(Received for publication, May 30, 1996, and in revised form, November 18, 1996)
From the Institute for Virus Research, Kyoto
University, Kyoto 606, Japan,
Precursory Research for
Embryonic Science and Technology, Japan Science and Technology
Corporation, Kyoto 619-02, Japan and § Center for Molecular
and Cellular Biology, Osaka University, Suita, Osaka 565, Japan
The versatility of integrin functions is mediated
by engagement of a number of proteins that assemble with integrins.
Among them, paxillin is one of the important molecules interacting with a variety of signaling molecules and cytoskeletal building blocks. We
report here that paxillin is not a single molecule with a unique physiological property. We identified two human paxillin isoforms, and
. These isoforms have distinct amino acid insertions; each consists of a distinct exon, at the same site of previously reported paxillin (paxillin
). Several proteins were co-precipitated with paxillin, and we found that
bound to focal adhesion kinase but weakly to vinculin, and
bound to vinculin but only weakly to focal
adhesion kinase, although both bound equally to talin. No additional
proteins were found to bind to
and
over those binding to
.
Unlike the
isoform,
and
mRNAs were not detected in normal tissues, but several cancer cells expressed both
and
proteins simultaneously. All three isoform proteins were expressed in
promonocytic cells with ratios comparable with each other, and the
expression patterns were altered during differentiation of floating
promonocytic cells into adherent macrophage-like cells. Therefore, each
isoform of paxillin exhibits distinct expression and different
biochemical as well as physiological properties and thereby appears to
act as a distinct module involved in different functions of
integrins.
Integrin heterodimers mediate cell adhesion to
extracellular matrixes (ECMs)1 and to other
cells. Integrin-mediated cell adhesion plays a critical role in
directing cell motility, growth, differentiation, survival, and
invasion through intracellular regulation of a variety of signals, such
as protein tyrosine phosphorylation, calcium ion mobilization, inositol
lipid metabolism, and proton efflux (for reviews, see Refs. 1-7).
Integrins themselves have no intrinsic enzymatic activity; integrins appear to function by interacting with other proteins possessing cytoskeletal as well as catalytic signaling properties, primarily with their cytoplasmic domains (for reviews, see Refs. 4 and 7-9). A number of proteins have been identified as able to associate with the cytoplasmic domains of integrins or are localized to focal adhesion plaques where integrins interact with the ECM to connect the extracellular environment with intracellular effectors, resulting in the organization of the actin-containing cytoskeleton (for reviews, see Refs. 7 and 10-12) (13-15). Conversely, intracellular events also affect integrin conformation and affinity to the ECM (for reviews, see Refs. 1 and 16).
It has been demonstrated that triggering of an integrin fibronectin
receptor response involves hierarchies of protein interactions that
assemble with the integrin (13, 14). Moreover, integrin signaling
pathways vary with integrin isoforms even within a single cell type
(17). Thus, to achieve a high diversity of integrin-mediated signals,
different cytoskeletal building blocks and signal transducers may be
engaged to each distinct integrin and focal assembly. Based on our
current knowledge of the biochemical properties of protein-protein interactions among focal adhesion proteins, several possible
architectures of complexes formed at focal adhesions or connecting
integrins to actin stress fibers can be drawn. In this regard,
-actinin, talin, vinculin, and tensin have been shown to bind
directly to actin; and
-actinin, talin, and focal adhesion kinase
(Fak) (15) bind directly to the cytoplasmic domain of integrin
1
(for review, see Ref. 7). Paxillin is also associated with the
cytoplasmic peptide of integrin
1 (15; also see below). These
proteins may interact with each other to connect integrin and actin.
For example, vinculin binds to talin, tensin, and
-actinin (for
review, see Ref. 7); Fak binds to paxillin (18-20), which binds to
vinculin (18, 21; also see below). Different combinations of these
actin- and integrin-binding proteins may then form different
architectures of focal assembly frameworks. Protein modification such
as tyrosine (and perhaps serine and threonine) phosphorylation, which
mediates protein binding to the src homology 2 (SH2) domains
could also participate in the alteration of the organization of the
focal adhesion assembly (for review, see Ref. 7). Moreover, a variety of alternative splicing products exist not only for integrins and ECM
proteins but also for focal adhesion proteins, such as vinculin
(22-25) and Fak-related nonkinase (26).
