From the Glaxo-IMCB Group, Institute of
Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore
and § Institute of Neurology, University College London,
London WC1N 1PJ, United Kingdom
Received for publication, March 22, 2001
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ABSTRACT |
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The myotonic dystrophy kinase-related kinases
RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding
kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we
uncovered two major substrates, p130 and p85, for MRCK The Rho subfamily of GTPases are biological regulators of actin
cytoskeleton. In adherent cells, RhoA induces stress fiber formation,
Rac-1 generates lamellipodia, and Cdc42 produces filopodia and actin
microspikes (1). A variety of effectors of these cytoskeletal switches
has been isolated and characterized (see Refs. 2 and 3 for reviews),
some of which are directly involved in regulation of actin dynamics. We
and others have reported serine/threonine kinases related to the
myotonic dystrophy kinase that play effector roles for the perspective
GTPase in cytoskeletal reorganization (4-7). ROKs or Rho kinases are
downstream effectors of RhoA in organizing stress fibers (4-6),
whereas MRCKs1 play an
important role in Cdc42 functions in regulating actinomyosin dynamics
in cultured cells (7). Precisely how these occur is not clear, although
a number of proteins are known to be effective substrates for these
kinases. These include the non-muscle myosin light chain 2 (MLC2),
whose phosphorylation state is crucial for actinomyosin contractility
and polymerization (7, 8), the myosin binding subunit p130 (9-11),
Ezrin, Radixin, and Moesin (ERM) family proteins (12), adducin (13),
and intermediate filament proteins (14-17), which are directly or
indirectly linked to the actin cytoskeleton.
In particular, the myosin binding subunit MBS130 appears to play a
unique role in the regulation of the activity of the associated PP1
catalytic subunit. The specific binding of MBS130 to RhoA may link this
regulatory subunit to Rho-dependent events (9). Indeed
chicken gizzard MBS130 is found to be effectively and specifically phosphorylated by ROK at threonine 695 and serine 854 (10, 11). Phosphorylation at threonine 695 resulted in inhibition of the intrinsic phosphatase activity. Other proteins that can interact with
and phosphorylate MBS130 include the cGMP-dependent protein kinase 1 As a first step toward identification of new substrates for
kinases such as MRCKs, we derived a filter assay to screen for potential candidates. This assay allows renatured proteins on the
filter to be phosphorylated by specific recombinant kinases. Using this
assay for MRCK Construction of Expression Vectors--
Full-length p85
cDNA was obtained by first performing a PCR reaction of the
presumed initiation codon from the first exon of the human genomic DNA
using two adaptor primers, 5'-CAGGATCCATGTCCGGAGAGGATGGC-3' and
5'-GCAGGCCTGGTGCAGG-3'. This was then joined to a
StuI/NotI fragment from EST clone AI1825921 that
contains the rest of the 3' end of p85. p85NT was derived
from a BamHI/PstI DNA fragment of p85 (encoding
residues 1-389), and p85CT was obtained by subcloning a
PCR fragment from a 5' primer 5'-CAGGATCCTGCCGCCTGCTGGCCG-3' (encompassing residues 346-782) into pXJ-40 FLAG-tagged vector as
previously described (4). A shorter C-terminal construct encompassing
the conserved Cell Culture, Transfection, and Cell Staining--
COS-7 cells
were cultured in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum, and HeLa cells were cultivated in modified Eagle's
medium with 10% fetal bovine serum (Hyclone). Subconfluent HeLa cells
plated on coverslips for 48 h were transfected with various HA- or
FLAG-tagged DNA constructs (1 µg/ml) with LipofectAMINE (Life
Technologies, Inc.) according to recommended protocol. 16 h after
transfection, cells were fixed with 4% paraformaldehyde and stained
with the combination of various primary antibodies: anti-HA
(12CA5; Roche Molecular Biochemicals) or anti-FLAG (M2; Sigma).
