(Received for publication, January 21, 1997)
From the Laboratory of Biochemistry and the § Biophysics
Unit, Site- and phosphorylation state-specific
antibodies are useful to analyze spatiotemporal distribution of
site-specific phosphorylation of target proteins in vivo.
Using several polyclonal and monoclonal antibodies that can
specifically recognize four phosphorylated sites on glial fibrillary
acidic protein (GFAP), we have previously reported that Thr-7, Ser-13,
and Ser-34 on this intermediate filament protein are phosphorylated at
the cleavage furrow during cytokinesis. This observation suggests that
there exists a protein kinase named cleavage furrow kinase specifically
activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa,
K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895-2902; Sekimata, M., Tsujimura, K.,
Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635-641). Here we report that GFAP is
phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by
Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho
in its GTP-bound active form. The kinase activity of Rho-kinase toward
GFAP is dramatically stimulated by guanosine
5 Intermediate filaments (IFs),1
major components of the cytoskeleton and the nuclear envelope in most
eukaryotic cells, undergo dramatic reorganization of their structure
during cell signaling and cell cycle (for review, see Refs. 1-3). This
IF reorganization is thought to be regulated by site-specific
phosphorylation of IF proteins at serine and threonine residues, and
several protein kinases have been shown to act as IF kinases in
vivo (for review, see Ref. 4). Site- and phosphorylation
state-specific antibodies that recognize a phosphorylated
serine/threonine residue and its flanking sequence can visualize
site-specific IF phosphorylation and thereby IF kinase activities
in situ by immunocytochemistry (Ref. 5; for review, see Ref.
6). Recently, we reported two distinct types of mitotic phosphorylation
of glial fibrillary acidic protein (GFAP), an IF protein expressed in
the cytoplasm of astroglia, using antibodies that react with four
distinct phosphorylated sites on GFAP (7, 8). One type is the
phosphorylation of Ser-8 on GFAP, which appeared at G2-M
phase transition in the entire cytoplasm. The other type is the
phosphorylation of Thr-7, Ser-13, and Ser-34, which appeared at
metaphase-anaphase transition at the cleavage furrow. This GFAP
phosphorylation specifically localized at the cleavage furrow was
observed not only in astroglial cells but also in other cultured cells
transfected with GFAP cDNA (8). These findings suggested the
existence of a protein kinase specifically activated at the cleavage
furrow and its important role in cytokinesis. We tentatively termed
this kinase cleavage furrow (CF) kinase (8). However, the molecular
identity, regulation, and function of CF kinase remained to be
examined.
The small GTP-binding protein Rho is implicated in the control of
cytoskeletal structures, cell adhesions, and cell morphology (for
review, see Ref. 9). Upon stimulation with certain signals, the
GDP-bound inactive form of Rho may be converted to the GTP-bound active
form, which binds to specific targets and thereby exerts its biological
functions. We have identified three putative targets for Rho, p128
protein kinase N (10, 11), p138 myosin-binding subunit (MBS) of myosin
phosphatase (12), and p164 Rho-kinase (13), which is also named ROK
(14). Rho-kinase phosphorylates MBS and consequently inactivates myosin
phosphatase (12). Rho-kinase also phosphorylates myosin light chain and
thereby activates myosin ATPase (15). Other putative targets for Rho
include rhophilin (11), p160 Rho-associated coiled-coil containing
protein kinase (16), and citron (17).
Recently, Rho was shown to play a critical role in cytokinesis by
inducing and maintaining the contractile ring, an actin-based cytoskeletal structure (18, 19). In addition, Rho was reported to be
translocated from the cytosol to the cleavage furrow during cytokinesis
(20). These results raise the possibility that Rho may also be
implicated in the regulation of CF kinase and thereby in the efficient
separation of IFs to daughter cells. As a first step toward defining
this possibility, we have examined whether protein kinase N and/or
Rho-kinase can phosphorylate GFAP at the same sites as CF kinase.
