(Received for publication, May 31, 1995; and in revised form, August 8, 1995)
From the
We have isolated a cDNA encoding a novel human intracytoplasmic
tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine
kinase). In addition, we have cloned and characterized the murine
homolog of the human RAFTK cDNA. Comparison of the deduced
amino acid sequences of human RAFTK and murine Raftk cDNAs revealed 95% homology, indicating that RAFTK is highly
conserved between these species. The RAFTK cDNA clone,
encoding a polypeptide of 1009 amino acids, has closest homology (48%
identity, 65% similarity) to the focal adhesion kinase
(pp125). Comparison of the deduced amino acid sequences
also indicates that RAFTK, like pp125
, lacks a
transmembrane region, myristylation sites, and SH2 and SH3 domains. In
addition, like pp125
, RAFTK contains a kinase domain
flanked by large N-terminal (426 residues) and C-terminal (331
residues) domains, and the C-terminal region contains a predicted
proline-rich stretch of residues. In fetal tissues, RAFTK expression
was abundant in brain, and low levels were observed in lung and liver.
In adult tissues, it was less restricted, indicating that RAFTK
expression is developmentally up-regulated. Expression of RAFTK was
also observed in human CD34
marrow cells, primary bone
marrow megakaryocytes, platelets, and various areas of brain. The human RAFTK gene was assigned to human chromosome 8 using genomic
DNAs from human/rodent somatic cell hybrid lines. The mouse Raftk gene was mapped to chromosome 14, closely linked to
gonadotropin-releasing hormone. Using specific antibodies for RAFTK, a
123-kDa protein from the human megakaryocytic CMK cell line was
immunoprecipitated. Treatment of the megakaryocytic CMK cells with
thrombin caused a rapid induction of tyrosine phosphorylation of RAFTK
protein. The structural features of RAFTK suggest that it is a member
of the focal adhesion kinase gene family and may participate in signal
transduction in human megakaryocytes and brain as well as in other cell
types.
Protein-tyrosine kinases participate in a variety of signal
transduction pathways that modulate cell growth and differentiation (1, 2, 3) . Signal transduction is triggered
by stimulation of a cell-surface receptor that either has kinase
activity itself or is physically and/or functionally linked to an
intracellular protein-tyrosine
kinase(4, 5, 6) . The integrins are also
capable of transducing cytoplasmic
signals(7, 8, 9) , and activation of this
pathway is linked to one or more protein-tyrosine
kinases(10, 11) . A nonreceptor cytosolic tyrosine
kinase, termed the focal adhesion kinase (pp125), has
been identified as one of the cellular proteins that is phosphorylated
in response to
- and
-integrin-mediated cell
adhesion(8, 12, 13, 14, 15) .
Induction of the kinase activity and tyrosine phosphorylation of
pp125
were observed following the adherence of
fibroblasts to
fibronectin(10, 11, 12, 13, 14, 15, 16) ;
the adherence of epidermal carcinoma cells to fibronectin, laminin, or
collagen type IV(17) ; and the aggregation of platelets in the
presence of fibrinogen, a ligand for
-integrin (glycoprotein
IIb/IIIa)(18) . Phosphorylated pp125
is localized
in focal adhesion contacts.
pp125 has been cloned from Xenopus laevis, avian, rodent, and human species and is
expressed in a wide range of cell
types(13, 14, 19, 20) . In addition
to activation of pp125
following stimulation of
-integrins (11) and glycoprotein IIb/IIIa
integrin(21) , it has been observed that pp125
is
activated in signaling mediated via high affinity IgE receptors (22, 23) and neuropeptide receptors (24) and
upon oncogenic transformation (16) in adherent cells. In cells
expressing both pp125
and src, pp60
formed a stable complex with
pp125
(25, 26) . Functional studies of
pp125
protein in various systems showed that bradykinin
stimulated tyrosine phosphorylation of pp125
, paxillin,
Ras GTPase-activating protein-associated p125, and src transformation-associated p130(27) . In addition, stable
association of pp125
with phosphatidylinositol 3-kinase
in NIH 3T3 mouse fibroblasts was observed(28) . pp125
expressed by nerve cell lines manifested increased tyrosine
phosphorylation in response to Alzheimer's A
peptide(24) . Recently, a novel pp125
-related
protein that is a substrate of tyrosine kinases in T and B lymphocytes
was reported(29) .
