(Received for publication, October 31, 1994; and in revised form, December 12, 1994)
From the
Paxillin is a 68-kDa focal adhesion protein that is
phosphorylated on tyrosine residues in fibroblasts in response to
transformation by v-src, treatment with platelet-derived
growth factor, or cross-linking of integrins. Paxillin has been shown
to have binding sites for the SH3 domain of Src and the SH2 domain of
Crk in vitro and to coprecipitate with two other focal
adhesion proteins, vinculin and focal adhesion kinase
(p125). After preliminary studies showed that
paxillin was a substrate for the hematopoietic oncogene
p210
, we investigated the role of this protein in
hematopoietic cell transformation and signal transduction. A
full-length cDNA encoding human paxillin was cloned, revealing multiple
protein domains, including four tandem LIM domains, a proline-rich
domain containing a consensus SH3 binding site, and three potential
Crk-SH2 binding sites. The paxillin gene was localized to chromosome
12q24 by fluorescence in situ hybridization analysis. A
chicken paxillin cDNA was also cloned and is predicted to encode a
protein approximately 90% identical to human paxil-lin. Paxillin
coprecipitated with p210
and mul-tiple other cellular
proteins in myeloid cell lines, suggesting the formation of multimeric
complexes. In normal hematopoietic cells and myeloid cell lines,
tyrosine phosphorylation of paxillin and coprecipitation with other
cellular proteins was rapidly and transiently induced by interleukin-3
and several other hematopoietic growth factors. The predicted structure
of paxillin implicates this molecule in protein-protein interactions
involved in signal transduction from growth factor receptors and the BCR/ABL oncogene fusion protein to the cytoskeleton.
Paxillin is a 68-kDa cytoskeletal protein found in specialized structures, termed focal adhesions, that occur at sites where cells adhere to the extracellular matrix(1, 2) . It is primarily within focal adhesions that transmembrane integrin molecules connect the actin cytoskeleton to the extracellular matrix. The formation of focal adhesions and the interaction of adhesion molecules with the cytoskeleton are dynamic processes that can be regulated by cytokines in normal cells, including epidermal growth factor and platelet-derived growth factor. Also, several oncogenes, such as v-src, are known to disrupt focal adhesions(3, 4) . The signaling pathways that regulate these events are not well understood.
A number of observations
suggest that paxillin is involved in transducing signals from growth
factor receptors to focal adhesions. In addition to epidermal growth
factor and platelet-derived growth factor, paxillin is transiently
tyrosine phosphorylated in response to several small peptide growth
factors such as bombesin, endothelin, and
vasopressin(5, 6) . Paxillin is also tyrosine
phosphorylated in response to integrin-mediated cell adhesion (7) and during embryonic development(8) . The tyrosine
kinase(s) that phosphorylate paxillin are unknown, but in vitro paxillin can be phosphorylated by the focal adhesion tyrosine
kinase p125(9) . Further evidence
linking paxillin to signal transduction pathways comes from the
findings that paxillin binds to the SH3 domain of p60
(10) and to the SH2 domain of v-Crk (11) in
vitro, as well as to the carboxyl terminus of vinculin in
vitro and in vivo(12) . Finally, paxillin is also
prominently tyrosine phosphorylated in Rous sarcoma virus-transformed
chick embryo fibroblasts(1) . Interestingly, paxillin is also
prominently tyrosine phosphorylated during transformation by
v-crk(11) .
In preliminary studies, we have found
that p210 colocalized with paxillin in punctate,
membrane-associated structures that also contained vinculin.
P210
is known to be localized in part in the
cytoskeleton, and this localization is believed to be critical for
transformation of myeloid cells, perhaps by allowing access to critical
substrates(14, 15, 16) . We found that
tyrosine and serine phosphorylation of paxillin is strikingly and
constitutively increased in myeloid cell lines transformed by BCR/ABL. In an effort to understand the structure and function
of paxillin in hematopoietic cells, we cloned a full-length paxillin
cDNA and investigated its interactions with other cellular proteins.
