COMMUNICATION
A Calcium-dependent Tyrosine Kinase Splice Variant in
Human Monocytes
ACTIVATION BY A TWO-STAGE PROCESS INVOLVING ADHERENCE AND A
SUBSEQUENT INTRACELLULAR SIGNAL*
Xiong
Li
§,
Deborah
Hunter
§,
John
Morris
,
J. Stephen
Haskill
¶, and
H. Shelton
Earp
**
From the
University of North Carolina Lineberger
Comprehensive Cancer Center, the ¶ Departments of Obstetrics and
Gynecology and Microbiology and Immunology, and the
Departments
of Medicine and Pharmacology, University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599
 |
ABSTRACT |
Freshly isolated human monocytes do
not express p125FAK but upon adherence to substrata
activate the highly related calcium-dependent tyrosine
kinase (CADTK), also known as Pyk2, CAK
, RAFTK, and FAK2. The
monocyte CADTK was 5 kDa smaller than protein from epithelial cells;
isolation and sequencing of the monocyte CADTK cDNA revealed a
predicted 42-amino acid deletion between the two proline-rich domains
of the enzyme. The nucleic acid sequence suggests that the deletion is
caused by alternative RNA splicing. This species was also found in T
and B lymphocytes and appears to be the predominant form of
cytoskeletal associated tyrosine kinase in non-neoplastic, circulating,
hematopoietic cells. CADTK was not activated when monocytes maintained
in suspension were treated with agents that produce an intracellular
calcium (thapsigargin) or protein kinase C (phorbol 12-myristate
13-acetate) signal including a chemokine, RANTES, that binds to the HIV
co-receptor, CCK5. In contrast, monocyte adherence to tissue culture
plastic-stimulated CADTK tyrosine phosphorylation, a process that was
enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES
but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or
collagen-coated surfaces produced only minimal CADTK activation but
permitted significant stimulation by added thapsigargin. These data
suggest that in a cell type that lacks p125FAK, CADTK plays
an early role in post-adherence signaling. Its activation involves two
stages, cytoskeletal engagement, which is permissive, and
co-stimulatory signals (calcium or protein kinase C) generated by
extensive cell surface engagement, agonists, or inflammatory chemokines.
 |
INTRODUCTION |
Peripheral monocytes circulate until they encounter an injured or
activated endothelial surface to which the receptors of the monocyte
adhere (1, 2). This results in shape changes initiating migration as
well as altered transcription and mRNA stability, which in turn
change gene expression and produce a more differentiated phenotype
(3-5). Adherence to extracellular matrix or engagement of fibroblast
or epithelial cell surface integrins activates the focal adhesion
kinase, p125FAK (6-8). We have purified (9) and then
sequenced (10) another member of the p125FAK family whose
regulation by calcium led us to call it the
calcium-dependent tyrosine kinase
(CADTK).1 Four other groups
isolated this kinase by molecular techniques, naming it Pyk2 (11),
CAK
(12), RAFTK (13), and FAK2 (14). CADTK is 45% identical and
66% similar to p125FAK, but unlike p125FAK,
CADTK is not tyrosine phosphorylated in adherent, epithelial (10),
neural (11), and smooth muscle cells (15). Rather CADTK is rapidly
activated and tyrosine phosphorylated when an intracellular calcium or
protein kinase C signal is generated (10, 11, 15). p125FAK,
but not CADTK, is detected in well studied fibroblast cell lines (e.g. NIH 3T3), whereas both enzymes are expressed in many
neural and epithelial cells (10, 11, 16). In this report we demonstrate a third type of cell exemplified by freshly isolated monocytes, which
express CADTK but not p125FAK. In addition, CADTK
activation in monocytes and epithelial cells is apparently a two-stage
process involving a permissive cytoskeletal engagement step and an
additional intracellular calcium or PKC signal. The concept of a
hierarchy in adherence-dependent signaling in monocyte
endothelial interactions is well established and may be reflected in
the two-stage activation of CADTK.
 |
EXPERIMENTAL PROCEDURES |
Isolation and Adherence of Monocytes--
Human monocytes were
isolated from randomly selected, healthy donors as described previously
(17). Purified monocytes were cultured in RPMI 1640 supplemented with
5% autologous serum at 37 °C under 5% CO2. When
cultured adherently, 5 × 106 to 5 × 107 monocytes were plated on polystyrene tissue culture
dishes (Corning) or fibronectin (Becton Dickenson) or collagen Type IV
(Sigma) coated culture dishes. Nonadherent monocytes were incubated in polystyrene tubes (Falcon) at cell concentration 106
cells/ml. Rat liver epithelial cells (GN4) were cultured as described (9).
