(Received for publication, September 4, 1996, and in revised form, October 28, 1996)
From the Diabetes Unit and Medical Services,
Massachusetts General Hospital and Harvard Medical School, Boston,
Massachusetts 021291, ¶ Division of Experimental Medicine, Brigham
and Women's Hospital and Harvard Medical School, Boston, Massachusetts
021152, and
National Center of Neurology and Psychiatry,
National Institute of Neuroscience, Tokyo, 187 Japan
SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segment of ABP-280, upstream of the ABP carboxyl-terminal dimerization domain. Activation of SEK-1 in situ increases the SEK-1 activity bound to ABP-280 without changing the amount of SEK-1 polypeptide bound.
The influence of ABP-280 on SAPK regulation was evaluated in human
melanoma cells that lack ABP-280 expression, and in stable transformants of these cells expressing wild type ABP, or an
actin-binding but dimerization-deficient mutant ABP (ABPCT109).
ABP-280-deficient cells show an activation of SAPK in response to most
stimuli that is comparable to that seen in ABP-280-replete cells;
ABP-280-deficient cells, however, fail to show the brisk tumor necrosis
factor-
(TNF-
) activation of SAPK seen in ABP-replete cells and
have an 80% reduction in SAPK activation by lysophosphatidic acid. Expression of the dimerization-deficient mutant ABP-280 fails to
correct the defective SAPK response to lysophosphatidic acid, but
essentially normalizes the TNF-
activation of SAPK. Thus, a lack of
ABP-280 in melanoma cells causes a defect in the regulation of SAPK
that is selective for TNF-
and is attributable to the lack of
ABP-280 polypeptide itself rather than to the disordered actin
cytoskeleton that results therefrom. ABP-280 participates in TNF-
signal transduction to SAPKs, in part through the binding of SEK-1.
SEK-1 (1) (also known as MKK-4 (2) and JNKK (3) is a dual
specificity kinase whose only known substrates are the ,
, and
isoforms of the p54/46 SAPK1 subfamily of ERKs, and the
p38 ERK subfamily. SEK-1 phosphorylates these proteins
on a tyrosine and (probably) a threonine residue in the motif TPY for
the SAPKs and TGY for p38. The status of SEK-1 as a SAPK activator
in situ is certain, whereas its ability to serve as an
activator of p38 under physiologic circumstances is unclear, inasmuch
as recombinant MEKK-1 activates SEK-1 and SAPK (3-5) but not p38 (3)
in situ. Moreover, two potent activators specific for p38
have been identified at a molecular level, namely MKK-3 and MKK-6 (6).
The existence of SAPK activators other than SEK-1 has been demonstrated
in extracts from osmotically stressed 3Y1 rat fibroblasts; multiple
chromatographic peaks of SAPK activator activity are evident, including
a minor peak corresponding with immunoreactive SEK-1 (7). No
information as to the molecular structure of other SAPK kinases is
available. SAPKs are activated in situ by a remarkably
diverse array of cell perturbations (8). Several of these stimuli,
e.g. anisomycin and TNF-
, have been shown to also
activate SEK-1; however, it is not known whether all of the SAPK
agonists activate SEK-1 or recruit SAPK kinases differentially.
An interesting question in the operation of these protein kinase cascades is how the signal is conveyed from each receptor and multiple upstream stimuli to the appropriate MEK. A classical view is that the specificity is inherent in the sequential interaction of each element with its upstream and downstream partners, i.e. the MEKK interacts with the MEK, which in turn interacts with the ERK. Stable, relatively high affinity interactions of this kind have been documented (9) (e.g. Raf-MEK-1). Recent work, however, has emphasized an important contribution by "targeting" proteins in signal transmission, i.e. noncatalytic polypeptides whose main function is to provide a binding site for one or more catalytic elements, which positions them proximate to their upstream regulators and/or substrates, so as to facilitate or perhaps enable a speedy, localized and productive interaction. Examples include the targeting subunits for protein phosphatase-1 catalytic subunit and the A kinase binding protein AKAP 79, which appears to bind simultaneously kinase A (through its RII regulatory subunit), the Ca2+-activated protein (Ser/Thr) phosphatase calcineurin and protein kinase C (10, 11). More relevant to the ERK-based kinase cascades is the Saccharomyces cerevisiae protein Ste5p, a noncatalytic 90-kDa LIM domain polypeptide that binds the MEKK (Ste11), the MEK (Ste7), and the ERKs (Fus3/KSS1) that constitute the core kinase cassette in the pheromone signal transduction pathway of haploid S. cerevisiae (12). Each kinase is bound to Ste5p at a different site, concomitantly. Although direct interactions between the kinase/substrate pairs in this cassette also can be demonstrated, both in vitro and by two-hybrid methods, selective ablation of the Ste5 gene abolishes the pheromone response in situ. The kinases Ste11 and Ste7 are necessary for the response of haploid cells to nutrient deprivation (called the "invasive response") as well as to pheromone, and deletion of either kinase abrogates both responses (13). In contrast, abolition of Ste5 has no effect on the response to nutrient deprivation (14). Thus, the noncatalytic function of Ste5p is indispensable for coupling the pheromone receptor to the kinase cascade but is unnecessary for signal transmission to the same kinases by the upstream elements involved in the invasive response. Although it seems likely that signal transmission in the invasive response employs a protein analogous in function to Ste5, proteins homologous in structure or function to the Ste5p have not been identified in yeast or mammalian systems.