The results indicate that paxillin has multiple protein-protein
interaction domains, consistent with its potential role as a signal
transduction molecule in the cytoskeleton. In contrast to its
constitutive phosphorylation in BCR/ABL-transformed cells,
paxillin phosphorylation in normal cells is regulated by numerous
hematopoietic growth factors, including IL-3. (
)
Figure 1: cDNA sequencing strategy and full-length cDNA structure of human and chicken paxillin. A, diagram of individual human and chicken paxillin cDNAs. The shadedbox represents the translation product from the deduced open reading frame. Pax-0 through Pax-8 represent individual cDNAs isolated from human libraries. B, BamHI; E, EcoRI. Pax-6 and Pax-7 were isolated by 5`-RACE from K562 and A2058 cell lines. Pax-8 was isolated by 3`-RACE from A2058 cell line. cPax-1, -4, and -5 are individual cDNAs isolated from chicken libraries. B, full-length cDNA structure of human paxillin. Shown are the nucleic acid base pairs with an open reading frame of 557 amino acids. Also shown is the starting Kozak sequence and the poly(A) adenylation sequence for the human paxillin cDNA. The potential v-Crk binding site(s) and potential SH3 binding site are identified. Noted are the four tandem LIM domains. C, comparison of predicted amino acid structures for human and chicken paxillin. The homology between human and chicken predicted amino acids is approximately 90%.
Chromosome somatic cell hybrid panel blot was purchased from Oncor,
Pax-4 cDNA probe was labeled with P random priming, and
Southern hybridization was performed as described(24) .
Comparison of the structure of paxillin with other proteins revealed an array of discrete protein domains (Fig. 2A). Within the carboxyl-terminal third of paxillin are four tandem cysteine- and histidine-rich sequences termed LIM domains(29) . Although LIM domains are found in a number of proteins, including the cytoskeletal proteins zyxin and cysteine-rich protein, paxillin is the first protein reported to have four LIM domains. The homology of the LIM domains in paxillin with LIM domains from other proteins is represented in Fig. 2B.
Figure 2:
Schematic diagram of human paxillin
protein and comparison of LIM domains with other known proteins
containing LIM domains. A, various domains of paxillin with
homologies to other proteins are depicted. There are four tandem LIM
domains. There are three YXXP motifs that may be binding sites
for v-Crk SH2. The proline-rich domain with potential Src-SH3 binding
site is also identified. From precipitations of lysates with various
constructs of GST-paxillin fusion proteins, a talin binding site is
identified. B, comparison of LIM domains of paxillin with
previously described proteins containing LIM domains. Consensus
sequence for LIM domains is cysteine/histidine-rich with conserved
repeats. The degreeofshading represents
the degree of homology to each of the proteins. Lin-11, Isl-1, and Mec-3 are classic LIM
homeodomain-containing proteins. RBTN-1 (also called rhombotin; Ttg-1)
and RBTN-2 (Ttg-2) are newly described members of the LIM family of
proteins. Zyxin (which contains three LIM domains, and for
representation sake only two domains are shown) and human cysteine-rich
protein (hCRP) are cytoskeletal associated proteins described
to contain the LIM domains. Each LIM domain is designated 1, 2, 3, or 4
depending on proximity to the NH terminus of the protein.
Note that the LIM domain 3 of paxillin is a perfect match with other
LIM domains, whereas there is similarity to domain 1, 2, and 4 of
paxillin LIM domains. Each of the known sequences is obtained from
GenBank.
In addition to LIM domains, paxillin has a proline-rich domain (amino acids 46-55) that contains the sequence PPPVPPPPSS, which may function as an SH3 domain binding site(30) . There are three YXXP motifs (Tyr-31, Tyr-118, and Tyr-181), each of which is a potential binding site for the SH2 domain of v-Crk(11) .