Protein kinase N was found to phosphorylate GFAP mainly at Ser-8, a
site that is not phosphorylated by CF kinase.2
In this report, we show that GFAP can serve as an excellent substrate
for Rho-kinase and that the GFAP phosphorylation by Rho-kinase prevents
its filament formation in vitro. Furthermore, we present
evidence that Rho-kinase phosphorylates GFAP at Thr-7, Ser-13, and
Ser-34 in vitro, the same sites that are phosphorylated by
CF kinase in vivo.
Recombinant human GFAP was prepared from
Escherichia coli as described previously (8). Mouse
monoclonal antibodies YC10, KT13, KT34, and MD389 were prepared as
described previously (8, 21). GST-RhoA was purified and loaded with
guanine nucleotides (22). Rho-kinase was purified from bovine brain
(13). Constitutively active GST-Rho-kinase, a GST fusion protein of the
catalytic fragment of Rho-kinase, was purified from Sf9 cells as
described previously (15). The catalytic subunit of
cAMP-dependent protein kinase (protein kinase A) was
prepared from bovine heart by the method of Beavo et al.
(23). Cdc2 kinase was prepared from FM3A cells by the method of
Kusubata et al. (24). Protein concentrations were measured
according to Bradford (25) using bovine serum albumin as a
standard.
The phosphorylation reaction for
Rho-kinase was performed for 30 min at 25 °C in 20 µl of 25 mM Tris-Cl (pH 7.5), 0.2% CHAPS, 4 mM
MgCl2, 3.6 mM EDTA, 100 µM
[ GFAP (130 µg) was
incubated with GST-Rho-kinase (8.5 µg) and [ All the procedures for immunoblotting have
been described elsewhere in detail (8, 21). Immunofluorescence
microscopy was performed as described previously (8). Amino acid
sequences were analyzed with an ABI 476A gas-phase sequencer.
Two-dimensional phosphoamino acid analysis was performed as described
(27). Electron microscopy was carried out as described (28).
We recently developed four monoclonal antibodies, YC10 (21), KT13,
KT34, and MO389 (8) against four distinct phosphopeptides corresponding
to the partial amino acid sequences of porcine GFAP. YC10, KT13, and
KT34 react with GFAP phosphorylated at Ser-8, Ser-13, and Ser-34,
respectively. MO389 reacts with both the phosphorylated and
unphosphorylated GFAPs and stains filamentous structures in both
mitotic and interphase cells. MO389 immunostained an intricate mesh of
glial filaments in the entire cytoplasm of both metaphase and anaphase
cells, but YC10 stained filamentous structures throughout the cytoplasm
of metaphase but not anaphase cells (Fig.
1A). In contrast, the immunoreactivities of
KT13 and KT34 were observed specifically between the daughter nuclei
and at the cleavage furrow of anaphase cells (Fig. 1A).
Immunocytochemical studies with KT13 (Fig. 1B) and KT34
(data not shown) using confocal laser scanning microscopy revealed that
GFAP phosphorylated at Ser-13 and Ser-34 is associated with the
cleavage furrow to form a ring-like structure but not a disc-like
structure, such as the telophase disc reported by Andreassen et
al. (29).
To search for the putative CF kinase responsible for the cleavage
furrow-specific phosphorylation described above, we examined whether
Rho-kinase purified from bovine brain can phosphorylate GFAP in
vitro. The results indicated clearly that Rho-kinase
phosphorylated GFAP in a GST-RhoA-dependent manner (Fig.
2A). GDP-bound GST-RhoA enhanced the
phosphorylation of GFAP by Rho-kinase 13-fold, and GTP
We then examined the phosphorylation sites on GFAP by Rho-kinase using
the anti-phosphoGFAP antibodies described above. After the
phosphorylation reaction, samples were resolved by SDS-PAGE and
immunoblotted with MO389, YC10, KT13, or KT34. As shown in Fig.
2B, Rho-kinase phosphorylated GFAP at Ser-13 and Ser-34 but not at Ser-8 in a GTP We also used the constitutively active GST-Rho-kinase, a fusion protein
between GST and the catalytic fragment of Rho-kinase produced in Sf9
cells by recombinant baculovirus infection. Analyses with the
anti-phosphoGFAP antibodies revealed that GST-Rho-kinase also
phosphorylated GFAP at Ser-13 and Ser-34 but not at Ser-8 (Fig.