Herein we present evidence that the actin binding protein-280 (ABP-280,
non-muscle filamin) (15) provides a function analogous to yeast Ste5p
(16) in at least some mammalian cells by functionally coupling TNF-
and perhaps lysophosphatidic acid (LPA) receptors to the activation of
the mammalian SAPK pathway, concomitant with binding selectively the
SAPK activator, SEK-1.
Two-hybrid expression cloning was carried out as described by Durfee et al. (17). The SEK-1 cDNA sequence starting at amino acid 40 was inserted into the pAS1-CYH2A plasmid at SalI site downstream of the Gal4 DNA-binding domain. Human B cell and murine T cell cDNA libraries, inserted downstream of the Gal4 activation domain in the vector pACTII were screened by co-expression in S. cerevisiae strain Y190. The latter contains two reporter genes, HIS3 and LacZ, which allow selection of cDNAs encoding proteins that interact with the product of the gene of interest based on growth and LacZ activity.
Yeast transformed by the method of Geitz et al. (18) were
grown on plates lacking at least tryptophan and leucine to select for
the presence of the bait and the prey plasmid, respectively. After 4 days, a -galactosidase assay was performed by a color filter assay
using 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside as
described previously (17).
Catalytic subunit of cAMP-dependent
protein kinase (protein kinase A), protein A, LPA, and myelin basic
protein were purchased from Sigma. EGF, anisomycin,
TNF- were obtained from Cell Biology Boehringer Mannheim.
Glutathione-Sepharose 4B and protein G were purchased from Pharmacia
Biotech Inc. and Life Technologies, Inc., respectively.
Anti-glutathione S-transferase (GST), anti-SEK-1, and anti-ERK-1,2 antibodies were from Santa Cruz Technologies. Goat anti-TNF receptor 1 antibody was purchased from R&D Systems, Minneapolis. Monoclonal antibody anti-EE was a gift from Dr. D. Templeton (4). Polyclonal anti-XMpk2 (p38) antibodies were kindly provided by Dr. A. Nebreda. The anti-SAPK antibodies (19), the monoclonal anti-ABP-280 antibody (20), and purification of ABP-280 (20) were as described.
Preparation of GST Fusion Proteins and in Vitro Binding AssayscDNAs encoding p54 SAPK (19), human MEK-1 (21),
MEKK-1 (4) catalytic domain (carboxyl-terminal 320 amino acids), and c-Jun (1-135) were expressed in Escherichia coli as GST
fusion protein using the p-GEXkg vector (22) and purified by
glutathione-Sepharose affinity chromatography. The free p54
SAPK or
MEKK1 polypeptides were obtained after thrombin cleavage. The GST-SEK-1
polypeptide containing a hexahistidine tag at its carboxyl terminus was
purified by sequential chromatography glutathione-Sepharose and nickel chelate resin.
Binding in vitro of various polypeptides to GST or GST fusion proteins immobilized on glutathione-Sepharose was carried out at 4 °C for 2 h in binding buffer containing 25 mM Tris-Cl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100, and proteinase inhibitors. The beads were washed in excess binding buffer five times and the retained polypeptides were eluted directly into SDS-containing buffer, separated by SDS-PAGE, transferred into a polyvinylidene difluoride membrane, and analyzed by immunoblot.