Paxillin was originally identified as a substrate for the v-Src
tyrosine kinase (27) and shown to be highly localized at focal adhesion
plaques (21). cDNA cloning of human and chicken paxillins has
revealed their primary structure (18, 28). At its COOH terminus,
paxillin contains four repeats of the LIM domain, which may mediate
protein-protein interactions, as in the case of zyxin (29). Proteins
interacting with these LIM domains are unknown. Besides putative
LIM-mediated binding, paxillin has been shown to bind to vinculin (18,
21, 30), talin (28), and Fak (18- 20). In addition, paxillin binds to
the SH3 domain of c-Src (31). Paxillin can also be recovered within a
complex of proteins binding to the cytoplasmic domain peptide of
integrin 1 (15). Although it is not clear whether this binding is
direct, Fak is not essential for this binding (15). Moreover, tyrosine phosphorylation of paxillin is accompanied by cell adhesion to the ECM
and disappears when the cell adhesions are not maintained (32). This
cell adhesion-dependent tyrosine phosphorylation of
paxillin creates binding sites for the SH2 domains of COOH-terminal Src
kinase (33) and v-Crk, an adapter molecule that consists only of SH2
and SH3 domains (34-36). Inhibition of cell adhesion-mediated protein
tyrosine phosphorylation has been shown to affect the formation of
focal adhesions and cell cycle progression to S phase (32). Therefore,
binding of paxillin to cytoskeletal building blocks, which may result
in connection of integrin with actin, and interaction of paxillin with
a variety of signaling molecules highlight the important role of
paxillin in focal adhesions. Paxillin and its modifications, such as
tyrosine phosphorylation, appear to be crucial in regulating focal
adhesion assemblies, as well as integrin-mediated signal
transduction.
When we analyzed paxillin on two-dimensional SDS-PAGE, we detected at
least eight different spots cross-reacting with the anti-paxillin
antibody.2 Several seemed to be generated
by tyrosine and serine phosphorylation (27, 33, 35). However, it is
also possible that paxillin consists of several isoforms of primary
protein structure. Thus, we were sensitive to this possibility when we
isolated paxillin cDNAs. Moreover, we chose U937 human promonocyte
cells as a source of mRNA for potential isolation of paxillin
isoforms, the expression of which might be restricted to floating cells
or change during the differentiation of the cells into macrophage-like
cells, accompanied with altered cell adhesion (37). Here, we report two
different isoforms of paxillin, and
, generated by insertion of
distinct exons. These two novel isoforms, along with the isoform
previously reported (isoform
; Ref. 28), were expressed
simultaneously in monocyte cells. Several cancer cells expressed both
and
isoforms, but not the
isoform. Biochemical analysis
revealed that these isoforms exhibit different binding properties to
several proteins, including Fak and vinculin. Moreover, during the
maturation of monocyte cells, the protein level of the
isoform was
increased severalfold. The pattern of the expression of the
isoform
was also changed during this process. Thus each isoform of paxillin appears to have distinct physiological functions, possibly through formation and maintenance of different architectures and signaling complexes of focal adhesions.
U937, Jurkat, and HPB-ALL cells were cultured
in RPMI 1640 medium supplemented with heat-inactivated 10% FCS; HeLa
and BOCS23 cells were in Dulbecco's modified Eagle's medium with 10%
FCS; NIH3T3 cells were in Dulbecco's modified Eagle's medium with 5% FCS; and K562 cells were in Ham's F-12 medium with 10% FCS. For the
differentiation of U937 cells in vitro, cells were seeded at
3 × 105 cell/ml and treated with 1.6 × 107 M 12-O-tetradecanoylphorbol
acetate (TPA) for 3 days (37). The differentiation was assessed by the
appearance of about 50% of cells with an adherent macrophage-like
phenotype as described (37), and those cells undifferentiated and still
floating after the TPA treatment were washed off before harvesting the
cells. For radiolabeling, HeLa cells were incubated in the presence of 0.1 mCi/ml L-[4,5-3H]leucine (80 Ci/mmol,
Moravek Biochemicals) in Dulbecco's modified Eagle's medium (without
L-leucine, Life Technologies, Inc.) containing 10%
dialyzed FCS for 7 h, after being starved for
L-leucine by incubating in Dulbecco's modified Eagle's
medium (without L-leucine) with 10% dialyzed FCS for 30 min.