To confirm the authenticity of the human paxillin cDNA, a GST fusion protein was constructed using the open reading frame of a 2.1-kb (Pax-0) fragment and used to produce a rabbit polyclonal antisera, which was then shown to recognize authentic paxillin by immunoblot (Fig. 3) and to stain focal adhesions of fibroblasts (data not shown). A partial chicken cDNA (approximately half of the full-length cDNA reported in this paper) was also reported during preparation of this manuscript that is contained within the chicken cDNA reported here(31) .
Figure 3: Confirmation that the cloned cDNA is paxillin. A New Zealand White female rabbit was immunized with GST-fusion protein constructed from Pax-0 (Fig. 1A), and third immunization serum was used to identify that the antibody generated reacted with the 68-kDa paxillin protein (Western blot, 1:2000 dilution). Represented are whole cell lysates (lanes1 and 3) and commercial paxillin monoclonal antibody immunoprecipitates of NIH3T3 lysates (lanes2 and 4). Lanes1 and 2 are blotted with rabbit polyclonal antibody, whereas lanes3 and 4 are blotted with commercial paxillin monoclonal antibody. Arrow shows p68 paxillin.
Figure 4: Northern analysis of various tissue samples and cell lines using human paxillin cDNA as probe. A, human tissue RNA is as follows: lane1, heart; lane2, brain; lane3, placenta; lane4, testis; lane5, ovary; lane6, small intestine; lane7, colon; lane8, peripheral blood leukocyte. B, cell line RNA is as follows: lane 1, chick embryo fibroblasts; lane2, MRC-5; lane3, FS-2; lane4, A498; lane5, A2058; lane6, Hela; lane7, CV-1; lane8, CCL223; lane9, NIH3T3; lane10, NIH3T3 containing tensin-transfected cDNA. Also shown is the actin hybridization of RNA for standard controls.
Figure 5: Chromosomal localization for paxillin gene. A, a normal human metaphase spread from phytohemagglutinin-stimulated peripheral blood was denatured and hybridized with paxillin P1 plasmid. Arrows point to the signals observed at the long arm end of a pair of C group chromosomes. B, same metaphase banded with DAPI localizing the signal in A to 12q24.
Figure 6:
IL-3 and P210 induces
paxillin migration in 32Dcl3 myeloid cells. Paxillin is a 68-kDa
protein that is recognized by the mouse monoclonal antibody. IL-3
stimulation time course is represented (lanes1-6: 0, 1, 5, 15, 30, and 60 min, respectively).
Four individual 32Dcl3.P210
subclones were also
evaluated for paxillin immunoblotting. Proteins were separated by 7.5%
SDS-PAGE and immunoblotted using anti-paxillin monoclonal antibody.
There is a migration of the paxillin band with IL-3 stimulation and
constitutively in P210
cells. The antibody also
recognizes a doublet at 44/46 kDa.
IL-3
stimulation of factor-deprived 32Dcl3 cells led to a rapid (1-5
min) shift to multiple slower migrating isoforms up to 80 kDa (Fig. 6). These slower migrating forms of paxillin have
increased phosphorylation of both serine and tyrosine residues (Fig. 7), and similar results were observed with other
hematopoietic cell lines and growth factors. For example, there is also
hyperphosphorylation in FDCP-1 and BAF-3 cells in response to IL-3,
human neutrophils and NIH3T3 /
1 fibroblasts in response to
GM-CSF, FDCP-1 in response to Steel factor, human neutrophils in
response to G-CSF, HL-60 in response to trans-retinoic acid, human
T-cells in response to phytohemagglutinin, human macrophages in
response to M-CSF, and BAF-3 cells in response to erythropoietin. These
shifts are due to principally or exclusively increased phosphorylation
because treatment of paxillin immunoprecipitates and whole cell lysates
with potato acid phosphatase reduced this complex set of bands to a
single 68-kDa band (data not shown). A similar pattern of paxillin
phosphorylation was observed in 32Dcl3 cells transformed to factor
independence by expression of p210
( Fig. 6and Fig. 7), except that this phosphorylation pattern is
constitutive. Finally, we did not observe a change in phosphorylation
status of paxillin with IL-2 in T-cells, IL-6 in U266 cells, IL-12 in
NK cells, and oncostatin-M in U266 cells.