Transfection and Preparation of Cell LysateThe association
of polypeptides in situ was assessed during transient
expression in COS M7 cells, transfected by the DEAE-dextran method
(23). GST was expressed using the vector pEBG(1); GST-SEK was expressed
by insertion of the cDNA encoding SEK-1 inframe downstream of the
GST coding sequence. The EE-tagged MEKK was expressed using the vector
pCMV5, as before (4). Subfragments derived from ABP carboxyl-terminal
411 amino acid fragment were constructed using the polymerase chain
reaction and were subcloned into the cytomegalovirus FLAG vector. All
transfections utilized a total of 20 µg of DNA; 48 h after
transfections, cells were extracted into a homogenization buffer
containing 20 mM Hepes (pH 7.4), 1 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 25 mM
-glycerophosphate, 1 mM sodium vanadate, 1% Triton
X-100, and proteinase inhibitors.
Extracts were equalized for protein prior to immunoprecipitation. After addition of antibody (monoclonal antibody anti-ABP-280 or monoclonal antibody for the EE-epitope) incubation was carried out for 2-4 h at 4 °C. Immune complexes were harvested with protein G-Sepharose, collected by centrifugation and washed four times with homogenization buffer. Bound protein was released from the particles by boiling for 5 min in SDS sample buffer. GST fusion protein were recovered using glutathione-Sepharose beads. Immunoblots were carried our using the ECL method (Amersham Corp.) according to the manufacturers' directions.
Activation of SAP Kinase, MAP Kinase, and p38 Kinase in Human Melanoma CellsHuman melanoma cell lines (parental line M2,
ABP, i.e. lacking ABP-280 expression; M2A7,
ABP+, stably transfected with full-length ABP-280; or M2
1.5, ABP
CT109 stably transfected with truncated
ABP
CT109) were grown in minimum essential medium supplemented
with 8% newborn calf serum as described previously (24, 25).
Confluent melanoma cells were deprived of serum overnight, and treated
at 37 °C with anisomycin (50 µg/ml, 40 min), EGF (50 ng/ml, 20 min), TNF- (50 ng/ml, 20 min), LPA (10 µg/ml, 10 min), or serum
(20%, 20 min). Cells were rinsed and extracted into homogenization buffer as above; cleared lysates were incubated with antisera for
4 h at 4 °C. Immunocomplexes were recovered by adsorption to
Sepharose-protein-G; the beads were washed three times with lysis
buffer, three times with lysis buffer containing 500 mM LiCl, and three washes with kinase reaction buffer (20 mM
MOPS (pH 7.2), 2 mM EGTA, 10 mM
MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100). Aliquots of beads were resuspended in 30 µl of kinase reaction buffer containing 1 µCi of [
-32P] ATP per
reaction and 20 µM of unlabeled ATP. Substrates used were
5 µg of purified GST-c-Jun(1-135) for SAPK (19) and 3 µg of myelin
basic protein for MAPK and p38 kinase activity. The reactions were
terminated after 20 min at 30 °C by addition of 10 µl of 5 times
concentrated SDS gel sample buffer. One unit of activity represents
transfer of 1 pmol of phosphate min
1 from ATP to protein
substrate.
Seeking proteins that interact with the SEK-1 protein kinase, we
used the SEK-1 coding region, starting at amino acid 40, to screen a
human B cell cDNA library constructed in the two-hybrid system
described by Durfee et al. (17). Thirteen LacZ+
His+ clones were isolated from 6.6 × 106
transformants; 10 gave very rapid complementation of LacZ+
activity and each encoded isoforms of the SAPKs. Among the remaining cDNA, one encoded polypeptide sequences identical to a
carboxyl-terminal segment of actin-binding protein-280 (20), starting
at amino acid residue 2236 of this 2647-amino acid polypeptide (see
Fig. 2A). In a separate screen of a mouse T cell library, a
cDNA encoding a carboxyl-terminal fragment of ABP-280 was isolated
independently. The specificity of the SEK-1 interaction with ABP-280
was evaluated by examining the ability of other protein kinases to bind
to the carboxyl-terminal fragment of ABP-280 in the two-hybrid assay. No evidence of binding of ABP-280 to MKK-3, p54 and p46 SAPK /
, Raf, or p70 S6 kinase was detected. Weak but definite complementation was observed consistently with MEK-1, MEK-2, and p38 (Table
I).
|
We attempted to verify the apparent interaction between SEK-1 and the
carboxyl-terminal region of ABP-280, and to examine its functional
significance. ABP-280 is an abundant cytoplasmic protein that contains
an amino-terminal actin binding domain of approximately 275 amino
acids, followed by an extended, rodlike structure created by 24 repeats
of a segment that is approximately 96 amino acids in length. Each of
these segments contains eight short sheet structures (A to H)
interrupted by turns. Sequence insertions of 23 and 35 amino acids,
probable hinge regions, precede repeats 16 and 24, respectively. The
carboxyl-terminal repeat (number 24) comprises a homodimerization
domain (15, 20). ABP-280 efficiently cross-links actin filaments into
orthogonal arrays. ABP-280 has also been shown to bind to the von
Willebrand factor (GPIb
/
IX) (26) and the high
affinity Fc (Fc
R1) (27) receptors through its
carboxyl-terminal rod domain.