Relatively little is known about the
repertoire of signal transduction molecules in human
megakaryocytes(30, 31) . Platelets, the progeny of
megakaryocytes, contain pp125 that is phosphorylated on
tyrosine following platelet activation(18, 32) . We
have identified and characterized a novel intracytoplasmic kinase
isolated from human megakaryocytic and brain cells with 48% identity
(65% similarity) to pp125
. We have also cloned the murine
homolog of this cDNA. Given its homology to pp125
, we
have termed this new gene a related adhesion focal tyrosine kinase (RAFTK). Based on its molecular structure, its pattern of
expression, and the induction of tyrosine phosphorylation of RAFTK
proteins by thrombin in megakaryocytic cells, it is likely that RAFTK
participates in signal transduction and may have a role in cell growth
and differentiation.
A 340-bp probe was prepared from the 5`-end of one of the CMK cDNA clones (termed 2-1) and used to screen the human brain (hippocampus) cDNA library. Twelve clones were isolated, and two clones were sequenced on both strands. In addition, a 248-bp probe was prepared from the 5`-end of one of the clones (termed 4C), and the human hippocampus cDNA library was rescreened. Twelve clones were identified and isolated, and of these, one clone (termed 3B) was sequenced on both strands.
The mouse brain cDNA library
(catalog No. ML1042b, CLONTECH, Palo Alto, CA) in gt11 vector was
screened (
5
10
recombinants/screening) using
the 381-bp 5`
KpnI fragment or the 764-bp ApaI
3`-fragment of human RAFTK cDNA as a probe, and the
filters were hybridized and washed under high stringency conditions.
Six clones were isolated. The DNA was isolated as described previously (38) , subcloned into pBSK, and thereafter sequenced.
Nucleotide sequences were determined by the automated laser fluorescent
DNA sequencer using Autoread (Pharmacia Biotech Inc.) and by manual
sequencing using the Sequenase kit.
Figure 1:
A, schematic
representation and restriction enzyme map of the RAFTK cDNA.
The various cDNA clones, obtained from the human hippocampus cDNA
library (in ZAPII vector) and the CMK phorbol 12-myristate
13-acetate cDNA library (in
gt10 vector), are shown as indicated.
Restriction enzyme sites are indicated along the length of the cDNA. B, nucleotide and deduced amino acid sequences of the RAFTK cDNA clone representing the full-length cDNA. Nucleotide
numbers are shown on the left. The amino acid numbers are shown on the
right. The putative initiation codon at nucleotides 294-296 is
shown in boldface type. The catalytic domain is boxed. The ATP-binding site is underlined, and the
putative phosphorylation sites are encircled. The asterisk refers to the stop codon.
For metabolic labeling,
10 cells were labeled with 100 µCi of
[
S]methionine in 1 ml of Dulbecco's
modified Eagle's medium minus methionine (Amersham Corp.) for 16
h. Immunoprecipitation of RAFTK protein from labeled cells with RAFTK
antiserum or with normal rabbit serum was performed as described
previously(31, 45) . For immunoprecipitation of Tyr(P)
proteins, cold soluble extracts were first incubated with RAFTK
antibodies (R-4250) overnight at 4 °C. The extracts were then
incubated with protein G-Sepharose beads precoupled to goat anti-rabbit
IgG for 1.5 h at 4 °C. Proteins were eluted from the beads by
heating the samples at 100 °C for 5 min in SDS-polyacrylamide gel
electrophoresis buffer. Proteins were separated by SDS-polyacrylamide
gel electrophoresis, transferred, and immunoblotted with PY-20 (diluted
1:5000). The immunoreactive bands were visualized using the ECL system.
Figure 2:
Alignment of the predicted amino acid
sequences (single-letter code) of mouse Raftk, human RAFTK, and the mouse pp125 gene translated
product. Amino acid residues found to be conserved are boxed.
