(Received for publication, January 17, 1996; and in revised form, March 12, 1996)
From the
Although Ras-related small GTPases are believed to control cell proliferation and motility through activation of protein kinase cascades, little is known about the intracellular protein targets of activated kinases. Here we show that the p90 ribosomal S6 kinase 2 (RSK2) phosphorylates actin-binding protein (ABP-280) in intact rat 3Y1 fibroblasts. Growth factors such as fetal calf serum, epidermal growth factor, phorbol 12-myristate 13-acetate, and lysophosphatidic acid stimulate the phosphorylation of serine residues in ABP-280 in quiescent 3Y1 cells. Extracts from 3Y1 cells prepared after stimulation by lysophosphatidic acid, fetal calf serum, and epidermal growth factor retain activated protein kinase activity(s) toward ABP-280 in vitro. ABP kinase activities in lysates from lysophosphatidic acid-stimulated 3Y1 cells can be fractionated by MonoQ anion exchange column chromatography into three peaks having ABP kinase activities. One (ABP kinase peak 1) coelutes with the peak of RSK2 as judged by immunoblotting and S6 peptide kinase assays. Two-dimensional phosphopeptide maps show RSK2 phosphorylated ABP-280 to be phosphorylated at the same site(s) as those stimulated by growth factors in vivo. Incubation of ABP kinase peak 1 fractionated from unstimulated cells with activated ERK2 activates latent ABP kinase activity. These results show RSK2 to phosphorylate ABP-280 in vivo.
Protein phosphorylation cascades are essential for many
processes in mammalian cells. In particular, transmission of mitogenic
signals to intracellular targets is mediated by the activation of
protein kinases. One pathway is the Ras-dependent or the
mitogen-activated protein kinase (MAPK) ()signaling cascade (1, 2, 3, 4, 5) . In this
pathway, binding of growth factors to their receptors activates the
small GTPase Ras through the GRB2 adapter protein and guanine
nucleotide exchange factor (SOS). This is followed by a sequential
activation of multiple protein kinases (raf-1, MEK, MAPK, and RSK).
This protein kinase cascade leads to the phosphorylation of factors
which initiate gene expression and modify metabolic and cytoskeletal
processes to sustain or promote cell growth and differentiation. Hence,
activation of the serine/threonine-specific protein kinases (i.e. MAPKs and RSKs) is believed to effect the phosphorylation of
targets that play important regulatory roles in a variety of cellular
processes(6, 7) . However, the identification of
intracellular protein targets and the effect of phosphorylation on the
function of these proteins are poorly understood.
ABP-280 is a
member of a large protein family that shares the ability to cross-link
actin filaments into orthogonal networks(8) . ABP-280 is an
elongated dimer composed of identical 280-kDa subunits, each composed
of long rod domains of repetitive -sheet motifs that connect an
amino-terminal actin-binding domain to a carboxyl-terminal
self-association site(9, 10) .
ABP-280 and its
muscle isoform (filamin) are phosphorylated in intact
cells(11, 12) , and cAMP-dependent protein kinase
(cAMP kinase), protein kinase C, and
Ca/calmodulin-dependent protein kinase II (CaM kinase
II) phosphorylate ABP-280 and/or filamin in
vitro(13, 14, 15, 16) . Some
effects of in vitro phosphorylation have been identified.
ABP-280 phosphorylated by cAMP kinase has increased resistance to the
calcium-activated protease calpain (14) and altered interaction
with GTP and/or unidentified small GTP-binding
protein(s)(17, 18) . ABP-280 in platelets is a
substrate for cAMP kinase in vivo. Compounds, such as
prostaglandins I
and E
or forskolin, which
increase the intracellular cAMP levels, stimulate ABP-280
phosphorylation in platelets(14) . It is unclear, however, if
protein kinases other than cAMP kinase phosphorylate ABP-280 in intact
cells. Phosphorylation of filamin by CaM kinase II decreases
filamin's actin filament cross-linking activity(16) .