Standard methods (38) were used
for DNA and RNA manipulations, unless otherwise stated. Polyadenylated
RNA was prepared from cultured U937 cells according to the
manufacture's instructions (QuickPrep mRNA purification kit,
Pharmacia Biotech Inc.), and cDNA was synthesized from poly(A)
mRNA templates using random primers and reverse transcriptase
(Pharmacia). PCR amplification of human paxillin transcripts was then
performed using a combination of oligonucleotides (primer 1-24,
5-CCGGATCCATGGACGACCTCGACGCCCTGCTG-3
; primer 922-942,
5
-CAGCTTGTTCAGGTCAGACTG-3
; and a combination of primer 844-865,
5
-GGGAAGACAGGGAGCAGCTCAC-3
; and primer 1648-1674, 5
-CCGAATTCCTAGCAGAAGAGCTTGAGGAAGCAGTT-3
), all synthesized based on the reported human paxillin cDNA sequence (28). Nucleotide numbers in this article correspond to the sequence of human paxillin isoform
, with nucleotide A at the first ATG codon as 1. After digestion of the former PCR products with BamHI and
SphI (located at nucleotide 905 of isoform
) and the
latter with SphI and EcoRI, these DNA fragments
were ligated with BamHI-EcoRI-cleaved pGEX-2T vector (Pharmacia) using T4 DNA ligase (Takara Shuzo). The resulting plasmids were transformed into Escherichia coli DH5, and
each clone was isolated and subjected to DNA sequencing analysis.
High
molecular weight DNA isolated from human placental tissue was subjected
to PCR amplification using oligonucleotides of primer 742-768
(5-GTCACCTCCACCCAACAGCAGACACGC-3
) and primer 889-915
(5
-CCCCAGCATGCTGTCCAGCTGGCTCCC-3
) to isolate a genomic DNA fragment
encompassing paxillin
- and
-specific exons. A DNA fragment
produced was isolated by separating on 0.7% agarose gel
electrophoresis and then ligated into pT7Blue(R) vector bearing single
3
-T overhangs at its EcoRV site (Novagen). After
transformation of the resulting plasmid into E. coli
NovaBlue cells (Novagen), a single clone was isolated and subjected to
restriction enzyme mapping and sequencing analysis.
For PCR amplification of - and
-specific exons from this genomic
DNA fragment, combinations of oligonucleotides of primer
-5
(5
-ATCCAGGACCTGGAGCAAAGAGCG-3
) and primer
-3
(5
-CCCTCCCTCGTCCTGCCCTCC-3
), specific for isoform
, and primer
-5
(5
-GGCTCCTGGCCCCTGGAGGAG-3
) and primer
-3
(5
-CTGTAGACACGGAGGGGGCTG-3
), specific for isoform
, were
used (see Fig. 1D).
Exogenous Expression of Paxillin Isoforms
For the exogenous
expression of paxillin in mammalian cells, pBabePuro vectors (39)
bearing paxillin cDNAs were constructed as follows.
BglI-EcoRI fragments of each paxillin isoform
were isolated from the pGEX constructs, ligated with a synthetic double strand DNA fragment containing the 5-end coding region as well as the
Kozak sequences (underlined) of paxillin (28):
5
-GATCT
ATGGACGACCTCGACGCCCTGC-3
and
3
-AGGCCGGTACCTGCTGGAGCTGCGGG-5
. The ligated fragments were then
ligated into pBabePuro vector cleaved with BamHI and
EcoRI. After isolation of single clones with each isoform,
constructs were confirmed by sequencing analysis.
The resulting plasmid DNAs were transfected into BOSC 23 cells (40) by the calcium phosphate precipitation method, and 48 h after transfection recombinant viruses bearing pBabePuro and paxillins were collected. NIH3T3 cells were then infected with these viruses. After culturing the infected cells for 1 week in the presence of 2 µg/ml puromycin (Sigma), cells were harvested and analyzed for expression of paxillin by immunoblotting as described above.
GST Fusion ProteinspGEX-2T vectors containing cDNA
sequences encoding the human paxillin isoforms were expressed in
E. coli as glutathione S-transferase (GST) fusion
proteins by isopropyl--D-thiogalactopyranoside
induction. Bacterial lysates were incubated with glutathione-Sepharose
beads (Pharmacia) for 2 h at 4 °C and then washed extensively
with 1% Nonidet P-40 buffer (1% Nonidet P-40, 150 mM
NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, and 2 µg/ml leupeptin), as described previously (33, 35).
Cell lysates were prepared at 4 °C by solubilizing cells with 1% Nonidet P-40 buffer and clarifying for 10 min at 10,000 × g as described previously (33). Each 500 µg of lysates was incubated with 5 µg of the GST fusion proteins bound to glutathione-Sepharose beads at 4 °C for 2 h. Beads were then washed four times, or six times for radiolabeled cell lysates, with 1% Nonidet P-40 buffer, resuspended in Laemmli SDS sample buffer, and incubated for 5 min at 100 °C.