Figure 7:
Phosphoamino acid analysis of in vivoP-labeled paxillin. After
immunoprecipitation of
P-labeled paxillin protein with the
monoclonal antibody, cell lysates were applied to a 7.5% SDS-PAGE gel
and electrophoresed. The appropriate paxillin bands were cut, and
phosphoamino acids were analyzed. Lane1, 32Dcl3,
unstimulated; lane2, 32Dcl3, IL-3 stimulated for 15
min; lane3: clone
26.32D.P210BCR/ABL.
Given the structure of
paxillin and the observation of increased phosphorylation following
growth factor stimulation or oncogenic transformation, we asked if
growth factor or p210-induced phosphorylation was
associated with changes in the interaction of paxillin with other
cellular proteins. Lysates of factor-deprived, IL-3-stimulated, or
p210
-transformed 32Dcl3 cells were immunoprecipitated
with antibody to paxillin and then blotted with antibody to
phosphotyrosine. Proteins of 210, 116, 94, 60, 55, and 44 kDa were
detected in lysates from factor-stimulated or
p210
-transformed cells but not from resting cells (Fig. 8A). Western blotting of these immunoprecipitates
identified the 210 kDa as p210
in
oncogene-transformed cells and the 116-kDa protein as vinculin (data
not shown).
Figure 8:
Identification of multiple proteins that
associate with paxillin. A, association of paxillin with other
phosphotyrosine proteins as determined by paxillin immunoprecipitation
and antiphosphotyrosine immunoblot. After immunoprecipitation of
paxillin protein with the monoclonal antibody, isolated proteins were
applied to a 7.5% SDS-PAGE gel and electrophoresed, and
phosphotyrosine-containing proteins were visualized by immunoblots. Lane1, 32Dcl3, unstimulated; lane2, 32Dcl3, IL-3 stimulated for 15`; lane3, clone 26.32D.P210BCR/ABL; lanes4-6 are 32Dcl3, unstimulated, 32Dcl3, IL-3 stimulated for 15`, and
clone 26.32D.P210BCR/ABL cell lysates, respectively, immunoprecipitated
with control antibody 3c11c8 (-interferon). B,
association of GST-paxillin fusion protein with talin. Various GST
fusion proteins of paxillin domains were constructed as described under
``Materials and Methods,'' and the expressed fusion proteins
were used to precipitate lysates from clone 26.32D.P210BCR/ABL cell
line. GST protein alone and GST-SH2 domain of tensin were used as
controls. Thereafter, isolated proteins were applied to a 7.5% SDS-PAGE
gel and electophoresed, and talin, vinculin, and p125
proteins were visualized by immunoblots. Lane1, GST-protein alone; lane2,
GST-SH2-tensin; lane3, Pax-0-GST-paxillin (amino
acids 100-557); lane4, Pax-1-GST-paxillin
(amino acids 227-557); lane5, LIM domains 1
through 4-GST-paxillin (amino acids 325-557); lane6, LIM domain 1-GST-paxillin (amino acids 325-374); lane7, pre-LIM domain-GST-paxillin (amino acids
303-327); lane8, whole cell
lysate.
Using various constructed GST-fusion proteins of
paxillin (Fig. 8B), it is seen that talin is
precipitated with Pax-0 GST-fusion protein (amino acids 100-557)
but not Pax-1 (amino acids 227-557), pre-LIM domain, LIM 1
domain, or LIM 1-4 domain GST-fusion proteins. None of these
fusion proteins precipitate vinculin or p125. As a
control, a tensin-SH2 GST-fusion protein did not precipitate talin,
vinculin, or p125
.