Immunoprecipitation and Immunoblotting--
Lysates were
immunoprecipitated with CADTK (21) or p125FAK antibody
(A-17 or C-20, Santa Cruz Biotechnology) and analyzed as described
(10).
PCR Analysis--
Total cellular RNA from purified monocytes was
isolated by the guanidimium isothiocyanate-CsCl method, and
subsequently reverse transcribed with random hexamers as primers (17).
Three sets of PCR primers, which cover N-terminal
(5'-CTTAGCTGCTGCCTGAGAGG-3', 5'-CAGCTGAAGTACTGCCTGGC-3'), catalytic
(5'-GCCAGGCAGTACTTCAGCTG-3', 5'-CCAGCAGCGGGTCATGAGGG-3'), and
C-terminal domains (5'-CCCTCATGACCCGCTGCTGG-3', 5'-GGTGGCCCCCACCCTCCGTC-3') of human CADTK (Pyk2), were used to amplify
the entire first strand cDNA from monocytes. PCR-amplified products
were then cloned and sequenced. Cellular RNA of T and B cell lines were
kindly provided by Drs. Beverly Mitchell and Nancy Raab-Traub,
respectively. First strand cDNAs were made by a SuperscriptTM
preamplification system according to the manufacturer's (Life
Technologies, Inc.) protocol. The C-terminal PCR primers were used to
distinguish between the full-length and 126-base pair-deleted
isoforms.
 |
RESULTS AND DISCUSSION |
Monocytes Do Not Express p125FAK but Activate CADTK on
Adherence--
We adhered freshly isolated, lymphocyte-depleted
monocytes (an approximately 95% pure population) to plastic tissue
culture dishes for 30 min and immunoprecipitated CADTK and
p125FAK. Comparison with parallel immunoprecipitates from
angiotensin II (Ang II)-treated GN4 rat liver epithelial cells revealed
tyrosine phosphorylated CADTK in both cell types; however, the monocyte CADTK immunoreactive species had a faster electrophoretic mobility (Fig. 1, A and B).
In contrast to GN4 cells, monocytes lacked p125FAK (Fig.
1C). We have noted, under less stringent conditions,
i.e. those not involving preplating with autologous serum,
that p125FAK-containing, large, platelet-like entities were
found in the monocyte preparations. With our technique, these were
eliminated and p125FAK was absent.

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Fig. 1.
CADTK is tyrosine phosphorylated in freshly
isolated adhered monocytes but p125FAK is absent.
CADTK or p125FAK was immunoprecipitated (IP)
from adhered (30 min) human monocytes or Ang II-treated rat liver
epithelial cells (GN4). A, anti-Tyr(P) immunoblot
(IB) showed tyrosine phosphorylated CADTK in adhered
monocytes and Ang II-treated GN4 cells, but Tyr(P) p125FAK
was only seen in GN4 cells. B, reprobing with anti-CADTK
antibody demonstrated a slight difference in the electrophoretic
mobility of CADTK protein from human monocytes and GN4 cells.
C, p125FAK immunoblotting demonstrated
p125FAK in GN4 cells but not in human monocytes.
|
|
Monocytes as Well as T and B Lymphocytes Express a Putative CADTK
Splice Variant--
Monocyte CADTK had an estimated molecular mass of
110 kDa versus 115 kDa in GN4 cells. This could result from
cell type-specific phosphorylation, antibody cross-reaction with yet
another member of the p125FAK family, proteolysis, or an
encoded isoform. We used RNA from freshly prepared human peripheral
monocytes to isolate and sequence the entire CADTK coding region. The
monocyte CADTK cDNA sequence was almost identical to human Pyk2
with an exception, a 126-base pair deletion resulting in a molecule
missing 42 amino acids between the two CADTK SH3 binding proline-rich
domains (Fig. 2, A and B). Examination of the nucleic acid sequence reveals
potential splice donor sites at the junctions of the deletion,
suggesting that this isoform may be generated by alternative RNA
splicing (Fig. 2C). A similar set of splice donor acceptor
sites is found in the rat CADTK sequence, suggesting the capability of
forming this splice variant in other species. CADTK (and
p125FAK) has two proline-rich domains; this deletion could
influence the local tertiary structure, thereby changing individual
CADTK-SH3 interactions (e.g. with p130Cas).