The selective binding of SEK-1 to ABP-280 as measured by the two-hybrid
method was verified by examining the ability of a prokaryotic
recombinant GST-SEK to bind in vitro to full-length ABP-280
polypeptide, either in a biochemically pure form, or in cytoplasmic
extracts of 293 cells, where ABP-280 is bound to endogenous actin.
ABP-280, purified to homogeneity, was incubated with purified immobilized prokaryotic recombinant GST and comparable amounts of
GST-SEK, GST-SAPK, and GST-ERK-1 fusion proteins; only GST-SEK bound
ABP-280 (Fig. 1A). ABP-280 also bound
specifically to GST-MEK-1, although weakly as compared to GST-SEK-1; in
the experiment shown in Fig. 1B, GST SEK-1 retained about
90% of the input ABP-280 and GST-MEK-1 approximately 6% (Fig.
1B). GST-SEK-1 also bound ABP-280 endogenous to an extract
of HEK-293 cells (Fig. 1C), wherein ABP-280 is bound to
actin and perhaps other cytosolic components. Neither GST-ERK-1 nor
GST-SAPK bound ABP-280, whether the latter was presented as a purified
polypeptide or in an extract of 293 cells (Fig. 1C).
Binding of APB-280 to GST-SEK-1 in
vitro. A, aliquots of GSH-Sepharose beads, each
containing 20 µg of GST (lane 1), GST-SEK-1 (lane
2), GST-SAPK (lane 3), or GST-Erk1 (lane 4),
were incubated with purified ABP-280 (4 µg) in 1 ml at 4 °C for
2 h, and washed, and the retained proteins were subjected to
SDS-PAGE and immunoblotting with the antibody indicated to the
right of each panel. Lane 5 contains 5 µg of
pure ABP-280 for reference. The upper panel is an
anti-ABP-280 immunoblot; the lower panel is an anti-GST
immunoblot B, 20 µg of immobilized GST (lane
2), GST-MEK-1 (lane 3), and GST-SEK-1 (lane
4), were incubated with pure ABP-280 and treated as in
A. The upper panel is an anti-ABP immunoblot of
the retained ABP-280; lane 1 contains 1 µg of pure ABP-280
for reference. The lower panel shows the recovered fusion proteins. C, 20 µg of immobilized GST-SEK-1 (lane
1), GST-SAPK (lane 2), and GST (lane 4)
(20 µg) were tumbled with 293 cell extract for 6 h
(approximately 1 mg of protein) in 1 ml of binding buffer containing 1% Triton X-100; the beads
were washed twice with buffer containing 500 mM LiCl.
Retained proteins were subjected to SDS-PAGE and anti-ABP (upper
panel) and anti-GST (lower panel) immunoblot.
Lane 1 contains an aliquot of 293 cell extract.
Specific binding in situ of recombinant GST-SEK to a carboxyl-terminal fragment of ABP-280 tagged with a FLAG epitope, was demonstrable by co-precipitation after transient expression in COS cells (Fig. 2). Truncation of the carboxyl-terminal 193 amino acid from the 365 amino acid carboxyl-terminal ABP-280 fragment did not impair its association with GST-SEK in situ (Fig. 2B); shorter fragments of ABP-280 were poorly expressed. Thus GST-SEK binds to ABP-280 between amino acids 2282 and 2454 (repeat 21 to 23C); by comparison, the von Willebrand factor receptor binds to a fragment of ABP-280 containing repeats 16-23.
Whereas the von Willebrand factor receptor appears to bind
constitutively to ABP-280, ligand engagement of the FcR1
results in its dissociation from ABP-280 (27). We therefore examined the effect of SEK activation on the association of SEK with ABP-280. MEKK-1 directly phosphorylates and activates SEK-1 in vitro;
when overexpressed in situ, MEKK-1 activates SEK-1 and
produces a marked and preferential activation of SAPK (3-5). Vectors
encoding GST-SEK or GST were cotransfected in COS cells with an EE
epitope-tagged, constitutively active MEKK-1 carboxyl-terminal
catalytic fragment. Purification of cell extracts on GSH-Sepharose
yielded ABP-280 from cells expressing GST-SEK-1, but not GST (Fig.