The cytoskeleton is essential for many cellular functions,
including regulation of cell shape, flexibility, mobility, and adhesive
properties(32) . The focal adhesion is a specialized structure
of the cytoskeleton and plasma membrane that forms at areas of contact
between the cell and extracellular matrix proteins such as
fibronectin(33) . Integrins serve as receptors for
extracellular matrix proteins, localize in focal adhesions, and their
intracellular domains interact directly with focal adhesion proteins
such as talin and -actinin and indirectly with actin(34) .
The expression and function of adhesion receptors and the formation of
focal adhesions can be modulated by various events, including exposure
to cytokines and growth factors, although the mechanisms are generally
not understood. Phosphorylation of proteins within focal adhesions is
closely associated with changes in the structure of the actin
cytoskeleton(7, 35, 36, 37, 38) .
Focal adhesions have very high concentrations of proteins
phosphorylated on tyrosine residues, and a tyrosine kinase unique to
this structure, p125
, has been
described(39, 40) . Further evidence implicating
tyrosine phosphorylation in the regulation of focal adhesion functions
comes from the observation that cross-linking of integrins induces
tyrosine phosphorylation of focal adhesion plaque
proteins(41) . Thus, the focal adhesion appears to be a
structure that receives signals from both sides of the membrane in a
complex manner.
Paxillin is a 68-kDa focal contact protein initially identified by making antibodies to phosphotyrosine-containing proteins in Rous sarcoma virus-transformed chick embryo fibroblasts(1) . The pI of paxillin ranges from pH 6.31-6.86, with increased acidic forms (consistent with multiple phosphorylation sites) migrating more slowly on SDS-polyacrylamide gels(2) . The antibody also recognizes two lower forms of 46 and 44 kDa, with pIs of pH 6.9. Up to 20-30% of paxillin is phosphorylated on tyrosine in Rous sarcoma virus-transformed cells and in embryonal tissues(1, 2, 8, 9) . The significance of tyrosine phosphorylation is unknown, although the phosphorylation of this protein appears to be a consequence of signal transduction from membrane receptors in normal fibroblasts. Paxillin is one of the major tyrosine phosphoproteins in cells transformed by v-src or v-crk(11) . Further, paxillin can interact directly with both oncogenes through the SH3 domain of Src (10) and the SH2 domain of Crk(11, 42) , based on in vitro binding studies. Binding of v-Src and v-Crk to paxillin may be a major determinant in concentrating both of these oncogenes in focal adhesions and therefore could be important in the altered cytoskeletal structure and adhesive properties that accompany transformation by both viral oncogenes.
To identify any role paxillin might play in regulating cytoskeletal structure in either transformed or normal hematopoietic cells, we cloned a full-length cDNA encoding human paxillin by expression and authenticated the clone by demonstrating that an antibody raised against the amino terminus of the protein precipitated paxillin. The cDNA has an open reading frame of 1671 base pairs, which encodes a predicted protein of 557 amino acids and molecular mass of 61 kDa. The expected molecular mass of paxillin is 68 kDa, and the difference between the expected and predicted molecular weights could be due to the high proline content of this protein or alternative splicing. The formal proof that paxillin described here shares identical amino acid sequences with native paxillin is still lacking since we have not purified the native paxillin and analyzed individual amino acid composition. We have also cloned chicken paxillin, using human paxillin cDNAs as probes to screen a chicken embryo fibroblast library. While this manuscript was in preparation, a partial cDNA of chicken paxillin (approximately half of the full-length cDNA reported here), identified initially by peptide sequencing of paxillin protein and thereafter using degenerate oligonucleotide primers to screen a chicken fibroblast library, was reported by Turner and Miller(31) . This published partial sequence is contained within the chicken paxillin sequence reported here, which predicts a protein 90% identical to human paxillin, further supporting the authenticity of our human cDNA. There is a major human RNA message on Northern blot of 3.7 kb, expressed in most tissues, although low in brain.