In this study, we find that stimulation of quiescent 3Y1 rat fibroblasts by growth factors such as epidermal growth factor (EGF) and lysophosphatidic acid (LPA) results in a rapid increase in the phosphorylation of ABP-280 through serine/threonine-specific protein kinases. We identified one of these protein kinases to be the p90 ribosomal S6 protein kinases (RSK2).
Figure 1:
Treatment of quiescent
rat 3Y1 fibroblasts with growth factors induces ABP-280
phosphorylation. A, quantitation of the effects of various
agents on the phosphorylation of ABP-280 in 3Y1 cells. 3Y1 fibroblasts
were labeled for 18 h in phosphate-free RPMI 1640 medium containing
[P]orthophosphate (0.2 mCi/ml).
P-Labeled cells were treated at 37 °C for 10 min with
Tris-buffered saline (control), 100 ng/ml PMA, 1.0 mM dibutyryl cAMP, 100 ng/ml EGF, 0.5 µM ionomycin, 10%
FCS, or 10 µg/ml LPA. Cell extracts were prepared and ABP-280
immunoprecipitated with monoclonal anti-ABP-280 antibody.
Phosphoproteins were analyzed by SDS-PAGE followed by autoradiography.
The extent of phosphate incorporation into ABP-280 band is expressed as
the percentage relative to the control value. The number of experiments
performed are indicated. Each value represents the mean ± S.E.
The inset shows the autoradiograph of
P-labeled
ABP-280 immunoprecipitated from 3Y1 cells stimulated by various agents.
The
P contents of the excised ABP-280 polypeptide from the
SDS-PAGE gel were determined by scintillation counting (Cerenkov). They
were 72 cpm (control), 157 cpm (LPA), 154 cpm (EGF), and 118 cpm (dBcAMP), respectively. The amount
of ABP-280 immunoprecipitated from each cell extract was identical
between control and stimulated cells as judged by Coomassie Blue
protein staining of the 280-kDa subunit of immunoprecipitated ABP-280. B, time course for the phosphorylation of ABP-280 after
exposure of quiescent 3Y1 cells to 10% FCS (
) or 10 µg/ml LPA
(
).
P-Incorporation is plotted relative to the
amount of phosphate incorporation into untreated cells. The
P content was determined by scintillation counting of
excised ABP-280 from SDS-PAGE gels. Each value represents the mean
± S.E. (n = 3)
Phosphoamino acid analysis of ABP-280 immunoprecipitated from lysates of LPA-treated cells reveals serine as the major phosphate-containing residue (Fig. 2A), demonstrating serine/threonine-specific protein kinase(s) to phosphorylate ABP-280 in intact cells. The phosphorylation site(s) are in the carboxyl-terminal third of the ABP-280 polypeptide (Fig. 2B), which is easily dissected from the amino end of ABP-280 using the calcium-activated enzyme calpain. There are only two calpain sites in ABP-280, and the most rapidly cleaved site generates 190- and 100-kDa subfragments. The 190-kDa fragment contains the amino termini F-actin binding domain of ABP-280. The 100-kDa carboxyl-terminal piece contains the second calpain site, and upon extended digestion, is cleaved into 90- and 10-kDa subfragments. This 90-kDa calpain fragment was phosphorylated when cells were incubated with FCS, EGF, and PMA (Fig. 2B). Two-dimensional phosphopeptide mapping analysis reveals that treatment with LPA (Fig. 5B) and other agents (data not shown) increases the phosphorylation of two major peptides of ABP-280 in intact cells. These results suggest that growth factors activate similar protein kinases which phosphorylate specific serine residues in the carboxyl-terminal tail of ABP-280.
Figure 2:
Phosphoamino acid analysis and
phosphopeptide map of ABP-280 isolated from P-labeled 3Y1
cells. A, two-dimensional phosphoamino acid analysis of
ABP-280.