Alternatively, the simultaneous interaction of SH3 domains from
different molecules with the two proline-rich regions of CADTK would be
influenced by the different spacing between the two regions due to the
42-amino acid deletion. In addition, the deleted sequence is rich in
prolines and serines characteristic of PEST-like sequences whose
deletion might change the susceptibility to proteolysis (18),
i.e. foreshortened CADTK could be more long-lived.

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Fig. 2.
Structure and expression of a putative
monocyte CADTK splice variant. A, schematic of CADTK showing
the N-terminal domain of unknown function, the tyrosine kinase domain,
the two proline-rich motifs separated by 63 amino acids, and a
potential focal adhesion targeting domain that is 70% similar by amino
acid sequence to p125FAK. The area of deletion in the
proline-rich domain is shown. B, the predicted monocyte
amino acid sequence is identical to the published human Pyk2 sequence
with the exception of the deletion shown aligned with the full-length
sequence. C, the nucleic acid sequence surrounding the
splice variant reveals potential RNA splice sites for acceptance and
donation. D, PCR primers designed to distinguish the
full-length and deleted isoforms were synthesized (see "Experimental
Procedures") and were predicted to yield PCR products of 1082 or 956 base pairs from the full-length and deleted isoform, respectively.
Monocytes and a B cell line express the deleted isoform; Jurkat cells
expressed both forms in roughly similar amounts. Subcloning and
sequencing confirmed the identity of both PCR products. First strand
cDNA from human peripheral blood mononuclear cells
(PBMC), a mixture of monocytes, and B and T cells
demonstrated only the 956-base pair fragment.
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|
Lymphocyte CADTK (Pyk2/RAFTK) is activated by T cell receptor
engagement (19, 20), a finding that we have replicated in mouse T
lymphocytes activated with specific
antigens.2 Therefore, we
investigated CADTK isoforms using first strand cDNA from human T,
B, and epithelial cell lines. A PCR strategy designed to detect
full-length and spliced CADTK revealed the short (splice variant) form
in monocytes as well as T and B cell lines (Fig. 2D). Jurkat
T cells also expressed a normal sized CADTK species (Fig.
2D). The identify of the PCR products from Jurkat and the B
cell line were confirmed by subcloning and sequencing. The short form
was identical to that of monocytes; the sequence of the larger Jurkat
product was identical to full-length human CADTK/Pyk2. Analysis of
multiple neoplastic T, B, and myelomonocytic leukemia cell lines
reveals the presence of both the deleted and full-length isoforms. The
epithelial lines (C33-1B cervical) and MCF10 (breast) do not exhibit
the deleted isoform (Fig. 3A).
Normal, monocyte-depleted, peripheral, blood, mononuclear cells exhibit either the deleted isoform alone (Fig. 2D) or a
preponderence of the deleted isoform with a smaller proportion of the
full-length isoform. The proportion of the full-length isoforms appears
to be donor-specific. In summary, the deleted isoform is found in all
hematopoietic cells tested with the amount of full-length isoform being
variable but appearing to be increased with neoplastic transformation.
Close examination of Figs. 2D and 3A reveals one other band in some T, B, and mononuclear cell line samples
(e.g. CEM-LD, H-9, BL41, RAJ1, and U937). Currently, we have
not identified a third isoform, but this possibility exists.
Furthermore, PCR analysis of adherent monocyte CADTK expression showed
that expression of the longer isoform increased somewhat with long term
monocyte adhesion (Fig. 3B).

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Fig. 3.
PCR analysis of CADTK isoform expression in
mononuclear cell lines and adherent monocytes. First strand
cDNA from monocyte-depleted, peripheral blood mononuclear cells
(PBMC, >70% T cells), human T cell lines (Jurkat, CEM-LD,
H-9, and KT-1), B cell lines (BL41, BL41-95, and RAJ1), myelomonocytic
leukemia lines (U937 and HL-60), and epithelial cell lines (C33-1B and
MCF-10) were amplified with C-terminal CADTK primers. As shown in
A, the deleted isoform of CADTK was expressed in all tested
cell lines of hematopoietic origin, but not in human epithelial cell
lines. Although the deleted isoform of CADTK predominates in monocytes,
a small amount of the full-length isoform is seen after one day of
monocyte adhered to plastic (B).