Figure 3:
Comparison of the deduced amino acid
sequence of RAFTK with those of m-pp125, Src, c-Fyn, Htk,
and Fgfr. Gaps (indicated by dashes) were introduced to
optimize the alignment. Amino acid residues found to be conserved are boxed.
Figure 4: Mapping of RAFTK in humans to chromosome 8 using human RAFTK cDNA. Shown are BamHI-digested genomic DNAs from hamster (h), human (H), and mouse (M) as well as 24 human/rodent somatic cell hybrids (labeled 1-22, X, and Y) probed with RAFTK cDNA. The human-specific RFLP is indicated with arrows and is seen in the human control lane and lane 8. A faint signal is observed in lane 20.
Figure 5:
Mapping of Raftk to mouse
chromosome 14. A, BamHI restriction enzyme pattern
for C57BL/6J (B) and M. spretus (S) genomic
DNAs probed with the 1.4-kb human RAFTK cDNA. The molecular
sizes of the fragments (in kilobase pairs) are indicated. B,
haplotype analysis of chromosome 14 genetic markers in (C57BL/6J
M. spretus)F
M. spretus BSS-type backcross mice showing linkage and relative position of Raftk. Closed boxes indicate the inheritance of the
C57BL/6J (B) allele, and open boxes indicate the
inheritance of the M. spretus (S) allele from the
(C57BL/6J
M. spretus)F
parent. Gene names
and references to these loci can be found in GBASE. The first two
columns indicate the number of backcross progeny with no
recombinations. The following columns indicate recombinational events
between adjacent loci (signified by a change from an open box to a
closed box). The number of recombinants is listed below each column,
and the recombination frequency (REC %) between adjacent loci
is indicated.
The position of Raftk on mouse chromosome 14 was confirmed by determining the segregation of a SacI RFLP for Raftk DNAs from BxD RI lines. The SacI RFLP for Raftk was indicated by the presence of a 16.5-kb genomic DNA band in C57BL/6J or a 6.2-kb fragment in DBA/2J (Fig. 6A). These alleles were characterized for 26 DNAs from the BxD RI line (Fig. 6B). The strain distribution patterns of Raftk and the locus coding for gonadotropin-releasing hormone, Gnrh(50) , indicate close linkage between these two loci on chromosome 14 (Fig. 6B). Perfect concordance was observed with the BxD strain distribution pattern for the Gnrh locus, indicating linkage of <1-map unit distance from Raftk Gnrh(51) . These mapping data place Raftk distal to Nfl and are a contradiction to the backcross data. However, backcross data are not as accurate as RI data since backcross mice were derived from an interspecies cross.
Figure 6: Cosegregation of Raftk and Gnrh in BxD RI lines and localization to chromosome 14. A, SacI restriction enzyme pattern for C57BL/6J (B) and DBA/2J (D) genomic DNAs probed with the 1.4-kb human RAFTK cDNA. The molecular sizes of the fragments (in kilobase pairs) are indicated. B, strain distribution pattern for Raftk in the BxD RI lines. The RI line distribution pattern is compared with that of the Gnrh locus. Map units are indicated between Raftk and Gnrh, as are 95% confidence limits.
Northern blot analysis of RNA from
human fetal heart, brain, lung, liver, and kidney revealed a weak
single major species of mRNA of 4.5 kb in brain, and it appears to be
expressed at low levels in lung and liver (Fig. 7A).
Expression in human adult tissues was assessed by hybridization of the
cDNA probe to a Northern blot of poly(A) RNA from
heart, brain, placenta, lung, liver, skeletal muscle, kidney, and
pancreas. While heart and skeletal muscle RNA samples were negative for
RAFTK, a single mRNA was observed in all other tissues, with the
highest levels expressed in brain (Fig. 7B). To further
characterize the distribution of RAFTK expression in other human
tissues, Northern blot analysis of spleen, thymus, prostate, testis,
ovary, intestine, colon, and peripheral blood leukocytes revealed a
high level of expression of RAFTK in thymus, spleen, and peripheral
blood leukocytes (Fig. 7C). Northern blot analysis of
different human brain regions (amygdala, caudate nucleus, corpus
callosum, hippocampus, hypothalamus, substantia nigra, subthalamic
nucleus, and thalamus) revealed that the highest level of expression of
RAFTK was in the amygdala and hippocampus (Fig. 8). A lower
level of expression was observed in the other brain regions, with the
exception of the corpus callosum and substantia nigra, where there was
no detectable signal. These results indicate that the brain has
abundant expression of RAFTK, especially in the amygdala and
hippocampus.
