(Received for publication, April 27, 1996, and in revised form, October 15, 1996)
From the Division of Hematologic Malignancies,
Dana-Farber Cancer Institute, and § Division of Hematology
and Oncology, Deaconess Hospital, Department of Medicine, Harvard
Medical School, Boston, Massachusetts 02115
Integrin ligation initiates intracellular
signaling events, among which are the activation of protein tyrosine
kinases. The related adhesion focal tyrosine kinase (RAFTK), also known
as PYK2 and CAK, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK
is similar to p125FAK in that it lacks a transmembrane
region, does not contain Src homology 2 or 3 domains, and has a
proline-rich region in its C terminus. Here we report that RAFTK is a
target for
1-integrin-mediated tyrosine phosphorylation in both
transformed and normal human B cells. Ligation of the B cell antigen
receptor also induced tyrosine phosphorylation of RAFTK.
Phosphorylation of RAFTK following integrin- or B cell antigen
receptor-mediated stimulation was decreased by prior treatment of cells
with cytochalasin B, indicating that this process was at least
partially cytoskeleton-dependent. One of the
tyrosine-phosphorylated substrates after integrin stimulation in
fibroblasts is p130cas, which can associate with
p125FAK. RAFTK also interacted constitutively with
p130cas in B cells, since p130cas was detected in RAFTK
immunoprecipitates. Although the function of RAFTK remains unknown,
these data suggest that RAFTK may have a significant function in
integrin-mediated signaling pathways in B cells.
Integrins are /
heterodimeric receptors that are involved in
cell-cell and cell-matrix interactions. Integrins play a role both in
adhesion and in transducing signals involved in a variety of cellular
functions. One of the intracellular signaling events initiated by
integrins is the activation of tyrosine kinases (1). In many cell
types, including fibroblasts, epithelial cells, and hematopoietic
cells, there is prominent tyrosine phosphorylation of proteins of
105-130 kDa following integrin cross-linking (2, 3, 4, 5, 6, 7). One of these
proteins has been identified as the focal adhesion kinase
(FAK)1 p125FAK in a large
number of different cell types (8, 9, 10, 11, 12, 13, 14). It has been proposed that this
kinase, which localizes to focal adhesion contacts (9, 10), is involved in linking adhesive events at the cell surface with intracellular pathways required for normal cellular function.
Recently, we and others have reported the identification and cloning of
another related focal adhesion tyrosine kinase, called RAFTK (also
known as PYK2 and CAK) (15, 16, 17). This kinase has 65% homology with
p125FAK but is clearly distinct and could be distinguished
using specific antibodies (18). Similar to p125FAK, RAFTK
lacks a transmembrane region and does not contain any SH2 or SH3
domains but does have a proline-rich region in its C terminus. RAFTK is
highly expressed in hematopoietic cells and coexpressed with
p125FAK in megakaryocytes and B lymphocytes. It has also
been reported that stimulation of platelets and megakaryocytes with
thrombin induces the tyrosine phosphorylation of RAFTK, suggesting that it plays an important role in platelet signal transduction (15).
We have previously reported that integrin-mediated tyrosine
phosphorylation of the 105-130-kDa substrates in B cells can occur in
the absence of detectable p125FAK in certain B cell lines
(SB cells) (14). In this report we demonstrate that RAFTK was
tyrosine-phosphorylated after 1-integrin stimulation in normal and
transformed human B cells. RAFTK was also tyrosine-phosphorylated in
normal B cells following B cell antigen receptor (BCR) cross-linking.
Furthermore, p130cas, one of the tyrosine-phosphorylated
substrates following integrin ligation (19, 20), bound constitutively
to RAFTK. These results suggest a potential role for RAFTK in integrin
and BCR signaling pathways in B cells.
Monoclonal antibodies directed against
CD29/1-integrin (K20, IgG2a), and CD18 (10F12) were obtained from
ascites. Antiphosphotyrosine monoclonal antibody (4G10, IgG2a) was
provided by Dr. Brian Druker (Oregon Health Sciences Center).
Affinity-purified Rabbit anti-mouse (R
M) was obtained from Jackson
Laboratories (West Grove, PA). Anti-human IgG and IgM was obtained from
Jackson Laboratories. Polyclonal antibodies directed against RAFTK were
prepared as described previously (15). Anti-p130cas
monoclonal antibody was obtained from Transduction Laboratories (Lexington, KY).