P-Labeled 3Y1 cells were incubated with 10% FCS
for 10 min at 37 °C. Cell extracts were prepared and ABP-280
immunoprecipitated with anti-ABP-280 antibody. After SDS-PAGE and
autoradiography, the phospho-ABP-280 subunits were excised and
incubated with trypsin (100 µg/ml) and
-chymotrypsin (50
µg/ml) for 24 h at 37 °C. The phosphoamino acids of the eluted
peptides were determined as described under ``Experimental
Procedures.'' The directions of electrophoresis are indicated. P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. B, one-dimensional
phosphopeptide map of ABP-280 after calpain digestion.
P-Labeled 3Y1 cells were treated at 37 °C for 10 min
with Tris-buffered saline (control), 10 µg/ml LPA, 10% FCS, 100
ng/ml EGF, or 100 ng/ml PMA. ABP-280 was immunoprecipitated from cell
lysate, the immunoprecipitates were washed and incubated with 0.01
unit/ml calpain in the presence of 5 mM CaCl
, as
described under ``Experimental Procedures.'' Phosphoproteins
were analyzed by SDS-PAGE followed by autoradiography. The position of
the well defined cleavage fragments of 190, 100, and 90 kDa are
indicated.
Figure 5:
Phosphorylation of ABP-280 by RSK2. A, purified ABP-280 (a) or 40 S ribosomal protein (b) were incubated with purified RSK2 in the presence of
[-
P]ATP. Proteins were separated and
analyzed by SDS-PAGE followed by autoradiography. The autoradiogram is
shown. The positions of the ABP-280 subunit and 40 S ribosomal
polypeptide are indicated by arrows. B, comparison of
two-dimensional phosphopeptide map of ABP-280 phosphorylated in
quiescent, LPA-treated cells or by RSK2 in vitro.
P-Labeled 3Y1 cells were incubated without (a) or
with (b) 10 µg/ml LPA for 10 min at 37 °C. The ABP-280
immunoprecipitate was displayed by SDS-PAGE. Purified ABP-280 was
phosphorylated by RSK2 in vitro with
[
-
P]ATP and separated by SDS-PAGE (c). Phosphorylated ABP-280 was excised from the gels and
incubated with trypsin (50 µg/ml) for 24 h at 37 °C. After
digestion, phosphopeptides were separated in two dimensions on
cellulose plates and radiolabel detected by autoradiography. The
orientation of electrophoresis (+, -) and the direction of
chromatography are shown.
Figure 3:
Characterization of the ABP kinase
activity present in extracts from LPA stimulated cells. Cytosolic
extracts from quiescent 3Y1 cells (control) or cells treated
with 2 µg/ml LPA, 10% FCS, or 100 ng/ml EGF for 5 min were
incubated with [-
P]ATP in the presence of
exogenous purified human ABP-280 for 20 min at 30 °C as described
under ``Experimental Procedures.'' The reactions were then
stopped, and the samples were analyzed by SDS-PAGE followed by
autoradiography. The amount of phosphate incorporated into ABP-280 in
quiescent cell extracts is defined as 100%. Autoradiographs were
scanned and quantitated using the NIH Image software program to
determine their relative
P content. The number of
experiments is indicated. Each value represents the mean ± S.E.
The inset shows the autoradiographs of
P-labeled
ABP-280 phosphorylated in vitro by cytosolic extracts from
quiescent 3Y1 cells (control) or cells treated with LPA for 5
min (+LAP) in the presence (upper panel) or
absence (lower panel) of exogenous ABP-280 with
[
-
P]ATP for 20 min at 30 °C. The
phosphorylation of human ABP-280 in vitro by extracts from
quiescent and agonist-stimulated cells were linear for 20
min.