Proteins recovered with the beads were separated by 8% SDS-PAGE and transferred to membrane filters (Immobilin P, Millipore). After blocking with Tris-buffered saline containing 0.1% Tween 20 (Sigma) and 5% bovine serum albumin (Sigma), filter membranes were probed with appropriate antibodies as described previously (33, 35). The antibodies retained on filter membranes were then detected by a peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) and visualized by an enzyme-linked chemiluminescence method according to the manufacture's instructions (Amersham Corp.). The same filter was subjected to immunoblotting analysis sequentially with different antibodies, according to the manufacture's instructions (Amersham). Monoclonal antibodies used were anti-talin (8d4, Sigma), anti-vinculin (VIN-11-5, Sigma), and anti-Fak (Transduction Laboratories). A polyclonal antibody against Fak was raised against the COOH-terminal one-third portion of mouse Fak.
In the case of radiolabeled cell lysates, protein recovered with the beads was resolved by 8% SDS-PAGE. Gels were fixed in isopropanol:water:acetic acid (25:65:10) solution, soaked in Amplify solution (Amersham), and then dried and subjected to fluorography. Prestained rainbow markers (Amersham) were used as size standards.
mRNA Expression AnalysisFor detection of mRNA
expression of human paxillin isoforms, premade Northern blots of
polyadenylated RNA (Multi Tissue Northern blots, Clontech) were used.
Northern blotting analyses were done using a
BamHI-BamHI DNA fragment of pGEX human paxillin
encompassing 1-1072 nucleotides of human paxillin
coding
region, which can detect all the isoforms. For detection of paxillin
- and
-specific mRNA, probe DNA fragments were synthesized by
PCR amplification of each cDNA of paxillin, using oligonucleotides
of
-5
and
-3
for isoform
and
-5
and
-3
for isoform
. A probe for
-actin (Clontech) was used as a standard
control.
For reverse transcription-PCR detection of paxillin isoform mRNAs,
primer 597-617 (5-AGAGAAGCCTAAGCGGAATGG-3
) and an antisense primer
of nucleotides 1101-1121 (5
-TCCTTTTCACAGTAGGGCTGT-3
) were used for
detection of the
isoform, primer 597-617 and an antisense primer
corresponding to an internal sequence of
-specific exon (antisense
primer, 5
-ATCCGCTCTTTGCTCCAGGTC-3
) for detection of
, and
primer 597-617 and an antisense primer corresponding to an internal
sequence of
-specific exon (antisense
primer, 5
-GTACTTTTCTCCCTCCTGGAC-3
) for detection of
.
Anti-paxillin antibodies were
generated by immunizing rabbits with a keyhole limpet
hemocyanin-conjugated synthetic peptide of
Lys-Glu-Lys-Pro-Lys-Arg-Asn-Gly-Gly-Arg-Gly-Leu-Glu-Asp-Val-Arg-Pro-Ser-Val-Cys, which corresponds to amino acids 199-217 of the isoform sequence. The COOH-terminal Cys residue is for conjugation with keyhole limpet
hemocyanin. Antibodies raised (Ab 199-217) were purified using the
peptide used for the immunization. For generation of antibodies
specific for paxillin isoforms
and
, rabbits were immunized with
synthetic peptides of
ILe-Gln-Asp-Leu-Glu-Gln-Arg-Ala-Asp-Gly-Glu-Arg-Cys-Trp-Ala and
Ala-Gly-Trp-Pro-Arg-Asp-Gly-Gly-Arg-Ser-Ser-Pro-Gly-Gly-Gln-Asp-Glu-Gly, each conjugated with a multiple antigenic peptide (41) for
antibodies specific for the
isoform, or with synthetic peptides of
Leu-Val-Ser-Ile-Ser-Ser-Ser-Val-Gln-Glu-Gly-Glu-Gly-Glu-Lys-Tyr-Pro-His-Pro-Cys-Ala and
Ala-Arg-His-Arg-Thr-Pro-Ser-Leu-Arg-Ser-Pro-Asp-Gln-Pro-Pro-Pro-Cys, each conjugated with multiple antigenic peptide for the
isoform. For immunoprecipitation of paxillin, a monoclonal antibody
against paxillin (Transduction Laboratories) was used, coupled with
protein G-Sepharose (Pharmacia). Mouse IgG1 protein used as a negative control was purchased from Sigma.