The human B cell lines ARH-77, SB, and Nalm-6 were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 units/ml penicillin, and 50 µg/ml streptomycin. Normal tonsillar B cells were enriched from single cell suspensions of tonsil by immunomagnetic bead depletion of T cells, monocytes, and natural killer cells as described previously (5). Tonsils were obtained according to appropriate Human Protection Committee validation and informed consent. Following immunomagnetic bead treatment, these cells were greater than 95% CD20+, and less than 5% CD3+, CD56+, and CD11b+ when analyzed by indirect immunofluorescence and flow cytometry.
Stimulation of CellsCells were washed two times and
resuspended in Iscove's modified Dulbecco's media (Life Technologies,
Inc.) at 5 × 106 cells/ml, starved at 37 °C for 30 min, and then cooled on ice for 15 min. Cells were then incubated for
15 min on ice with a saturating amount of antibodies, washed once with
Iscove's modified Dulbecco's media before addition of RM (5 µg/ml), and then incubated for various periods at 37 °C. After
stimulation, cells were solubilized in cold lysis buffer (1% Nonidet
P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 5 µM aprotinin, 3 µM pepstatin, 10 mM NaF, 1 mM Na
vanadate) for 15 min on ice. After removing insoluble material by
centrifugation at 10,000 × g, either postnuclear
supernatants were stored at
70 °C, or immunoprecipitation studies
were directly performed. In some experiments, cells were pretreated
with cytochalasin B (2 µM) for 30 min at 37 °C before
stimulation.
For immunoprecipitation studies, cell lysates were precleared with 50 µl of 50% (v/v) protein A-Sepharose (Pharmacia Biotech Inc.) and incubated with specific antibodies for 2 h at 4 °C. Immune complexes were then collected with 25 µl of protein A-Sepharose for 1 h at 4 °C and washed four times with cold lysis buffer. Immunoprecipitated proteins were eluted by boiling in 50 µl of sample buffer (2% SDS, 10% glycerol, 0.1 M Tris, pH 6.8, 0.02% bromphenol blue), boiled at 95 °C for 3 min, and analyzed by 7% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked using either 5% bovine serum albumin for antiphosphotyrosine studies or 5% nonfat dried milk (for RAFTK and Cas blotting) in TBS-T and incubated for 1 h with the first antibody. Secondary horseradish peroxidase-conjugated antibodies (Promega, Madison, WI) were then incubated for 30 min and detected by enhanced chemiluminescence (ECL, Amersham Corp.).
Stimulation of human B cells and B cell
lines with mAbs directed against 1-integrin induces tyrosine
phosphorylation of phosphoproteins ranging from 105 to 130 kDa. Since
in certain cell lines such as SB, these substrates could be
phosphorylated in the absence of any detectable p125FAK, we
investigated whether RAFTK could be involved in the
1-integrin signaling pathway in human B cells. Normal tonsillar B cells or B cell
lines (SB and ARH-77) were stimulated with anti-
1-integrin or
anti-
2-integrin mAb followed by R
M for 30 min. Cellular lysates were subjected to immunoprecipitation with anti-RAFTK antibody followed
by antiphosphotyrosine immunoblotting. Neither
phosphotyrosine-containing proteins nor RAFTK were detected in
immunoprecipitates formed using preimmune serum (Fig.
1A, left panel).
As seen in Fig. 1A, an increase in the tyrosine
phosphorylation of RAFTK could be specifically observed in the B cell
lines following 1-integrin stimulation. Stimulation by anti-
2
integrin antibodies did not induce any significant increase in tyrosine phosphorylation. Stimulation of tonsillar B cells with anti-
1 mAb as
well as anti-IgM and -IgG antibodies induced an increase in the
tyrosine phosphorylation of RAFTK (Fig. 1A, right panel). The increase in tyrosine phosphorylation of RAFTK in tonsillar B cells
was less than that seen in cell lines, possibly due to an activation of
the cells during their purification. The band migrating at
approximately 50 kDa in Fig. 1A represents an Ig heavy
chain. The membrane was then stripped and reprobed with anti-RAFTK
antibody to confirm that equivalent amounts of RAFTK were loaded in
each lane (Fig. 1A, lower panels). Depending on the
resolution of the gels, RAFTK was seen to migrate either as a single
band or as a doublet. To examine this further, we immunoprecipitated RAFTK from three B cell lines (ARH-77, SB, and Nalm-6) and normal tonsillar B cells. As seen in Fig. 1B, left
panel, RAFTK was seen to migrate as two distinct bands in
different ratios. Furthermore, following
1 stimulation of Nalm-6
cells, both bands were tyrosine-phosphorylated (Fig. 1B, right
panel).