Figure 7:
RAFTK expression. A, expression
of RAFTK by Northern blot analysis in human fetal tissues; B,
expression of RAFTK by Northern blot analysis in human adult tissues; C, expression of RAFTK by Northern blot analysis in various
human tissues. The RNA blots were hybridized with a P-labeled RAFTK gene-specific probe, followed by
hybridization with
-actin (A and B) or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (C)
as the control for uniform RNA loading. Skel. Musc., skeletal
muscle; PBL, peripheral blood
leukocytes.
Figure 8: Expression of RAFTK by Northern blot analysis in human brain regions. The RNA blot was hybridized as described in the legend of Fig. 6. Caud. Nucl., caudate nucleus; Corp. Callos., corpus callosum; Hippoc., hippocampus; Hypothal., hypothalamus; Subst. Nigra, substantia nigra; Subthal. Nucl., subthalamic nucleus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Expression of RAFTK was observed in several
megakaryocytic cell lines such as CMK, Mo7e, HEL, and DAMI (data not
shown). In addition, expression of RAFTK was detected in Ramos, FHS,
and HeLa cells, but a low level of expression was detected in Jurkat,
Hep3B, and CCL75 cells (data not shown). Using PCR techniques,
expression of RAFTK was also found in primary bone marrow
megakaryocytes, blood platelets, and CD34 marrow
progenitor cells. Interestingly, it appears that the level of
expression of RAFTK mRNA is similar to that of FAK in
CD34
cells and is higher than that of FAK in bone
marrow megakaryocytes. In platelets, the level of expression of RAFTK mRNA is lower than that of FAK, as observed by PCR under
the same experimental conditions. It appears also that RAFTK mRNA expression in bone marrow megakaryocytes is higher than that
in CD34
cells (Fig. 9). Taken together, these
results indicate that RAFTK is abundantly expressed in brain and
hematopoietic cells. The restricted expression observed in fetal versus adult tissues indicates that its expression is
up-regulated during development.
Figure 9:
Expression of RAFTK in hematopoietic
cells. Shown is RAFTK expression by PCR in human CD34 marrow cells (lane 3), human bone marrow megakaryocytes (lane 4), and human platelets (lane 5).
CD34
cells, platelets, and bone marrow megakaryocytes
were processed as described under ``Experimental
Procedures.'' The PCR products were electrophoresed on a 2%
agarose gel and hybridized with gene-specific probes for RAFTK, FAK, and actin. Actin was used as an internal
control in this set of experiments. PCR controls were as follows: lane 1, RNA alone; lane 2, primers
alone.
The specificity of this antiserum was examined by
immunoprecipitation. The CMK cell line was metabolically labeled with
[S]methionine, and extracts were
immunoprecipitated with anti-RAFTK antiserum. A major protein species
of
123 kDa was detected in CMK cells (Fig. 10A). A
similar species was observed in other human megakaryocytic cell lines
such as DAMI (data not shown). This band was not observed when normal
rabbit serum or preimmune rabbit serum was used for
immunoprecipitation. Incubation of R-4250 with 1 or 10 µg of the
GST-RAFTK C terminus fusion protein abolished the appearance of the
123-kDa protein, while incubation with 10 µg of the GST-MATK
(where MATK is megakaryocyte tyrosine kinase) SH2 domain fusion protein
did not have any effects. These results indicate that R-4250 polyclonal
antibodies specifically recognize RAFTK protein of
123 kDa in
size. Furthermore, thrombin (1 unit/ml) stimulated a rapid increase in
the amount of RAFTK protein immunoreactivity in anti-Tyr(P)
immunoprecipitates (Fig. 10B). These results indicate
that RAFTK is a protein-tyrosine kinase and that thrombin can induce
its tyrosine phosphorylation.
Figure 10:
The
RAFTK protein. A, detection of RAFTK protein in vivo.