3A). Despite substantial overall activation
of GST-SEK (not shown), coexpression of MEKK-1 with GST-SEK did not
alter the amount of ABP-280 polypeptide associated with GST-SEK-1 (Fig.
3A). The SAPK kinase activity of an ABP-280
immunoprecipitate prepared from extracts of cells expressing GST-SEK
was greatly increased by coexpression of MEKK with GST-SEK (Fig.
3B). Thus MEKK-1 activates the SAPK kinase activity bound to
ABP-280 (Fig. 3B) without altering the amount of associated
GST-SEK-1 protein (Fig. 3A). The MEKK catalytic fragment
does not associate directly with ABP-280 but can form a ternary complex
with SEK and ABP-280. Thus, a catalytically active prokaryotic
GST-MEKK-1 fusion protein does not bind pure ABP-280 in
vitro (Fig. 3C). Nevertheless, immobilized GST-SEK-1, bound to an essentially stoichiometric amount of prokaryotic
recombinant MEKK-1, still binds ABP-280 as efficiently as in the
absence of MEKK-1 (Fig. 3D). Moreover, immunoprecipitates of
the EE-tagged MEKK-1 catalytic fragment, expressed (singly) in COS
cells, are enriched in endogenous ABP-280 as well as endogenous SEK
(Fig. 3B). Thus it is likely that the MEKK-1 catalytic
fragment is able to bind to, and activate, SEK-1 while the latter is
bound to ABP-280, without displacement of the activated SEK-1 from
ABP-280 (e.g. see Fig. 3). By contrast, SAPK
immunoreactivity was not detected in association with ABP-280 (not
shown). Finally, immunoprecipitation of ABP-280 from 293 cells results
in the specific coprecipitation of a portion of the immunoreactive
SEK-1 endogenous to these cells; activation of SEK-1 by treatment of
the cells with anisomycin, prior to harvest, does not alter the amount
of endogenous SEK-1 polypeptide recovered with ABP-280 (Fig.
4).
The effect of SEK-1 activation in situ
on binding to ABP-280. A, endogenous ABP-280
associates with recombinant SEK. cDNAs encoding GST-SEK-1 (or GST)
and an EE-tagged MEKK-1 catalytic domain (or EE vector) were
co-transfected in COS cells. Extracts prepared 48 h after
transfection were purified by GSH-Sepharose affinity chromatography;
bound proteins were subjected to SDS-PAGE and immunoblot with anti-ABP 280 monoclonal antibody (upper panel) and
with anti-GST polyclonal antibody (lower panel).
B, SAPK kinase activity coprecipitates with ABP-280.
Extracts of COS cells co-transfected with GST-SEK-1 and EE vector
(lane 1) or EE MEKK1 (lane 2), were subjected to
anti-ABP immunoprecipitation. The washed ABP-280 immunoprecipitates
(Coomassie Blue stain, left panel) were assayed for SAPK
kinase activity by addition of prokaryotic recombinant p54 SAPK and
[
32P]ATP (autoradiogram, right panel).
C, ABP-280 does not bind directly to recombinant GST-MEKK1.
Immobilized prokaryotic recombinant GST (lane 1), GST-MEKK1
(lane 2), or GST-SEK-1 (lane 3) (20 µg) were
incubated with purified ABP-280 (4 µg). The retained proteins were
subjected to SDS-PAGE and immunoblot using anti-ABP (upper panel) or anti-GST antibodies (lower panel).
D, GST-SEK-1 binds ABP-280 and MEKK1 simultaneously.
Purified ABP-280 (2 µg; lanes 1-3) was incubated with
immobilized prokaryotic recombinant GST (10 µg; lane 3) or
GST-SEK-1 (10 µg; lanes 1 and 2) in the
presence of 10 µg of purified recombinant MEKK-1 catalytic fragment
(45 kDa, lanes 1 and 3), about a 50-fold molar
excess over ABP. After washing, bound proteins were subjected to
SDS-PAGE and transferred to polyvinylidene difluoride membranes, which
were subjected to Coomassie Blue stain (lower panel) and
anti-ABP-280 immunoblot (upper panel). E,
co-precipitation of recombinant EE-MEKK1 with endogenous SEK-1 and
ABP-280 from COS cell extracts. COS cells were transfected with
EE-tagged MEKK-1 catalytic domain (lanes 1 and 3)
or EE-vector (lane 2). Cells were extracted after 48 h,
and aliquots matched for protein were subjected to anti-EE immunoprecipitation. The immunoprecipitates were subjected to SDS-PAGE
and immunoblot with anti-ABP (upper panel) or anti-SEK-1 (lower panel).