Comparison of the predicted sequence with
known proteins reveals several interesting domains. There is a series
of 4 motifs identical or very closely related to LIM domains, with the
general consensus sequence
CX2CX16-23(W/Y/F)HX2CX2CX16-21CX2-3(C/H/D) (29) . The LIM motif is named for three homeodomain proteins,
each containing two LIM domains, lin-11, a Caenorhabditis
elegans cell lineage gene(43) ; isl-1,
preferentially expressed in pancreatic islet cells(44) ; and mec-3, involved in neuron differentiation in C. elegans(43) . A number of other LIM domain proteins
have been identified, including rhombotin 1 and
2(29, 45, 46, 47, 48, 49, 50, 51) ,
identified in chromosomal translocations in T-cell acute leukemias and
two cytoskeletal proteins, zyxin and cysteine-rich
protein(52, 53, 54) . LIM domains are
predicted to bind two molecules of Zn and could form
two zinc finger-like structures. Theoretically, therefore, one molecule
of paxillin could bind up to eight molecules of zinc and form eight
zinc fingers. One of the three LIM domains of zyxin has been shown to
bind cysteine-rich proteins(55) . In cysteine-rich protein,
metal binds to the two sites within a single LIM domain is
sequential(56) , with preferential occupancy of a site
involving the 4 carboxyl-terminal cysteines(57) . Proteins
containing LIM domains tend to be highly conserved among different
species. For example, rat, hamster, and human Isl-1 amino acid
sequences are identical(58) , while chicken and human
cysteine-rich proteins are 91% identical(54) . The high degree
of homology (90%) between chicken and human paxillin is therefore not
surprising. It is noteworthy that many LIM proteins are nuclear and are
involved in regulating development or differentiation. However, zyxin
and cysteine-rich proteins are cytoskeletal proteins, and like
paxillin, neither has been detected in the nucleus.
Analysis of paxillin structure reveals several other potential motifs of interest. Paxillin has been shown to bind to the SH2 domain of v-Crk in vitro, and recent studies indicate that phosphopeptides with pYXXP motifs will bind to Crk SH2 domains(11) . Human paxillin has three of these motifs, and it will be worthwhile to determine which interact with c-CRK and v-Crk. Cells transformed by v-crk have increased tyrosine phosphorylation, which is of interest because v-Crk does not contain a tyrosine kinase itself. Recent data from Feller et al.(59) indicate that c-Abl binds to the first c-Crk SH3 domain and can phosphorylate Tyr-221 in the spacer region between the two SH3 domains in c-Crk. This creates a high affinity binding motif for Crk-SH2, and possible intramolecular binding of Crk-SH2 to Tyr-221. This model of Crk function predicts that the interaction of c-Crk and paxillin would be controlled by the relative degrees of phosphorylation of Crk Tyr-221 and paxillin YXXP motifs, both potentially competing for c-Crk-SH2 binding. The transient phosphorylation of paxillin induced by growth factors, if it occurs on the appropriate tyrosine residues, could increase paxillin-Crk interaction, facilitating signal transduction to (or from) focal adhesions.
In BCR/ABL-transformed cells, paxillin is
phosphorylated, and interaction of c-CRK and paxillin might be
increased. However, CRK-L, a CRK-related protein, has been shown to be
tyrosine phosphorylated in CML cells(60) , and the interactions
of c-CRK, CRK-L, and paxillin may be complex. In the CML cell lines
studied here, paxillin coprecipitates with p210,
suggesting that a paxillin-CRK-L-P210
complex could
be formed, although these studies are preliminary. The formation of
such a complex may not only anchor p210
at the cell
membrane, adjacent to other critical substrates, but also interrupt the
normal signaling pathways that utilize paxillin and CRK-L. Alterated
regulation of integrin molecule function would be a potential outcome.
In v-Crk, Tyr-221 is deleted, and constitutive complexes of v-Abl and
v-Crk exist. It would be predicted that these complexes could also
contain paxillin, and this possibility is currently being tested.
Overall, the available data suggest that the interaction between Crk or
CRK-L and paxillin may be regulated by phosphorylation and could
contribute to signaling in focal adhesions.