To determine the time course of the tyrosine phosphorylation of RAFTK,
ARH-77 and SB cells were cultured with anti-1-integrin mAb followed
by R
M for increasing periods; RAFTK was then immunoprecipitated and
analyzed by antiphosphotyrosine immunoblotting. As shown in Fig.
2A, there was an increase in the tyrosine
phosphorylation of RAFTK in a time-dependent manner
following specific
1 stimulation, which began after 5 min, increased
until 60 min, and declined thereafter. A similar time course of
tyrosine phosphorylation was seen following BCR cross-linking in normal
tonsillar B cells (Fig. 2B).
Tyrosine Phosphorylation of RAFTK in Normal Tonsillar B Cells Requires an Intact Cytoskeleton following
In B cells, the 1-integrin-induced increase in
tyrosine phosphorylation of substrates from 105 to 130 kDa was
inhibited by cytochalasin B (14). We thus investigated the cytoskeletal
dependence of tyrosine phosphorylation of RAFTK. ARH-77 cells and
normal tonsillar B cells were preincubated for 30 min at 37 °C with
media alone (
CB) or cytochalasin B (+CB) prior to
1 or BCR
stimulation, respectively. As shown in Fig. 3, the
increase in RAFTK phosphorylation induced by stimulating cells with
anti-
1 or anti-Ig antibodies was reduced by cytochalasin B
pretreatment (+CB). These results suggest that the phosphorylation of
RAFTK induced by ligation of
1 integrin or BCR was partially
dependent on the integrity of the actin cytoskeleton.
RAFTK Associates in Vivo with the crk-associated Protein p130cas
One of the tyrosine-phosphorylated substrates
after integrin stimulation is p130cas (19, 20). Since
p130cas has been reported to bind to p125FAK (21),
we investigated whether p130cas could also associate with
RAFTK. Membranes containing immunoprecipitated RAFTK from the B cell
line SB, which were either unstimulated or anti-1-integrin- or
anti-
2-integrin-stimulated, were reprobed with antiphosphotyrosine
antibodies (4G10; Fig. 4A), anti-RAFTK antibodies (Fig. 4B), or anti-Cas antibodies (Fig.
4C). RAFTK was tyrosine-phosphorylated after anti-
1
stimulation, as detected by antiphosphotyrosine blotting (Fig.
4A). p130cas could be detected in RAFTK
immunoprecipitates (Fig. 4C); however, the association
appeared to be constitutive, since no increase was observed after
1
stimulation, despite an increase in the tyrosine phosphorylation of
RAFTK after such stimulation. Conversely, membranes containing
immunoprecipitated p130cas from SB cells were probed with
anti-RAFTK antibodies (Fig. 4B) or with anti-Cas antibodies
(Fig. 4C) and demonstrated the presence of RAFTK in the Cas
immunoprecipitate. No Cas was detected in the preimmune serum
immunoprecipitation of RAFTK. The band migrating at approximately 50 kDa in Fig. 4A represents the Ig heavy chain. A similar
in vivo association could also be observed in the B cell
lines Nalm-6 and ARH-77 (data not shown).
The signal transduction pathways initiated by integrin ligation involve cytoskeletal-dependent activation of tyrosine kinases and phosphorylation of a number of substrates (2, 3, 4). One of the kinases involved in integrin signaling is the focal adhesion kinase p125FAK. Tyrosine phosphorylation of p125FAK has been observed in a variety of cell types (8, 9, 10, 11, 12, 13, 22), which suggests that this kinase is part of a general signaling pathway for integrin signaling. In previous studies we have observed that certain B cell lines remain responsive to integrin ligation, as determined by tyrosine phosphorylations of various substrates, in the absence of detectable p125FAK (14). This suggests that in these cells other kinases may provide functions similar to p125FAK. In this study we provide evidence in human B cells that a tyrosine kinase that is closely homologous to p125FAK, known as RAFTK (15), also participates in integrin signaling.
Since 1-integrin-induced tyrosine phosphorylation of 105-130-kDa
substrates is observed in certain p125FAK-negative B cell
lines, we investigated whether RAFTK was involved in this pathway.