Fig. 4A shows the relationship between time of
exposure of fibroblasts to LPA and the amount of ABP kinase activity
present in extracts from lysates of these cells. The half-maximal
activation of protein kinase activity occurred 2 min after the addition
of 2 µg/ml of LPA to the cells; maximal stimulation (5-fold
relative to untreated cell extracts) was reached in
5 min. ABP
kinase activity declined thereafter and returned to near basal activity
after 30 min. ABP kinase activity was not affected by the addition of
specific inhibitors to protein kinase C or cAMP kinase (Table 1)
and was reduced about 50% in the presence of 1 mM CaCl
or 50 mM NaF. Direct addition of LPA into the extracts
from quiescent cells did not increase ABP kinase activity, showing that
the activation of ABP kinase activity depends on the receptor-mediated
signaling (data not shown).
Figure 4:
Characterization of the ABP kinase
activity present in extracts from LPA stimulated cells. A,
time course for the phosphorylation of exogenously added ABP-280 by ABP
kinase activity in extracts. Cell extracts were prepared at the
indicated time points after exposure to 2 µg/ml LPA, and ABP kinase
activity was determined using purified human ABP-280 as substrate. ABP
kinase activity was normalized to basal incorporation by subtracting
background labeling without added exogenous ABP-280. Each value
represents the mean ± S.E. of at least three determinations. B, one-dimensional phosphoamino acid analysis of ABP-280
phosphorylated in vitro by lysate from 2 µg/ml LPA-treated
cell. After SDS-PAGE and autoradiography, the phospho-ABP-280 subunit
was excised and incubated with trypsin (100 µg/ml) and
-chymotrypsin (50 µg/ml) for 24 h at 37 °C. The
phosphoamino acids of the eluted peptides were determined as described
under ``Experimental Procedures.'' P-Ser,
phosphoserine; P-Thr, phosphothreonine; P-Tyr,
phosphotyrosine. C, one-dimensional phosphopeptide map of
ABP-280 phosphorylated in vitro using a extract from 2
µg/ml LPA-treated cells. Phosphorylated ABP-280 was
immunoprecipitated with anti-ABP-280 antibody, the immunoprecipitates
were washed and incubated without or with calpain (0.01 unit) in the
presence of 5 mM CaCl
, as described.
Phosphoproteins were analyzed by SDS-PAGE followed by autoradiography.
The migration of the ABP-280 subunit and its calpain subfragments are
indicated.
ABP-280 is phosphorylated in vitro at the same sites as it is in vivo after growth factor treatment of cells. Phosphoamino acid analysis of ABP-280 phosphorylation using clarified extract from LPA-treated cells shows serine to be the major residue phosphorylated (Fig. 4B). Extracts isolated from cells treated with FCS or EGF also phosphorylate serine residues in ABP-280 (data not shown). These data demonstrate that serine/threonine-specific protein kinases present in cell extracts are activated by growth factors and are responsible for the phosphorylation of ABP-280 in vitro. ABP-280 was phosphorylated using LPA-treated cell extracts and immunoprecipitated with anti-ABP-280 antibody. The immune complex was washed extensively, and bound ABP-280 was cleaved using calpain. The carboxyl-terminal tail of ABP-280 is phosphorylated in vitro as is ABP-280 phosphorylated in vivo (Fig. 4C). A two-dimensional phosphopeptide mapping analysis of ABP-280 phosphorylated by extracts from LPA-treated cells in vitro also showed two major phosphopeptides whose positions were identical to those generated from ABP-280 phosphorylated in vivo in response to LPA (data not shown). These results strongly suggest that the ABP kinase activity measured in vitro is also responsible for the phosphorylation of ABP-280 in intact cells.