Paxillin has previously
been shown to bind to the SH3 domain of Src, and analysis of the
primary structure of paxillin reveals a proline-rich motif that could
serve as an SH3 binding domain. The sequence PPPVPPPPSS (amino acids
46-55) is consistent with a consensus SH3 binding site, related,
but not identical to, the SH3 binding sites previously identified in
SOS, p59, 3BP1, and 3BP2(30) . It has been
suggested that the colocalization of Src to focal adhesions may be the
result of this interaction(10) . The precise function of SH3
domains is uncertain, but SH3 domains are found in a variety of
cytoskeletal and membrane proteins and have been suggested to
participate in protein interactions in these structures. In addition,
some SH3 domains have recently been shown to increase enzyme activities
of binding partners. Notably, the GTPase activity of dynamin is
increased by SH3 domain binding(61) , and it will be of
interest to determine if the binding of paxillin to Src-SH3 affects Src
kinase activity.
Using FISH analysis, we have localized the gene for paxillin on chromosome 12q24. From known genomic data, there are several genes known to reside in this region. For example, the gene for Darier's disease (12q23-24.1), a rare autosomal dominant skin disorder in which there is abnormal adhesion between keratinocytes(62) , and autosomal dominant cerebellar ataxia (second locus, 12q23-24.1) (63) appear to be in the vicinity of the paxillin gene. At this time, the functional relationships to any or all of these genes is not known and is being investigated.
We have begun to generate fusion proteins containing
different domains of paxillin fused to GST to map potential binding
sites. Here, we show that GST-fusion proteins containing amino acids
100-557 bind to talin while amino acids 227-557 do not,
suggesting that a talin binding site resides between amino acids
100-227. In similar studies, Turner and Miller (31) have
shown that the non-LIM domains of chicken paxillin (amino acids
56-313 in our full-length predicted paxillin structure) bind
vinculin and p125. Since a fusion protein containing
amino acids 100-557 did not bind vinculin or p125
,
amino acids 56-100 could have critical residues for binding
vinculin and/or p125
, and this possibility is currently
being tested.
Our studies show that paxillin is tyrosine
phosphorylated in myeloid cell lines transformed by the human oncogene
P210. This is of interest in its own right, as very
few potential substrates of the p210
tyrosine kinase
have been identified(18) . We also found that paxillin is
transiently tyrosine phosphorylated in response to several
hematopoietic cytokines. Hematopoietic cells adhere to extracellular
matrix proteins through integrins, and these adhesive events are likely
to be critically important for migration and homing properties of
hematopoietic cells(13) . For example, GM-CSF has been shown to
induce rapid polymerization of F-actin in neutrophils, up-regulation of
integrin expression and function, and enhanced adhesion and chemotaxis
of neutrophils(22) . We found that growth factors such as
GM-CSF and IL-3 increase the tyrosine phosphorylation of proteins
within these structures. The aberrent tyrosine phosphorylation of
paxillin or other focal adhesion plaque proteins, caused by
p210
, could contribute to the known alteration in
adhesive properties of CML cells(13) . P210
reduces ability to bind to a number of extracellular matrix
proteins in the marrow microenvironment, and this is believed to
contribute to the extreme leukocytosis and extramedullary hematopoiesis
characteristic of this disorder.
In summary, we have shown that
paxillin is a common target for a tyrosine kinase(s) in hematopoietic
cells involved in IL-3 signal transduction and thus may be involved in
mediating signals from various receptors to focal adhesion-like
structures in this lineage as well as in others. The structure of
paxillin suggests that it could interact with other proteins through
many different motifs, including a series of LIM domains and SH2 and
SH3 binding domains, and thus contribute to the changes in cytoskeletal
structure that take place in response to cytokines, integrin
cross-linking, and differentiation. It will now be possible to
determine the nature of the interaction of vinculin, Crk, Src, and Fak
with paxillin. Finally, the phosphorylation of paxillin by
p210 and the reported interaction of p210
with CRK-L suggest that the role of paxillin in CML be examined.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14588 [GenBank]and U14589[GenBank].