2C). These results suggest that catalytic characteristics of
GST-Rho-kinase are similar to those of native Rho-kinase activated by
GTP To confirm phosphorylation sites on GFAP by Rho-kinase, GFAP (130 µg)
was phosphorylated by GST-Rho-kinase in the presence of
[
We then examined the effect of phosphorylation of GFAP by Rho-kinase on
the filament forming ability of GFAP. Soluble GFAP was preincubated
with or without GST-Rho-kinase for 30 min, and the samples were
incubated under conditions of polymerization (25 mM
imidazole-HCl, pH 6.75, and 100 mM NaCl at 37 °C) (28) for a further hour. Then the NaCl- and pH-dependent
filament formation of GFAP in these samples was analyzed by
centrifugation (Fig. 5A) and electron
microscopy (Fig. 5B). As shown in Fig. 5, the phosphorylation of GFAP by GST-Rho-kinase resulted in a nearly complete
inhibition of its filament formation. These results increase the
possibility that GFAP phosphorylation at Thr-7, Ser-13, and Ser-34
during cytokinesis may induce the fragmentation of glial filaments at
the cleavage furrow.
In the present study, we obtained evidence that GFAP can serve as an
excellent substrate for Rho-kinase in a GTP·Rho-dependent manner. So far, MBS (12) and myosin (15) were the only preferred substrates for Rho-kinase. The phosphorylated GFAP lost its ability to
form filaments in vitro. The in vitro
phosphorylation sites on GFAP by Rho-kinase were Thr-7, Ser-13, and
Ser-34, which are the same sites that CF kinase phosphorylates at the
cleavage furrow during cytokinesis. We are considering that Rho-kinase
may be CF kinase itself, and if so it may play an important role in the cleavage furrow-specific reorganization of IFs during
cytokinesis.
Because Rho-kinase was recently reported to act downstream of Rho in
the regulation of myosin phosphorylation (12, 15) and the formation of
stress fibers and focal adhesion complexes (32, 33), Rho-kinase may
also mediate the regulation of cytokinesis by Rho (18, 19). Whether
Rho-kinase is activated during cytokinesis is the subject of ongoing
studies. Because Rho-kinase belongs to a family of related
serine/threonine kinases including myotonic dystrophy kinase, these
kinases may phosphorylate the similar sites on GFAP. Further
investigations are necessary to elucidate the relationship between CF
kinase and Rho-kinase or its family members.
We are grateful to Dr. H. Goto (our
laboratory) for kind help with the reverse-phase HPLC and Dr. K. Tsujimura (Aichi Cancer Center Research Institute) for helpful
discussions and comments. M. Ohara provided critical comments on the
manuscript.
Division of Signal Transduction,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the
phosphorylation of GFAP by Rho-kinase results in a nearly complete
inhibition of its filament formation in vitro. The
possibility that Rho-kinase is a candidate for cleavage furrow kinase
is discussed.
Materials
-32P]ATP (5 µCi), 0.1 µM calyculin A,
130 µg/ml GFAP, and 0.5 µg/ml purified Rho-kinase in the presence
of GST, GDP·GST-RhoA, or GTP
S·GST-RhoA (each 1 µM). The phosphorylation reaction for GST-Rho-kinase, protein kinase A, or Cdc2 kinase was performed for 30 min at 25 °C in 100 µl of the reaction mixture (25 mM Tris-Cl (pH
7.5), 0.4 mM MgCl2, 100 µM ATP,
0.1 µM calyculin A, and 130 µg/ml GFAP) in the presence
of 8.5 µg/ml GST-Rho-kinase, 5 µg/ml protein kinase A, or 0.5 µg/ml Cdc2 kinase. The reaction was stopped by the addition of
Laemmli's sample buffer and boiling.