Figure 6:
Fractionation of ABP kinase activities by
MonoQ FPLC. Cytosolic extracts from untreated () or 2 µg/ml
LPA treated 3Y1 cells for 5 min (
) were fractionated by MonoQ
FPLC as described under ``Experimental Procedures.'' Eluted
fractions were assayed for ABP kinase activity (A) and S6
peptide kinase activity (B). C, aliquots of eluted
fractions (shown in A) were immunoblotted with
affinity-purified anti-RSK1, anti-RSK2, and anti-p70 S6 kinase
antibodies following SDS-PAGE and transfer to polyvinylidene
difluoride. The fraction number is indicated. D, cytosolic
extracts from unstimulated 3Y1 cells were fractionated by MonoQ FPLC.
Fractions were preincubated with (
) or without (
) ERK2 in
the presence of MgCl
and ATP for 20 min at 30 °C. They
were then incubated with ABP-280 and
[
-
P]ATP (0.25 µCi/tube) for 20 min at
30 °C. The reaction was stopped by the addition of 10 µl of
SDS-sample buffer, boiled for 2 min, and analyzed by SDS-PAGE followed
by autoradiography. The autoradiogram was digitized using the NIH Image
analysis program. Phosphorylation of ABP-280 by ERK-GST alone was
negligible in this experiment. The extent of ABP-280 phosphorylation
mediated by ABP kinase peak 1 (fraction 12) in the presence of
GST-beads was defined as 100%, and from this value, the relative
intensity of phosphorylated ABP-280 was
compared.
To definitely show that RSK kinase(s) phosphorylate ABP-280 in response to LPA, we determined if the activity of ABP kinases in the cell extracts could be stimulated by MAP kinase (ERK2). Cytosolic extracts from unstimulated 3Y1 cells were chromatographed by MonoQ column and eluted fractions incubated in the presence or absence of active ERK2 before addition of the ABP-280 substrate. Fig. 6D shows that only ABP kinase activity in peak 1 was activated by preincubation with ERK2.
ABP-280 is phosphorylated in vivo in various cells (11, 12, 13, 14) . In normal and Rous sarcoma virus-transformed chick fibroblasts, 5-10% of total ABP-280 is phosphorylated at serine residues suggesting serine-specific protein kinases to be responsible for its phosphorylation in vivo, but the identities of the protein kinases were undefined(11) . We have now demonstrated that growth factors such as LPA, FCS, and EGF increase the phosphorylation of ABP-280 in quiescent rat 3Y1 fibroblastic cells through the activation of serine/threonine-specific protein kinases.
ABP-280 phosphorylating activity was fractionated by MonoQ column chromatography from LPA-stimulated 3Y1 cell extracts. Several lines of evidence suggest that one of the ABP protein kinases (ABP kinase peak 1) is p90 RSK2. First, RSK2 coelutes with the peak of ABP kinase activity in peak 1 in MonoQ column as judged by S6 peptide kinase activity and immunoblotting (Fig. 6). Second, incubation of the MonoQ fractions from extracts from quiescent cells eluting at the same salt concentration as ABP kinase activity in peak 1 with activated MAP kinase (ERK2) activates ABP kinase activity in peak 1 (Fig. 6D). This indicates the ABP kinase in peak 1 is activated by ERK2 and suggests it is downstream of ERK. Third, purified RSK2 phosphorylates purified ABP-280 in vitro at the same serine residues that are phosphorylated in vivo as shown by two-dimensional phosphopeptide mapping (Fig. 5B). Phosphorylation sites are located at the 90-kDa carboxyl-terminal tail domain of ABP-280 which contains four consensus phosphorylation sequences for RSK2 (i.e. (R)(R)RXXSX) (25) . Although RSK2 derives its name from its ability to phosphorylate ribosomal protein S6 in vitro, S6 is not the physiological substrate for RSK2 in vivo, and only a limited number of substrates for RSK2 have been identified(6, 26) . Our present study demonstrates that ABP-280 is a physiological substrate for RSK2 and suggests a possible linkage between MAPK/RSK signaling pathway and actin cytoskeleton.