Among four
paxillin cDNA clones we isolated using mRNA prepared from U937
cells, two cDNA clones showed the same sequence as the human
paxillin reported previously (Ref. 28 and Fig. 1).
However, the other two cDNA clones appeared to have additional sequences, as assessed by restriction enzyme mapping analysis. Sequence
analysis revealed that each of these longer cDNAs contains distinct
sequences, both of which were inserted at the same site (just after
amino acid Lys277, i.e. between nucleotides 831 and 832) of the reported paxillin sequences (Fig. 1B). The
inserted sequences are shown in Fig. 1D. Both of them were
linked in frame to both the 3- and 5
-ends of the flanking sequences
of human paxillin. Except for these inserted sequences, both cDNAs
showed the same sequence as that previously reported for human paxillin
(data not shown). We hereafter call the reported paxillin isoform
and our longer isoforms
and
, respectively. Isoform
contains
a 34-amino acid insert, and
contains a 48-amino acid insert
(Fig. 1, B and D).
To examine how these isoforms are generated, we isolated a
human genomic DNA fragment containing these inserted sequences. We have
amplified high molecular weight human genomic DNA using a combination
of two oligonucleotides, each corresponding to nucleotides 742-768 and
889-915 of human paxillin (Fig. 1B, primers 742-768 and 889-915). This amplification generated a single DNA band of 6.4 kb
in length (Fig. 1C). PCR amplification of high molecular weight genomic DNA with
and
exon-specific primers (Fig.
1B, combinations of
-5
and
-3
, and
-5
and
-3
), which were synthesized based on the inserted sequences of
and
isoforms, also generated a single band each, of the expected
size (Fig. 1C). The 6.4-kb DNA fragment was isolated from
the gel and cloned directly into the pT7Blue(R) vector. PCR
amplification of this cloned DNA fragment using combinations of
oligonucleotides specific for isoforms
and
generated precise
sizes of DNA fragments, each corresponding to the inserted sequences of
isoforms
and
, respectively (data not shown). We then sequenced
the DNA fragment using
- and
-specific oligonucleotides, as well
as the two oligonucleotides used for the generation of the DNA
fragment. As shown in Fig. 1D, just after the sequences
corresponding to the codon for Lys277 of paxillin
began
sequences unrelated to the paxillin cDNAs. An exon encoding the
inserted sequence of isoform
was found near the 3
-end of the
genomic DNA fragment. Downstream of the 3
-end of this exon are again
unrelated sequences of 142 nucleotides in length. After this region,
another exon encoding the inserted sequence of isoform
began.
Interestingly, this exon was directly connected to the coding region of
Phe278 and thereafter of paxillin
sequences (Fig. 1,
B and D).
To
verify the protein products of these cDNAs, we expressed these
cDNAs in fibroblasts. We used a virus infection system with BOSC 23 virus-packaging cells and the pBabe vector, which uses the murine
retrovirus long terminal repeat as a promoter. As shown in Fig.
2, each isoform cDNA produced a protein of the
expected size in NIH3T3 cells that reacted with polyclonal
anti-paxillin antibodies, which were raised against amino acids
Lys199-Val217 of isoform (Ab 199-217).
Diffuse bands in each isoform may represent tyrosine and serine
phosphorylation of paxillin, as was shown with the
isoform of
paxillin (27, 35). With the expression of these isoforms of paxillin in
NIH3T3 cells, no significant morphological change or
anchorage-independent growth of cells was observed (data not
shown).
Isoforms
To examine possible functional differences in
integrin-mediated cell adhesions or focal adhesion assemblies, we then
assessed the protein binding properties of the paxillin isoforms. We
expressed GST fusion proteins of each isoform in E. coli.
Cellular proteins that co-precipitated with these recombinant proteins
coupled to glutathione beads were then analyzed. Isoform has been
shown to bind to Fak, talin, and vinculin (18-21, 28), and these
proteins were indeed co-precipitated with our GST fusion protein of
paxillin
(Fig. 3). It should be noted that there
were large differences in the relative affinities of the GST-paxillin
protein toward each of these proteins; with our conditions in the
presence of an excess amount of the fusion protein, more than 50% of
Fak protein in the cell lysates appeared to be recovered, whereas only
less than several percent of proteins were recovered in the case of vinculin and talin (Fig. 3). With fusion proteins of
and
isoforms, amounts of these focal adhesion proteins co-precipitated were different from those with the
isoform (Fig. 3). Compared with the
binding of isoform
, isoform
binds to Fak and talin similarly but exhibits only marginal binding to vinculin; isoform
binds to
vinculin and talin similarly but only weakly binds to Fak. Essentially
the same results were obtained with cell lysates prepared from HeLa and
U937 cells (Fig. 3).