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|
Characterization of CADTK Tyrosine Phosphorylation Following
Monocyte Adherence--
CADTK tyrosine phosphorylation occurs within 5 min of adherence to tissue culture plastic (data not shown) and
appeared maximal at 30 min. In adherent epithelial (10), neural (11,
16), and smooth muscle cells (15), CADTK is tyrosine phosphorylated upon addition of agonists. Thus, it was surprising that we failed to
activate CADTK in freshly isolated monocytes in suspension with PMA
treatment (Fig. 4, A and
C). However, addition of PMA to adherent monocytes produced
an additional increment in CADTK tyrosine phosphorylation above that of
adherence alone (Fig. 4A). Continued adherence for 20 h
or 4 days resulted in persistent CADTK tyrosine phosphorylation (Fig.
4A). We repeated the adherent/nonadherent experiment using
PMA, the chemokine RANTES (which produces a distinct calcium signal)
(21, 22), and the tumor promoter thapsigargin, which results in an
intracellular calcium signal by blocking the intracellular calcium
reuptake mechanism. Again, in suspended monocytes, agonists failed to
stimulate significant tyrosine phosphorylation, although, in an
occasional experiment, low but detectable levels of CADTK tyrosine
phosphorylation were seen with thapsigargin (e.g. Fig.
4C). In adherent cells, the addition of PMA or,
particularly, thapsigargin resulted in increased CADTK tyrosine
phosphorylation (usually from 50% to 2-3-fold above that seen with
adherence alone). The increase caused by thapsigargin was present
whether thapsigargin was present during the entire 30 min adherence or
during the last 5 min of the 30-min adherence protocol (data not
shown). These results suggest two phases of activation, adherence
followed by additional amplifying signals. Each experiment was
performed with individual donors, and there were some donors in which
adherence to tissue culture plastic produced near maximal CADTK
tyrosine phosphorylation without additional agonists (data not
shown).

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Fig. 4.
CADTK is tyrosine phosphorylated in adherent
but not nonadherent monocytes. Freshly isolated monocytes were
prepared and either kept suspended or adhered to tissue culture dishes
for 30 min, 20 h, or 4 days. Some samples were treated with PMA
(100 nM), thapsigargin (Thaps, 2 µM), or RANTES (1 µM). Cells were lysed,
immunoprecipitated (IP) with anti-CADTK antibody, and
subjected to SDS-PAGE and immunoblotting. In one experiment, the
anti-Tyr(P) immunoblot (IB, A) was reprobed with
anti-CADTK (B), which revealed that PMA slightly increased
CADTK tyrosine phosphorylation in adherent (Ad) but not in
nonadherent (Non-Ad) monocytes. In a second experiment, the
Anti-Tyr(P) (C) and reprobed anti-CADTK immunoblots
(D) revealed that PMA, RANTES, and thapsigargin increased
tyrosine phosphorylation of CADTK in adherent cells to a much greater
extent than in nonadherent cells. In a third experiment, monocytes were
adhered in the presence or absence of cytochalasin D (Cyto
D, 2 µM, 30 min). Immunoblotting (E)
revealed that cytochalasin D blocked adherence-dependent
CADTK tyrosine phosphorylation.
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|
The importance of cytoskeletal engagement was emphasized by a complete
absence of CADTK tyrosine phosphorylation in cells adhered to plastic
in the presence of cytochalasin D (Fig. 4E), even though
similar amounts of CADTK were immunoprecipitated from both samples
(Fig. 4E). In contrast, adherence in the presence of the
microtubular inhibitor, colchicine (10 µM, 30 min), did not inhibit CADTK tyrosine phosphorylation (data not shown). Thus, it
appears that engagement or involvement of the actin cytoskeleton but
not the microtubules is necessary for the CADTK activation.
Adherence to activated or injured endothelium is a multistage process
that appears to involve several sets of monocyte-endothelial surface
protein interactions. Experimentally, monocyte activation and
subsequent gene expression differ depending on the substrata to which
cells adhere (3-5, 17). To investigate any hierarchy in adherence
signaling, we compared adherence to tissue culture plastic, a strong
stimulus, with a more physiologic substratum, culture dishes coated
with fibronectin or collagen. Adherence to the latter substrate
produced low level CADTK tyrosine phosphorylation, but addition of
thapsigargin to monocytes on fibronectin and collagen stimulated CADTK
tyrosine phosphorylation to the level near that seen with adherence to
plastic (Fig. 5A). Adherence
to fibronectin and collagen appears to be a weak stimulus per se but is
permissive, allowing thapsigargin, which has little or no effect in
nonadhered monocytes, to fully promote CADTK tyrosine phosphorylation
(Fig. 5A, Exp. 1). Similarly, adherence to
fibronectin followed by RANTES resulted in enhanced CADTK tyrosine
phosphorylation (Fig. 5A, Exp. 2). To further
test the idea that cytoskeletal engagement is necessary for CADTK
activation, we examined the effect of agonists on rat liver epithelial
cells adhered to culture dishes and then suspended in same tissue
culture medium for 15 min. Ang II, thapsigargin, and TPA stimulate
CADTK in adherent GN4 cells but fail to do so when added to GN4 cells
in suspension, even though CADTK expression is very similar in these
two conditions (Fig. 5, C and D). These data
further support the hypothesis that cytoskeleton engagement is required
for CADTK activation.