We next attempted to evaluate the biologic significance of the
ABP-280/SEK association. The SAPK cascade might regulate the functions
of ABP-280 through phosphorylation of ABP-280 itself or of one or more
of the proteins to which it is bound or closely apposed. Although
ABP-280 can be phosphorylated in vitro by numerous protein
kinases (18), neither SEK (Fig. 5, lane 6)
nor SAPK (Fig. 5, lane 5) phosphorylate ABP-280 in
vitro under conditions wherein ABP-280 is well phosphorylated by
kinase A (Fig. 5, lane 7). Nevertheless, it remains possible
that as yet unidentified kinases downstream of SEK-1 other than SAPKs
are responsible for a portion of ABP-280 phosphorylation in
situ.
Another possibility is that ABP-280, rather than serving as a substrate
or positioning SEK near its substrates, is involved in the upstream
regulation of the SAPK pathway. Overexpression of the ABP-280
carboxyl-terminal 365 amino acid fragment does not abrogate activation
of a cotransfected SAPK reporter induced by MEKK-1 or V12 Rac, or by
treatment of cells with EGF, TNF-, or LPA (not shown). This negative
result, however, is tempered by the knowledge that multiple, perhaps
redundant, SAPK activators in addition to SEK-1 have been demonstrated
in other systems (7); their expression in COS cells and the ability of
ABP-280 to bind these elements is unknown.
Another approach to the role of ABP-280 in SEK-1 function is enabled by
the availability of human melanoma cell lines that have spontaneously
lost expression of ABP-280 (24, 25). These cells show prolonged
membrane blebbing after plating, poor spreading in cell culture, and a
deficit in directed cell movement. Stable expression of recombinant
ABP-280 in these cells such that the ratio of ABP-280 to endogenous
actin (1:160) is comparable to that found in motile cells, such as
macrophages, corrects these defects (24, 25). Extensive membrane
blebbing (over 50% of the cell circumference) in the ABP-280-replete
cells disappears in 90% of cells over the first 12 h after
plating, presumably as a consequence of the stabilization of the
cortical actin network (25). In contrast the percentage of cells
showing extensive membrane blebbing in the parental cells, which lack
ABP-280 expression, subsides to less than 20% slowly over the first
2-3 days after plating, coincident with a progressive increase in the
relative content of F-actin over that in the ABP-280-replete cells; the increase in F-actin is proposed to reflect an alternative mechanism for
cortical actin stabilization. Despite the loss of ABP-280 expression,
these lines express similar levels of SAPK and MAPK (ERK-1,2)
polypeptides (Fig. 6A). The regulation of the
SAP kinases in these two cell lines was therefore examined at 4-5 days
after plating, a time at which a comparable, low (<10%) fraction of cells continue to show blebbing. SAPK-specific activity 16 h after serum deprivation is similar in the two lines. Anisomycin, an inhibitor
of protein synthesis initiation, induces a marked SAPK activation
(range from 10- to 20-fold in three experiments) in both lines; EGF
yields a more modest but comparable 3-3.6-fold activation (averaged
over three experiments) in both lines, and in single experiments,
comparable SAPK activation was elicited by 20% fetal calf serum
(ABP, 5.3-fold; ABP+, 6.8-fold), 50 µM sodium arsenite (ABP
, 8.3-fold;
ABP+, 5.8-fold), and 0.7 M NaCl
(ABP
, 6.6-fold; ABP+, 10.2-fold). In
contrast, the average 4.7-fold activation of SAPK by TNF-
in the
ABP-280-replete line is lacking almost entirely in the
ABP-280-deficient cells, and the 5.7-fold SAPK activation elicited by
LPA in the ABP-280-replete cells is reduced by 80% in the
ABP-280-deficient, parental cells (Fig. 6B). These findings suggest that the SAPK activation in response to TNF-
and/or LPA involves the direct participation of ABP-280 or is influenced in a more
general way by the state of cortical actin organization (or both).
