands approximately the size of Ku70/Ku80.
However, the 80 kDa band was insufficient in amount for MS analysis, while the
large amount of HSP 70 could cover the weaker signal of Ku70. This might be due
to the distinct expression levels of Ku70/Ku80 in different cell lines.Although the interaction between Par3 and importin-a4, PA28b or PA28g was confirmed
by several methods, we were unable to co-precipitate importin-a4 or PA28b with Par3. This
may be due to several reasons. First, the interaction between importin-a4 or PA28b and Par3 in
cells could be transient and/or stimulation dependant. Second, the conformation
of Par3 in cells might be different from that of the recombinant GST-Par3.
Therefore, the association between importin-a4 or PA28b and Par3 in
cells might be rare or transient. This could suggest certain functions of Pars
are stimulation dependant. Consistent with this, Par3 was recently identified
as a novel component of the DNA-dependent protein kinase complex, and its
nuclear signal could be clearly enhanced by certain stimuli [39]. In a
constitutive situation, the Par3-importin-a4 association might be very
loose. Upon certain stimulation, their interaction would be greatly enhanced,
and Par3 could go into the nucleus to enhance DNA-dependent protein kinase
complex activity for efficient DNA repair. Comparably, Par3 may only interact
with PA28b during cell scattering to help in proteasome-dependent degradation
of tight junction proteins or to be degraded itself. Yet, most likely, this is
because importin-a4/Par3 and PA28b/Par3 complexes were not stable under the conditions that we used
for immunoprecipitation. This assumption is supported by the co-localization of
these proteins detected by immunofluorescence in cells (Fig. 5). We attempted to elucidate protein functions by unraveling
interactions that occur in cells. In this paper, we identified three novel
Par3-interacting partners, importin-a4, PA28b and PA28. Our findings may
shed light on the novel functions of Par3 in blood cells.
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