CMK cells were metabolically labeled with 100 µCi of
[S]methionine for 16 h, and the cell lysate was
immunoprecipitated by normal rabbit serum (lane 1), preimmune
rabbit (pre-R-4250) serum (lane 2), polyclonal antiserum for
RAFTK (R-4250) (lane 3), or anti-RAFTK antiserum(4250)
incubated with 1 µg (lane 4) or 10 µg (lane
5) of GST-RAFTK C terminus fusion protein or with 10 µg of
GST-MATK SH2 domain fusion protein (31) as a control (lane
6). B, tyrosine phosphorylation of thrombin-stimulated
CMK cells. CMK cells from whole cell lysates untreated (lane
1) or stimulated with thrombin (1 (lane 2) and 2 (lane 3) units/ml) for 5 min were immunoprecipitated with
RAFTK polyclonal antiserum R-4250 and then immunoblotted with the same
antibody (B) or immunoblotted with anti-Tyr(P) antibody PY-20 (C). Bands were visualized using the ECL
system.
The method of PCR cloning has been successfully employed by
many laboratories to identify novel members of the protein-tyrosine
kinase family. Using this strategy, we have identified a novel
intracytoplasmic tyrosine kinase in human megakaryocytic cells that we
have termed RAFTK. Sequence analysis of RAFTK revealed 48%
identity (65% similarity) to pp125
, suggesting that RAFTK
belongs to this subfamily of cytoplasmic tyrosine kinases. RAFTK does
not appear to be the recently described FAKB protein(29) , also
related to pp125
, since the specific amino acid sequence
used to make antisera that recognized FAKB protein is missing in the
predicted amino acid sequence of RAFTK protein (Fig. 2).
Furthermore, unlike FAKB, RAFTK protein did not form stable complexes
with the T-cell antigen receptor/CD3-linked tyrosine kinase ZAP 70 in
T-cells,
indicating that RAFTK and FAKB are different
proteins.
The chicken, human, and mouse focal adhesion kinases have
been recently implicated as playing key roles in signal transduction
pathways associated with extracellular adhesion molecules and with
receptors for neuropeptide growth
factors(14, 15, 52, 53) . Thus,
based on its homology to pp125, one would expect RAFTK to
participate in signaling pathways as well. The deduced 1009-amino acid
sequence of RAFTK (with a calculated molecular mass of 120 kDa)
contains a kinase domain and lacks a transmembrane region,
myristylation sites, and SH2 and SH3 domains (Fig. 1B).
To identify conserved regions within RAFTK between species that may
have important functions, we have cloned the murine homolog of the
human RAFTK cDNA. The sequence identity between the human RAFTK and murine Raftk cDNAs is 90% at the nucleotide
level and 95.6% at the predicted amino acid level. In the kinase
domain, 98.5% of the amino acids are identical (Fig. 2).
Therefore, the RAFTK gene is highly conserved in human and
rodent, again suggesting an important role in cell signaling functions.
RAFTK has an insertion of an additional 4 amino acids between positions
76 and 81
(Gly
-Arg
-Ile
-Gly
)
compared with chicken, murine, and human pp125
sequences (Fig. 2)(13, 14, 19) . Amino acids
corresponding to positions 292-320 of human pp125
and amino acids corresponding to positions 850-864 and
901-926 of chicken pp125
are absent in the
predicted RAFTK protein. Interestingly, like chicken
pp125
, the C terminus region of human RAFTK and
mouse Raftk contains a proline-rich stretch (residues
690-767). It has been shown that proteins containing proline-rich
peptide motifs (such as Shc, p62, and ribonucleoprotein K) could serve
as SH3 domain ligands and that the binding of these proteins to the Src
SH3 domain was inhibited with a proline-rich peptide
ligand(54) . Furthermore, the predicted RAFTK protein, like the
pp125
protein, displays several unique features among the
known tyrosine kinases. The primary sequence of RAFTK does not contain
a signal peptide or a membrane-spanning region, and the protein is
therefore presumed to be located in the cytoplasm. RAFTK lacks SH2 and
SH3 domains, which are structural elements involved in protein-protein
interactions(2, 47, 55, 56, 57) ,
and does not exhibit significant homology to any known protein-tyrosine
kinase beyond pp125
outside of the catalytic domain ( Fig. 2and Fig. 3). Lack of SH2 and SH3 domains suggests
that other regions within RAFTK protein are important for protein
interaction and targeting. In the case of the pp125
protein, it has been demonstrated by structural-functional
analysis that 159 amino acids within the C terminus are essential as a
``focal adhesion targeting'' sequence(58) . The
homology between RAFTK and pp125
within this region is
52%. The overall structure of RAFTK is characteristic of the
pp125
gene, with the catalytic domain flanked by large N-
and C-terminal domains (Fig. 2). It has recently been reported
that deletions of the N- or C-terminal noncatalytic domain of
pp125
including Tyr
did not abolish the
kinase activity of pp125
(59) . Moreover, there
is conservation of several tyrosine residues between RAFTK and
pp125
( Fig. 2and Fig. 3), including
Tyr
, which has been shown to be the major site of
tyrosine phosphorylation in pp125
protein(25) .