The existence of specific role for ABP-280 in the regulation of SAPK,
apart from indirect effects arising from of ABP-280's influence on
cortical actin structure was examined by stable expression in the
ABP-280-deficient cells of a dimerization-deficient of ABP-280 mutant
(ABP-CT109) that lacks the carboxyl-terminal 109 amino acids and is
able to bind, but not cross-link, F-actin. Cells expressing
ABP-
CT109 exhibit prolonged membrane blebbing similar to that
occurring in the ABP-280-deficient parental cells, indicating (as
expected) that the ability of ABP-280 to cross-link actin filaments is
crucial to its ability to stabilize the cortical actin network.
Expression of this ABP-280 carboxyl-terminal deletion mutant does not
alter SAPK activation by anisomycin, and has little effect on the
defective SAPK activation by LPA; in contrast, TNF-
activation by
SAPK is largely restored (Fig. 7). This response indicates that, while some aspect of the overall organization of
cortical actin influences SAPK regulation in response to LPA, the
monomeric ABP-
CT109, which contains the binding sites for actin,
SEK, various receptors, and perhaps other as yet unidentified elements,
but lacks the ability to cross-link F-actin, nevertheless contributes
in a specific way to the TNF-
regulation of SAPK.
The effects of ABP-280 deficiency on the regulation of two other well
characterized ERK isoforms was determined (Fig. 6, B and
C). In striking contrast to SAPK, whose specific activity in
serum-deprived cells is quite similar in the ABP-280-deficient and
-replete melanoma cells, the basal activity of MAPK and the p38 kinase
is 5-10-fold higher in the ABP-280-replete cells as compared to the
ABP-deficient cells despite similar levels of immunoreactive MAPK
polypeptide (Fig. 6A). EGF and serum increase the low basal
MAPK activity in the ABP-280-deficient cells to level approaching those
seen in the ABP-replete cells. TNF- gives little activation of p38
in either cell line; however, the 2-3-fold increment in MAPK induced
by TNF-
and LPA in the ABP-280-replete cell line is markedly reduced
in the ABP-280-deficient cells. The abundance of the p55 TNF-
receptor is comparable in the ABP-280- deficient and -replete cells
(Fig. 6A). Thus ABP-280 deficiency affects the regulation of
the various Erk cascades in a differential way, but in the case of both
the MAPKs and SAPKs, TNF-
activation is substantially reduced by
ABP-280 deficiency.
These data show that the dual specificity kinase SEK-1 binds directly and specifically to the actin-binding protein ABP-280. SEK-1 binding to ABP-280 occurs in vitro and in situ, and the association is not altered detectably when SEK-1 is activated in situ by anisomycin or by coexpression with an immediate upstream activator, MEKK-1. As a consequence, active SEK-1 remains associated with ABP-280, after ABP-280 immunoprecipitation, and is capable of phosphorylating and activating in vitro added bacterial recombinant SAPK. Neither SAPK itself nor MEKK-1, one of the candidate physiologic SEK activators, binds directly to ABP-280, but recombinant MEKK-1 overexpressed in COS cells coprecipitates a small fraction of cellular ABP-280 and SEK-1, presumably reflecting a complex wherein SEK-1 binds both ABP-280 and MEKK-1 concurrently. SAPK itself was not detected in immunoprecipitates of ABP-280, although we can not conclude on present evidence that SAPK is not associated (indirectly) with ABP-280 in situ, inasmuch as SEK-1 and SAPK exhibit an interaction in the two-hybrid format that is stronger than that between ABP-280 and SEK-1.
The interaction of SEK-1 with ABP-280 is specific in the sense that most of the other protein kinases we tested in the two-hybrid assay with ABP-280 failed to exhibit interaction; however, binding was detected between ABP-280 and MEK-1, and MEK-2 and the p38 kinase. The latter interactions of ABP-280 were much weaker than that observed with SEK-1 and were not characterized further, except for the demonstration that bacterial GST MEK-1 fusion protein is capable of weak but specific binding of ABP-280 in vitro.
ABP-280 appears to behave as a rodlike structure that binds actin
through its amino terminus, homodimerizes at its carboxyl terminus, and
contains two flexible hinges, one located two-thirds along its length,
and the second situated immediately before the carboxyl-terminal
homodimerization domain. As such ABP-280 is capable of cross-linking
actin filaments into orthogonal arrays, and contributes substantially
to the formation and structure of the actin meshwork situated
immediately subjacent to the surface membrane. This function may be
regulated, directly and indirectly, through cell surface receptors.