At present, the identities of the two other ABP kinase activities (i.e. ABP kinase activity 2 and 3) revealed by MonoQ fractionation are unknown. Although ABP kinase peak 3 coelutes with RSK1 as shown by S6 peptide kinase activity assay and immunoblotting (Fig. 6), we cannot conclude that RSK1 is the only kinase in ABP kinase peak 3. First, ABP kinase peak 3 from quiescent cells is not activated by preincubation with ERK2 in vitro (Fig. 6D). Second, recombinant RSK1 purified from Cos cells transfected with epitope-tagged RSK1 gene (27) failed to phosphorylate ABP-280 in vitro although LPA did stimulate the S6 peptide kinase activity of RSK1 in the Cos cells, as determined by immune complex kinase assay, but did not stimulate ABP-280 phosphorylation (data not shown).
MAP kinase (HOG-1) is also known to activate MAPK-activated protein kinase-2 (MAPKAP kinase-2) which phosphorylates heat shock protein 25 (28, 29) . However, MAPKAP-kinase-2 is unlikely to correspond to ABP kinase peaks 2 or 3. First, growth factors do not stimulate the HOG-1/MAPKAP kinase-2 pathway. Second, MAPKAP kinase-2 activity is stimulated in vitro by preincubation with ERK while ABP kinase peaks 2 and 3 are not. Lastly, human ABP-280 does not contain a consensus sequence for phosphorylation by MAPKAP kinase-2 (XX-Hyd-XRXXS, where Hyd is a bulky hydrophobic residue(30) ).
Although LPA is known to activate
protein kinase C (31) and protein kinase C phosphorylates
ABP-280 in vitro(15) , protein kinase C is unlikely to
correspond to one of the identified ABP kinase fractions. First, ABP
kinase activity isolated from LPA-stimulated cell extracts is
insensitive to specific inhibitors of protein kinase C (Table 1).
Second, phosphorylation of ABP-280 elicited by LPA occurs in the 90-kDa
carboxyl-terminal tail domain (Fig. 2B), while protein
kinase C phosphorylates ABP-280 mainly in the 190-kDa amino-terminal
domain in vitro (data not shown). It is also not likely that
LPA-stimulated ABP kinases correspond to cAMP kinase, although a number
of studies have suggested that cAMP kinase phosphorylates ABP-280 in vitro and in vivo(13, 14, 32, 33) and treatment
with dBcAMP increases the phosphorylation of ABP-280 in intact 3Y1
cells (Fig. 1A). LPA has been shown to inhibit
adenylate cyclase via pertussis-toxin-sensitive G and
reduce cellular cAMP levels in Rat 1 cells and human foreskin
fibroblasts(31) , and ABP kinase activity in LPA-stimulated
cell extracts is insensitive to a specific inhibitor of cAMP kinase (Table 1).
Since the phosphorylation sites in ABP-280 phosphorylated by ABP kinase peaks 2 and 3 are similar or identical to those by RSK2, it is likely that ABP kinase(s) in these peaks are unidentified serine/threonine-specific protein kinases which locate downstream of MAP kinase like protein kinases. Further studies are necessary to determine the identities of ABP kinase activity 2 and 3.
The specific physiological function of the phosphorylation of ABP-280 by RSK2 is not known. Studies suggest that the MAPK/RSK pathway is involved in the control of the cellular actin cytoskeleton. ERK2 and p90 S6 kinase activate rapidly during thrombin-induced platelet activation and aggregation(34) . Overexpression of dominant negative MEK in NIH 3T3 cells suppresses p90 S6 kinase activity and alters actin cytoskeleton and cell morphology(35) . We have, however, failed to detect any changes of the affinity of ABP-280 to actin filaments in vitro (data not shown). This indicates that regulation of the ABP-280 function by phosphorylation occurs at a different level. One possibility is that phosphorylation of ABP-280 could regulate the interaction of ABP-280 with other molecules such as small GTP binding proteins (17, 18) and membrane receptors(36, 37) . Further study is necessary to define the role of phosphorylation of ABP-280 in intact cells.