RAFTK was present in normal mature B cells and all B cell lines
examined and was tyrosine-phosphorylated after
1- but not
2-integrin stimulation. However, we did not detect a significant
increase in the autophosphorylation kinase activity in RAFTK following
1-integrin or BCR stimulation (not shown). This is somewhat
analogous to p125FAK, in which phosphorylation of Tyr-397
is not necessary for kinase activity (23, 24). Moreover, RAFTK
phosphorylation also occurred in the SB and RPMI 8866 (not shown) B
cell lines, which do not express detectable p125FAK,
indicating that tyrosine phosphorylation of RAFTK may occur independently of p125FAK. We also investigated whether FAK
could become tyrosine-phosphorylated in these studies, and no
consistent phosphorylation of p125FAK was observed (data
not shown). Further support of the nongeneralized involvement of
p125FAK is the finding that ligation of integrins on human
monocytes and T cell clones can lead to tyrosine phosphorylation of
various substrates in the absence of detectable expression of
p125FAK or tyrosine phosphorylation of p125FAK,
respectively (25, 26). Although homologous to p125FAK,
stimulation of fibroblasts through
1-integrins did not induce tyrosine phosphorylation of CAK
(identical to RAFTK; Ref. 17), suggesting that in different cell lineages the function of RAFTK may
differ.
Ligation of integrins in association with stimulation of the T cell
antigen receptor (TCR) provides a costimulatory signal to T lymphocytes
(27). This suggests that there may be common links between these
pathways. Following cross-linking of the TCR, p125FAK is
tyrosine-phosphorylated. We have observed that stimulation of B cells
through cross-linking the BCR induced tyrosine phosphorylation of
RAFTK. Furthermore, both integrin- and BCR-induced RAFTK
phosphorylation were partially decreased by pretreatment of cells with
cytochalasin B, suggesting that RAFTK tyrosine phosphorylation may
require the formation of a cytoskeletal complex, which provides a
foundation for the compartmentalization and interactions of kinases and
substrates. The functional effects of simultaneous ligation of integrin
and antigen receptors on T and B cells suggest that there are shared pathways between TCR (or BCR) and 1-integrin signaling and that one
of the common components may be the focal adhesion kinases.
The presence of potential SH2 binding motifs and a proline-rich region mediates the association of p125FAK with a number of other proteins. These include tyrosine kinases, including Fyn (28), Src (29), Csk (30), and the p85 subunit of phosphatidylinositol-3 kinase (31), through their SH2 domains. It has also been reported that the SH3 domain of p130cas binds to the proline-rich region of p125FAK (21). p130cas is one of the tyrosine-phosphorylated substrates following integrin ligation in a variety of cell types (19, 20). Furthermore, the SH3 binding motifs in p125FAK (APPKPSR) and RAFTK (PPPKPSR) appear almost identical (15).
We observed an in vivo association between RAFTK and
p130cas. This association was constitutive, since there was no
increase in the amount of p130cas coimmunoprecipitated with
RAFTK after 1 stimulation. This association was seen in B cell lines
that both expressed or lacked p125FAK, indicating that in
these B cells, p130cas bound to RAFTK independently of
p125FAK. The interaction p130cas and RAFTK is also
likely to be mediated through the SH3 domain of p130cas with
the C-terminal proline-rich region of RAFTK, and further studies are
currently directed toward identifying the binding site. The
p130cas observed to be associated with RAFTK did not appear to
be tyrosine-phosphorylated. p130cas is
tyrosine-phosphorylated in B cells following
1
stimulation,2 and this suggests that the
pool of tyrosine-phosphorylated p130cas may be distinct from
that associated with RAFTK. The precise role, if any, of RAFTK in
p130cas phosphorylation is presently under investigation. The
structure of p130cas suggests that it may act as a docking
protein and could function to bring various potential substrates into
proximity of RAFTK. The tyrosine phosphorylation of RAFTK may induce
such associations, since RAFTK contains a consensus high affinity
binding site for the SH2 domains of Src kinases (17). Further studies
of the various kinases and proteins associated with RAFTK should
provide new insights into integrin signaling events.
The tyrosine kinase PYK2, which is identical to RAFTK, is involved in calcium release and mitogen-activated protein kinase function in neuronal cells (16). In addition, stimulation of megakaryocytes with thrombin leads to tyrosine phosphorylation of RAFTK (15). The evidence that RAFTK is involved in both integrin and BCR stimulation in B cells further supports the potentially broad function of this kinase in signaling pathways. However, RAFTK may serve as a link between these two distinct stimuli. Evidence for this is from studies of ligation of both integrins and BCR, in which there appears to be a functional cross-talk, leading to modulation of normal B cell proliferation and differentiation (32). Future studies will be directed toward understanding the function of RAFTK in integrin and BCR signaling pathways and gaining insight into the association of adhesion with antigen-induced activation of B cells.
We thank Drs. Brian Druker and Alain Bernard for the generous gifts of antibodies.