We also examined paxillin-binding proteins using radiolabeled cell
lysates. As shown in Fig. 4, at least eight different
protein bands were detected as co-precipitating with the three isoforms over those seen with negative control GST protein. Among these isoforms, exhibits much decreased binding toward cellular proteins. Judging from the molecular sizes, protein band E may correspond to Fak,
and band B may be talin. Visualization of vinculin, which bound only
weakly, as assessed by the above experiments, may be hindered by band
E. In addition to these proteins, six bands were detected: A, C, D, F,
G, and H, all of which bound to isoform
. Compared with isoform
,
isoform
bound to bands A, C, D, G, and H similarly but not to band
F; isoform
bound to band A similarly, weakly to band H, and with
marginal levels to bands C, D, E, and G. No additional protein bands
were seen with isoforms
and
compared with those observed with
isoform
. Again, with radiolabeled cell lysates prepared from U937
cells, essentially the same patterns of protein binding were obtained
as with HeLa cell lysates (data not shown).
Expression of Paxillin
Expression
of mRNA has been studied in normal human tissues, as well as
several cultured cells (28). Consistent with the previous report, our
analysis showed that paxillin, as detected by a DNA probe encompassing
a 1-1072-nucleotide sequence of the
isoform, is expressed
ubiquitously in most normal tissues (Fig. 5). Among
cancer cells examined using the same DNA probe, high levels of paxillin
mRNA were detected in HeLa S3 epithelial carcinoma cells, K562
chronic myelogenous leukemia cells, SW480 colorectal adenocarcinoma
cells, A549 lung carcinoma cells, and G361 melanoma cells; marginal
levels of expression were detected in Molt 4 lymphoblastic leukemia
cells and Raji Burkitt's lymphoma cells, and paxillin expression was
not detected in HL-60 promyelocytic leukemia cells (Fig.
5A). On the other hand, with DNA probes each specific for
and
isoforms, expression of
and
mRNAs were not
detected in any of the normal tissues we have examined (Fig.
5B; data not shown). Expression of
mRNA, however,
was clearly detected in SW480 cells, with low levels in HeLa, K562, and
A549 cells.
mRNA was not detected in HL-60, Molt 4, Raji, and
G361 cells.
mRNA was not detected in any of above cell lines in
the same mRNA blot filters (data not shown). mRNAs each
specific for
,
, and
isoforms, on the other hand, were
clearly detected in U937 cells using a reverse transcription-PCR
amplification method (data not shown).
Expression of
We have generated
three types of polyclonal antibodies; one was Ab 199-217, which
recognizes all the three isoforms, and the other two were types of
polyclonal antibodies each against and
isoforms. The
specificity and sensitivity of these antibodies were assessed by the
use of GST fusion proteins of each paxillin isoform (Fig.
6). To detect isoform expression, paxillin was first immunoprecipitated with the anti-paxillin monoclonal antibody and then
blotted with antibodies specific for
and
. As shown in Fig.
6A,
and
isoforms were clearly detected in U937
cells, the mRNA of which was used to isolate these isoform
cDNAs. The
isoform in U937 cells appeared to consist of three
bands, one major and two minor, and the
isoform consisted of two
bands. The
isoform in fibroblasts is known to be highly
phosphorylated and thereby detected as multiple protein bands when
separated on SDS-PAGE (27, 33, 35). Thus, multiple protein bands of
and
isoforms may also represent phosphorylation of certain fractions of these isoforms in vivo. The protein bands that
reacted with Ab 199-217 but not with
- and
-specific antibodies
may correspond to the
isoform (Fig. 6A). Judging from
the immunoblot, the expression of
and
isoforms at protein
levels appears to be at a ratio of about 2:1 in U937 cells. The amount
of the
isoform seemed to be equivalent to or a little higher than
that of isoform
in U937 cells.
We also examined the isoform expression in several human cell lines. As
shown in Fig. 6B, protein expression of the isoform was
detected in Jurkat, HeLa, and K562 cells but not in HPB-ALL lymphoma
cells. The
isoform was almost undetectable in these cells (Fig.
6A; data not shown). Again, protein bands detected by Ab
199-217 but not by
- and
-specific antibodies in these cells may
correspond to those of the
isoform.