Direct regulation of ABP-280 function may occur through the association
of certain receptors with the carboxyl-terminal region of ABP-280. Thus
the integrin GPIb-IX, which binds von Willebrand factor on its
extracellular domain, associates constitutively through its
intracellular extension with ABP-280, binding to carboxyl-terminal
region of ABP-280 that includes the SEK-1 binding site (15, 26). The
likelihood that this binding is important to the organization of the
platelet cytoskeleton is indicated by the observation that platelets
lacking GPIb-IX (Bernard-Soulier syndrome) are enlarged and exhibit a disorganized cortical actin meshwork (28). The high affinity IgG Fc
receptor (Fc1R) also binds to ABP-280 in situ; however, this association is inhibited when ligand binds to FcR
(27).
Indirect regulation of ABP-280 function may occur through its phosphorylation inasmuch as ABP-280 undergoes phosphorylation in situ in response to serum, LPA, and other stimuli (29, 30). One of the candidate protein kinases responsible for a portion of ABP-280 phosphorylation in situ is the MAPK-regulated kinase, Rsk-2 (30); at this time, however, the functional consequences of ABP-280 phosphorylation in situ are not known. It is clear that neither SEK-1 nor SAPKs phosphorylate ABP-280. It is conceivable, however, that one of the elements downstream of SEK-1 (e.g. SAPK or one of its substrates) acts on a protein that also associates with ABP-280. Exploring this idea will require selective inhibition or knockout of SEK-1, an approach not yet technically attainable.
Conversely, the availability of human melanoma cells that have lost
selectively expression of ABP-280 (24) enabled an examination of the
possibility that the association of SEK with ABP-280 might be
consequential to the regulation of SEK-1. Inasmuch as reliably immunoprecipitating anti SEK-1 antibodies are not available, we chose
to monitor the activity in situ of a SEK substrate, SAPK. Our results show that, whereas SAPK activation in the serum-deprived state and in response to most perturbations is not altered
significantly by the absence of ABP-280, SAPK activation by LPA is
inhibited by 80%, and activation by TNF- is essentially abolished.
Expression of an ABP-280 fragment lacking the nomodimerization domain
does not restore SAPK responsiveness to LPA, but essentially corrects the loss of TNF-
regulation. Thus it appears that the decrease in
LPA responsiveness may be a consequence of the reorganization in
cellular actin caused by the absence of ABP-280, but the loss of
TNF-
regulation is directly attributable to the loss of the carboxyl-terminal, actin-independent region of ABP-280. Defective TNF-
signaling in the ABP-280-deficient cells is also evident in the
lack of MAPK activation by TNF-
, a response that is mediated by
MEK-1 and MEK-2, elements completely distinct from SEK-1. Notably, specific albeit weak binding of MEK-1 to ABP-280 is detectable in
vitro. The data presented provide good evidence that ABP-280 is a
participant in TNF-
signaling to SAPKs (and probably MAPKs), but
does not provide direct evidence that the loss of the ABP-280 binding
site for SEK-1 (and perhaps MEK-1) underlies the loss in TNF-
activation of SAPK and MAPK. Convincing support for this conclusion
will require the elucidation of the biochemical steps linking the TNF
receptor and other upstream inputs (e.g. UV-C ionizing
radiation, heat shock, and so forth) to SEK-1/SAPK, pathways that are
still poorly understood. SEK is activated by phosphorylation in
catalytic subdomain VIII, and two subfamilies of protein kinases, the
MEKKs and the MLKs, have been shown to be capable of catalyzing this
reaction, both in vitro and in situ (by
co-transfection). Regulation of the MEKKs is unclear; the MLK known as
SPRK/MLK-3 contains an SH3 domain and a binding site for
the small GTPases, Rac-1 and Cdc42. Overexpression of the
GTPase-deficient forms of Rac-1 and Cdc42 results in SAPK activation.
It may be relevant that ABP-280 has been reported to bind a small
GTPase (31, 32). In unpublished experiments we have observed specific
but GTP-independent binding of RhoA, Cdc42, and Rac-1 to biochemically
purified ABP-280. It is not known, however, whether TNF-
activation
of SAPK involves the participation of a small GTPase; this and other
aspects of TNF-
signaling to SAPKs are currently under
investigation. Nevertheless, the present results point to an important
role for the actin cytoskeleton in TNF-
signaling, at least in
melanoma cells, and suggest that the direct binding of one or more
signaling elements to ABP-280 may be important for this role.
We thank M. Kozlowski for valuable suggestions and J. Prendable for preparation of the manuscript.