RAFTK-specific mRNA expression was observed in human fetal tissues,
being most abundant in brain (predominantly in the amygdala and
hippocampus regions), and appeared to be developmentally up-regulated
as demonstrated in the pattern of adult tissue expression ( Fig. 7(A-C) and 8). Within the hematopoietic
system, in addition to peripheral blood leukocytes, a high level of
specific mRNA expression of RAFTK was detected in B-cells and various
megakaryocytic cell lines (data not shown). By using PCR, the specific
mRNA expression of RAFTK was also detected in CD34
primary bone marrow progenitor cells, primary bone marrow
megakaryocytes, and platelets (Fig. 9).
RAFTK is
phosphorylated after thrombin treatment of CMK cells. Interestingly,
FAK protein was also found phosphorylated on tyrosine after thrombin or
collagen treatment of platelets(18) . There is considerable
homology in the thrombin receptors and considerable signal similarities
in transduction mechanisms between platelets and
megakaryocytes(60) . Furthermore, bone marrow megakaryocytes in
liquid culture stimulated with thrombin for 5 min revealed dramatic
morphological changes reminiscent of those found in platelets,
including shape change and organelle centralization that involved
immature as well as mature cells(61) . Megakaryocytes were also
able to secrete -granule proteins in the dilated cisternae of the
demarcation membrane system(61) .
The human RAFTK gene was found on chromosome 8 using DNAs from the somatic cell
hybrid lines (Fig. 4). The signal observed in cell line 20 in
mapping panel 2 suggested that a fragment of chromosome 8 is in the
chromosome 20 cell line. Indeed, although cell line 20 contained the
human NEFL gene, there was no evidence of chromosome 20 or a
fragment of chromosome 20 in cell line 8 (Coriel Cell Institute for
Medical Research). Indeed, the localization of RAFTK to
chromosome 8 was confirmed using mapping panel 1. The human NEFL gene has been localized to chromosome 8p21 (62) . Nfl, the murine homolog of human NEFL, has been
mapped to mouse chromosome 14 and is within 3 cM of the Gnrh locus (GBASE). The close linkage of the mouse Raftk gene
to Nfl (whose NEFL homolog is on human chromosome
8p21) suggested that the human RAFTK gene may be mapped to
chromosome 8 based on homology between human and mouse
chromosomes(62) . Therefore, we predict that the human RAFTK gene will be localized to chromosome 8p21. We have
mapped the mouse Raftk gene to chromosome 14 using a (C57BL/6J
M. spretus)F
M. spretus backcross. The position of mouse Raftk was confirmed by
RI line mapping using the BxD RI lines. The Raftk gene was
also shown to be closely linked to Gnrh, whose human homolog (LHRH (luteinizing hormone-releasing hormone)) has been mapped
to human chromosome 8p21-11.2(42) .
There has been
considerable interest in the role of pp125 in signaling
pathways in a variety of normal and transformed cells in response to
different soluble and cell-surface stimuli. Future studies will aim to
gain insights into the function of RAFTK in these signal transduction
pathways, particularly distinguishing a role for RAFTK versus pp125
in these systems.
This paper is dedicated to Ronald Ansin for his friendship and support for our research program.