-32P]ATP
(50 µCi) for 120 min at 25 °C in 1 ml of the reaction mixture described above. The phosphorylated GFAP was precipitated with 10%
trichloroacetic acid and digested with 5 µg of lysyl endopeptidase (Wako) in 100 µl of 50 mM Tris-Cl (pH 8.0) and 4 M urea at 30 °C for 2 h. The phosphorylated head
domain of GFAP was isolated by reverse-phase HPLC and treated with 1/50
(w/w) L-1-tosylamide-2-phenylethyl chloromethyl
ketone-treated trypsin (Sigma), as described by Tsujimura et
al. (26). The obtained peptides were separated by HPLC on a Zorbax
C8 (0.46 × 25 cm) column equilibrated with 5% (v/v)
2-propanol/acetonitrile (7:3) containing 0.1% trifluoroacetic acid.
Elution was carried out with a 60-min linear gradient of 5-50%
2-propanol/acetonitrile followed by a further 10-min linear gradient of
50-80% 2-propanol/acetonitrile at a flow rate of 0.8 ml/min.
Fig. 1.
Immunoreactivity of the anti-GFAP antibody
(MO389) and the anti-phosphoGFAP antibodies (YC10, KT13, and KT34) in
U251 human astrocytoma cells. A, fluorescent
photomicrographs of mitotic U251 cells stained with the antibody MO389,
YC10, KT13, or KT34 (green). Chromosomes were stained with
propidium iodide (red). The bar represents 10 µm. B, confocal images on nine serial focal planes of an
anaphase U251 cell stained with KT13 and propidium iodide were obtained
by confocal laser scanning microscopy (Olympus GB200).
[View Larger Version of this Image (51K GIF file)]
S-bound
GST-RhoA enhanced it 291-fold (Fig. 2A).
Fig. 2.
Phosphorylation of GFAP by Rho-kinase.
A, GFAP was phosphorylated by Rho-kinase in the presence of
GST (lanes 1 and 4), GDP·GST-RhoA (lanes
2 and 5), or GTPS·GST-RhoA (lanes 3 and 6) as described under "Experimental Procedures." Samples
(each 10 µl) were electrophoresed in a 10% acrylamide gel. The gel
was stained with Coomassie Blue (C.B.B., lanes
1-3) and subjected to autoradiography (lanes 4-6).
B, GFAP phosphorylated as in A in the absence of
32P-labeled ATP was resolved by SDS-PAGE and immunoblotted
with MO389, YC10, KT13, or KT34 using the ECL Western blotting
detection system (Amersham Corp.). The chemiluminescence was detected
by exposure for 1 min. C, GFAP was incubated with
GST-Rho-kinase, buffer (control), protein kinase A
(A-kinase), or Cdc2 kinase as described under
"Experimental Procedures." Samples (each 2 µl) were analyzed by
immunoblotting with YC10, KT13, or KT34. The chemiluminescence was
detected by exposure for 5 s.
[View Larger Version of this Image (28K GIF file)]
S·GST-RhoA-dependent
manner.
S·GST-RhoA. In contrast, the catalytic subunit of
cAMP-dependent protein kinase phosphorylated all three
serine residues, and Cdc2 kinase weakly phosphorylated only Ser-8 (Fig.
2C).
-32P]ATP to approximately 2.7 mol of phosphate/mol of
GFAP (Fig. 3A). The radioactive GFAP was then
digested with lysyl endopeptidase to generate about a 6.5-kDa fragment
consisting mainly of the amino-terminal head domain. Tricine-SDS-PAGE
analysis (30) revealed that all radioactivity associated with GFAP was
retained in this 6.5-kDa head domain (Fig. 3B). This
phosphorylated head domain was isolated by reverse-phase HPLC, digested
with trypsin, and then again subjected to reverse-phase HPLC. As shown
in Fig. 4A, three major radioactive peaks, R1
to R3, were obtained. Phosphoamino acid analysis showed the presence of
phosphothreonine in R1 and phosphoserine in both R2 and R3 (Fig.
4B). Amino acid sequence analysis revealed that R1 was the
peptide containing Thr-7, R2 was the peptide containing Ser-34, and R3
was the peptide containing Ser-13 (Fig. 4A). Ethanethiol
treatment of R2 and R3, a procedure that converts specifically
phosphoserine into S-ethylcysteine (31), suggested that
phosphates were located on Ser-34 and Ser-13, respectively (data not
shown). Therefore, GFAP was shown to be phosphorylated at Thr-7,
Ser-13, and Ser-34 by GST-Rho-kinase. By using a rabbit polyclonal
antibody recognizing phosphorylated Thr-7, we have previously reported
that Thr-7 is also phosphorylated at the cleavage furrow (7).