To explore further the
possible physiological differences of these paxillin isoforms, we
examined the isoform expression during monocyte maturation. U937 cells
were treated with TPA for 3 days to differentiate the cells into
macrophage-like cells. Changes in the expression of integrins as well
as changes in the binding and spreading of cells toward different ECMs
has been documented in this process (42, 43). As shown in Fig.
6A, expression of the isoform was increased severalfold
after the differentiation, whereas the isoform consisted of two protein
bands in either stage of the cells. With the
isoform, one
additional protein band appeared, and the slow-migrating fractions of
the isoform were increased markedly, which may represent increased
phosphorylation of
isoform after the differentiation and adherence
of U937 cells.
We characterized two novel isoforms of human paxillin generated by exon insertion. These novel isoforms exhibited different expression, and the patterns of the expression were altered during monocyte maturation. Moreover, biochemical analysis using recombinant proteins revealed differences in binding to several proteins, including focal adhesion proteins.
Sequence analysis of the novel paxillin cDNAs revealed that both
the and
isoforms have distinct insertions just after Lys277 of human paxillin
. Both of these insertions are
followed by Phe278 and the remainder of the isoform
sequence. Analysis of the genomic structure revealed that there is
indeed an exon breakpoint just after the codon for Lys277.
After this codon begins an intron, and the exon encoding the sequence
inserted in isoform
lies about 6 kb 3
of this junction. This
-specific exon is followed by a short intron and then by an exon
encoding the sequence inserted in isoform
. The
-specific exon
connects directly to the sequence encoding Phe278. All of
these introns have consensus 5
-GT and 3
-AG splice sites. The
-specific exon ends with AG, followed by the TTC codon for Phe278 of isoform
. This kind of intronless exon-exon
junction is found in other genes, such as caldesmon (44, 45). There may
be additional exons, other than
- and
-specific exons, within
this 6.4-kb DNA fragment. So far, however, we have not detected other
transcripts by reverse transcription-PCR amplification of cellular
mRNAs using the 5
- and 3
-flanking sequences (primers 742-768 and
889-915) of the 6.4-kb DNA fragments as primers.
We showed that each GST fusion form of paxillin isoforms
co-precipitates focal adhesion proteins with a different affinity. Our
experiments using radiolabeled cell lysates suggested that no
additional proteins are bound to the and
isoforms over those
binding to paxillin isoform
, and the
isoform exhibits much
decreased affinity toward cellular proteins. The
and
isoforms
bind to talin with affinities comparable to that of isoform
.
However, binding to Fak and vinculin is altered greatly;
binds to
Fak strongly but quite weakly to vinculin, whereas
binds to
vinculin but only weakly to Fak. Moreover, several proteins, such as
Fak, vinculin, and bands D, F, and G in Fig. 4,
appear to bind selectively to either
or
isoforms. Deletion
analysis of paxillin
has suggested that amino acids 56-100 are
responsible for Fak and vinculin binding, and amino acids 100-277 bind
to talin (18, 28). Our
and
isoforms contained inserts between Lys277 and Phe278 of isoform
. Thus,
structures governing the binding sites for Fak and vinculin appear to
be much more complicated in isoforms
and
than those expected
from the analysis of isoform
, and primary binding sites for Fak and
vinculin may be distinct from each other in both
and
isoforms.
Moreover, one of our stocks of U937 cells lacked Fak expression. With
cell lysates prepared from this cell line, each paxillin isoform showed
essentially the same patterns of binding to vinculin and talin as in
Fak-positive cell lysates (data not shown). Thus, Fak is not essential
for the binding of paxillin isoforms to vinculin and talin.
The architecture of the focal adhesion assembly may not be unique. It
has not yet been established which focal adhesion proteins are used
with which integrins or focal adhesions. The different and selective
binding of isoforms to Fak and vinculin, for example, may involve
localization of vinculin to certain types of focal adhesions
independent of Fak. Indeed, the fact that focal adhesions are present
in Fak-deficient fibroblasts indicates that Fak is not essential for
certain types of adhesions (46). We also showed that the binding of the
isoform to vinculin is weak compared with those of isoforms
and
. However, it should be noted that the amounts of vinculin as well
as talin that bound to paxillin were quite low, compared with Fak
binding to paxillin (see Fig. 3). Thus, it should be studied further
whether the weak interaction of paxillin with vinculin and talin,
detected in vitro, could have certain physiological
relevance.