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Fig. 5.
Adherence on fibronectin and collagen is less
efficacious in activating CADTK tyrosine phosphorylation. Freshly
isolated monocytes were adhered on tissue culture plastic or dishes
coated with fibronectin (Fn) or collagen. Adherent cells,
treated with or without 2 µM thapsigargin (Exp.
1 and Exp. 2) or RANTES (Exp. 2) were lysed,
and CADTK immunoprecipitates (IP) were analyzed. The
anti-Tyr(P) (A) and reprobed anti-CADTK (B)
immunoblots (IB) revealed that adherence on plastic induced
CADTK tyrosine autophosphorylation to a greater extent than adherence
to fibronectin or collagen. Thapsigargin-stimulated CADTK tyrosine
phosphorylation in monocytes adhered on all three substratum, but the
effect was greater on fibronectin and collagen because basal tyrosine
phosphorylation was lower. On fibronectin, RANTES also resulted in
CADTK tyrosine phosphorylation. Adherent GN4 cells were suspended after
a short trypsin treatment and washing three times. With culture medium,
they were kept in suspension for 15 min. Both suspended and adherent
GN4 cells treated with Ang II (1 µM), thapsigargin
(Thaps, 2 µM), and TPA (100 nM)
for the indicated time were lysed and CADTK immunoprecipitates were
analyzed. The anti-Tyr(P) (C) and reprobed anti-CADTK
(D) immunoblots showed that calcium and/or PKC signal
significantly activate CADTK only in adherent but not in suspended
cultured GN4 cells. Ctrl, control.
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The discovery of a second member of the p125FAK tyrosine
kinase family independently by five groups (10-14) has raised
questions as to their similarities and differences between these
proteins. In epithelial and smooth muscle cells, adherence results in
sustained p125FAK tyrosine phosphorylation, whereas CADTK
is dephosphorylated. Agonists stimulate CADTK, and additional paxillin
and p130Cas tyrosine phosphorylation follows (23-25). The
monocyte provides a slightly different model. CADTK is expressed but is
not activated by thapsigargin, PMA, or RANTES in nonadhered peripheral
monocytes. Adherence by itself produces a range in monocyte CADTK
tyrosine phosphorylation depending on the substratum and, to some
extent, the donor, which was enhanced by acute treatment with
thapsigargin, PMA, or RANTES. The effect of cytochalasin D and the
minimal tyrosine phosphorylation when monocytes were adhered to
fibronectin and collagen support a potential two-stage process for
CADTK tyrosine phosphorylation (Figs. 4 and 5). A similar deficit in
thapsigargin or TPA-dependent activation is observed in GN4
epithelial cells (Fig. 5). This indicates that in adherent cells the
first stage, engagement of the cytoskeleton, has already occurred and
CADTK activation simply awaits a second cue, calcium or PKC activation. Whether this two-stage hypothesis implies distinct mechanisms, alignment to a cellular locale or structure followed by a calcium or
PKC-dependent activating phosphorylation, or a continuum in which cytoskeletal engagement is followed by another cytoskeletal step
that is indirectly influenced by calcium or PKC remains to be
determined.
 |
ACKNOWLEDGEMENTS |
We thank Ruth Dy and Tim Harding for
excellent technical assistance and Darla Nichols for manuscript
preparation.
 |
FOOTNOTES |
*
This work was supported in part by grants from the American
Cancer Society (to H. S. E.) and the National Institutes of
Health (to J. S. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
These two authors contributed equally to this project.
**
To whom correspondence should be addressed: Lineberger
Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Tel.: 919-966-2335; Fax:
919-966-3015; E-mail: hse{at}med.unc.edu.
1
The abbreviations used are: CADTK,
calcium-dependent tyrosine kinase; Ang II, angiotensin;
PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain
reaction; TPA, 12-O-tetradecanoyl-
phorbol-13-acetate.
2
A. Villette and H. S. Earp, unpublished
results.
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