Fig. 3.
Phosphorylation of the head domain of GFAP by
GST-Rho-kinase. A, GFAP (130 µg) was phosphorylated by
GST-Rho-kinase for 120 min as described under "Experimental
Procedures." Aliquots (each 5 µl) of the reaction mixture were
removed at the indicated times and added into Laemmli's sample buffer.
After SDS-PAGE, the radioactivity incorporated into GFAP was quantified
by using an image analyzer (Fujix BAS2000). B, after the
phosphorylation reaction in A, GFAP was precipitated with
trichloroacetic acid and subjected to Tricine-SDS-PAGE (30) before
(lane 1) or after (lane 2) the digestion with
lysyl endopeptidase. After electrophoresis, the gel (16.5% acrylamide)
was autoradiographed. The positions of molecular mass standards (in
kilodaltons) are indicated on the left.
[View Larger Version of this Image (17K GIF file)]
Fig. 4.
Phosphorylation of Thr-7, Ser-13, and Ser-34
on GFAP by GST-Rho-kinase. A, the phosphorylated 6.5-kDa
fragment derived from GFAP was digested with trypsin and subjected to
reverse-phase HPLC as described under "Experimental Procedures."
Three radioactive peaks (R1, R2, and
R3) were analyzed with a gas-phase sequencer. The determined
amino acid sequences of R1 (residues 5-11), R2 (residues 37-41), and
R3 (residues 13-29) are indicated on the right. Note that
we describe Ser-38 of human GFAP as Ser-34 because Ser-38 of human GFAP
corresponds to Ser-34 of porcine GFAP. B, aliquots of R1,
R2, and R3 in A were subjected to two-dimensional phosphoamino acid analysis.
[View Larger Version of this Image (37K GIF file)]
Fig. 5.
Inhibition of filament formation from soluble
GFAP by GST-Rho-kinase. A, GFAP (170 µg/ml) was
preincubated with (Rho-K) or without (control)
GST-Rho-kinase (8.5 µg/ml) for 30 min at 25 °C in 50 µl of 25 mM Tris-Cl (pH 7.5), 0.4 mM MgCl2,
100 µM ATP, and 0.1 µM calyculin A. Then,
the mixtures were incubated with 25 mM imidazole-HCl (pH
6.75) and 100 mM NaCl for further 1 h at 37 °C and
subjected to centrifugation at 15,000 × g for 30 min
at 2 °C. The supernatants (s) and precipitates
(p) were electrophoresed in a 10% acrylamide gel. The gel
was stained with Coomassie Blue. B, after the polymerization
reaction in A, an aliquot of each sample was negatively
stained and examined by electron microscopy. The bar
represents 200 nm.
[View Larger Version of this Image (51K GIF file)]
*
This work was supported in part by grants-in-aid for
scientific research and cancer research from the Ministry of Education, Science, Sports, and Culture of Japan and by special coordination funds
from the Science and Technology Agency of the Government of Japan.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.
¶
To whom correspondence should be addressed: Laboratory of
Biochemistry, Aichi Cancer Center Research Inst., Chikusa-ku, Nagoya, Aichi 464, Japan. Tel.: 81-52-762-6111 (Ext. 8824); Fax:
81-52-763-5233.
1
The abbreviations used are: IF, intermediate
filament; GFAP, glial fibrillary acidic protein; CF, cleavage furrow;
MBS, myosin-binding subunit; GST, glutathione S-transferase;
GTPS, guanosine 5
-(3-O-thio)-triphosphate; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE,
polyacrylamide gel electrophoresis; HPLC, high performance liquid
chromatography; Rho-kinase, Rho-associated kinase.
2
K. Matsuzawa, H. Kosako, N. Inagaki, M. Amano,
Y. Nishi, H. Mukai, Y. Ono, Y. Matsuura, K. Kaibuchi, I. Azuma, and M. Inagaki, manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.