Northern blotting analysis showed that mRNAs for both the and
isoforms were undetectable in normal tissues, whereas paxillin mRNA was clearly detected in most of normal tissues using a DNA probe encompassing nucleotides 1-1072 of the
isoform. This result indicates that isoform
is indeed expressed in normal tissues, as
reported previously (28). Since DNA probes specific for the
and
exons were relatively short (102 and 144 nucleotides in length,
respectively), it may have been difficult to detect any low levels of
the
and
mRNA. However, a DNA probe of similar length
prepared from isoform
cDNA using primers 742-768 and 889-915
(see Fig. 1B) did detect paxillin mRNA expression
clearly in normal tissues as well as several cancer cells (data not
shown).
Although the isoform mRNA is not detected in normal tissues, it
is expressed in several cancer cells, such as SW480, HeLa, K562, and
K549 cells. These
mRNA expression results recall the similar
results of the increased levels of Fak mRNA observed only in
metastatic and invasive cancers with no detectable expression in normal
adult tissues or most benign tumors (47). Although the cancer cells
expressing the
isoform are highly tumorigenic in vivo,
SW480 cells, in which the
isoform was detected at the highest level
among cancer cells examined, do not exhibit the highest metastatic and
invasive activity in vitro (for example, see Refs. 48 and
49). Thus, the correlation between the expression of the paxillin
isoforms and tumorigenesis, metastasis, and invasiveness, processes in
which integrins are deeply involved (50-52; for review, see Ref. 53),
is interesting but should be studied in more detail.
Expression of the isoform is much restricted. The
isoform was
not detected in any of samples we examined, except in U937 promonocytic
cells. Since other cells with hematopoietc lineages, such as Jurkat,
HPB-ALL, Raji, HL-60, Molt 4, and K562, were negative in its
expression, expression of the
isoform seems not to be specific for
every hematopoietic lineage but more restricted for certain
differentiation stages, including that of monocyte maturation.
All three isoform proteins were expressed in U937 cells at levels
comparable with each other. We found that the protein level of the isoform was markedly increased when U937 cells were differentiated into
adherent monocyte- and macrophage-like cells by TPA. Modification of
the
isoform was also accompanied by this process. In
undifferentiated U937 cells, the amount of the
isoform was smallest
among the three isoforms. On the other hand, the amount of the
isoform appeared to become higher than that of the
isoform after
the differentiation and adherence of cells onto culture dishes. Human monocyte precursor cells express several families of integrins, and
patterns of their expression are greatly altered during the monocyte
differentiation that accompanies the changes in the binding properties
of the cell toward different ECMs (54, 55). These properties are
important in the homing and positioning of monocytic cells at the sites
of inflammation. Indeed, in U937 cells, TPA treatment, which mimics
most of the effects of tumor growth factor
1, enhances the
expression of integrins
2 and
v, with a concomitant decrease in
integrin
4 expression (42, 43). Although the expression of integrin
types varies greatly during the monocyte maturation, altered expression
of integrins per se does not seem to be enough to account
for the changes of cell adherence and motility (56). It is, thus, quite
important to examine how much the changes in the expression of paxillin
isoforms contribute to this process. Also, it would be quite
interesting to see whether each isoform of paxillin assembles
selectively with different types of integrins in monocyte cells.
In conclusion, these paxillin isoform clones are useful tools with
which to explore possible differences in the components, regulatory
mechanisms, and signaling cascades of different types of focal
adhesions within a single cell as well as among different types of
integrins and cells. Although paxillin expression in brains of day 10 or day 12 chicken embryos, as well as in the human brain, is quite low
(21, 28, 57), we have detected high levels of paxillin mRNA in
neural tubes during the early stages (stages 15-20, 50-72 h) of
chicken embryogenesis,3 when neural crest
cells actively migrate along their ECMs. Although the and
isoforms were undetectable in normal tissues, they may have been
expressed at embryonic stages, at which cell movements and
differentiation actively take place for morphogenesis. More precise
analysis of the differences in the expression and localization of each
paxillin isoform will contribute to clarifying our understanding of the
role of paxillin and focal adhesions during embryogenesis, cell
adhesion, movement and invasion, and cancer metastasis.
We are grateful to Goro Eguchi for support and encouragement throughout this work, Tetsuya Taga for U937 cells, Warren S. Pear and David Baltimore for BOSC 23 cells, Hartmut Land for pBabe vector, Heidi Greulich and Tomohoro Kurosaki for critical reading of the manuscript, and Manami Hiraishi for technical assistance.