Stained cells were analyzed with an MRC 600 confocal imager adapted to
a ZEISS Axioplan microscope. For localization studies, transfected HeLa
cells were serum-starved for 4-6 h before treatment with
lysophosphatidic acid (300 ng/ml; Sigma) or phorbol myristic acetate
(100 ng/ml; Sigma). COS-7 cells grown in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum were similarly transfected with
various constructs. 24 h after transfection, cell extracts were
obtained with lysis buffer ((25 mM HEPES, pH 7.7, 0.15 M NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 1 mM sodium vanadate, 20 mM Filter Substrate Binding--
Tissue extracts from rat brain and
other tissues or cultured cells were resolved on either one- or
two-dimensional gels, electro-transferred onto PVDF filters, and
renatured in buffer containing phosphate-buffer saline with 0.5 mM MgCl2, 1 mM dithiothreitol, 1%
bovine serum albumin, and 0.1% Triton X-100 for 2 h. Filters were
washed with phosphate-buffered saline, 0.1% Triton, incubated with a
phosphorylation mixture containing 20 mM Tris-HCl, 75 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 5 µg/ml GST-MRCK Enzymatic Measurements--
Kinase assays were carried out in
kinase buffer containing 20 mM Tris-HCl pH 7.5, 75 mM NaCl, and 10 mM MgCl2 as
previously described (5). For labeling of GST-MLC2, purified fusion
protein (10 µg) on glutathione beads was subjected to phosphorylation using 20 µg/ml GST-MRCK- Immunoprecipitation and in Vitro Binding Assays--
COS-7 cells
co-expressing various FLAG-p85 and HA-PP1 RNA and Protein Analysis--
Northern blots containing mRNA
from various human tissues were obtained from
CLONTECH and were hybridized with a full-length 32P-labeled p85 probe as previously described (5). One- and
two-dimensional gel analyses were carried out according to standard
protocol, and separated proteins were transferred to PVDF membranes
(PerkinElmer Life Sciences) and probed with various antibodies,
including an antibody to a phosphorylated threonine 695 of chicken
gizzard M130 (which is identical to the flanking sequence around
threonine 560 of p85). Analysis of tryptic peptides and phosphopeptides were performed with a QSTAR Mass-spectrometer (PerkinElmer).
Identification of p130 and p85 as Major in Vitro Substrates for
MRCK
Two proteins of 130 and 85 kDa were prominently and specifically
phosphorylated by MRCK P85 Is a Novel Protein Related to Myosin Binding Subunit Protein
MBS130--
Both peptide sequencing and subsequent immunoblotting
analyses showed that the p130 corresponds to the previously reported MBS130 (data not shown). Peptide sequence analysis also indicated that
the p85 is a novel protein (Fig.
2A). A 2.8-kilobase human EST
clone AI825921 contains most of the coding sequence except for the
extreme N terminus. The complete match of this cDNA with two
overlapping genomic clones, S51329 and AC005782, from human chromosome
19 (Fig. 2C) suggested that the N terminus is confined
within the first exon. A full-length cDNA for p85 was thus
constructed from the PCR product of the first exon of human p85 genomic
DNA and subsequently joined to the truncated cDNA. The amino acid
sequence derived from this cDNA indicated that p85 is structurally
related to MBS130 (Fig. 2B). The N terminus of p85 consists
of a closely related structure with 6 ankyrin repeats and 48% identity
to MBS130, which has been reported to have 7-8 repeats (23, 24). A
putative PP1 binding consensus sequence, RTVRF (25), was also present
immediately before the ankyrin repeats. The C terminus contains a
conserved
The genomic organization of p85 showed that the mRNA is derived
from 22 exons (Fig. 2C). Intriguingly, a number of
integration hotspots (AAVS1) of adenovirus-associated virus (AAV) was
found in the first exon/intron regions of p85 genomic sequence (Fig. 2D; Refs. 27 and 28). The consequence of these integrations is not known but is expected to disrupt the expression of this gene.
Rearrangements and disruption of a nearby troponin gene were also
observed upon adenovirus-associated virus integration (29). Northern
blot analysis indicated that the 3-kilobase p85 mRNA was
ubiquitously expressed and is especially high in the heart (Fig.
2E).
P85 protein expressed in serum-starved HeLa cells mainly showed
cytoplasmic punctate distribution (Fig. 2F, a)
but was readily redistributed to cell peripheral upon treatment with
lysophosphatidic acid and phorbol myristic acetate (Fig. 2F,
b and c).
Identification of the Phosphorylation Site of p85 on Threonine 560 by MRCK
Further evidence was obtained by probing p85 protein expressed from
COS-7 cells that were co-transfected with either MRCK p85 Is Specifically Associated with PP1
The N terminus of p85 could also interact with MLC2 (Fig.
4B), and the C terminus is totally ineffective. Hence PP1
Next, to examine if the phosphorylation of threonine 560 by MRCK N and C Termini of MBS85 Show Independent Morphological Effects
Reflecting Their Biochemical Activities--
Because PP1 activities
are essential for regulating the phosphorylation states of myosin, it
is likely that the biochemical interaction of MBS85 with PP1 Phosphorylation Inhibitory Motif (PIM50) of MBS85 Binds PP1
To examine if such interaction is functional, we measured the catalytic
activities of the immuno-complex in the presence of in vitro
phosphorylated or nonphosphorylated GST-PIM50. Phosphorylated GST-PIM50, but not the nonphosphorylated form, was likewise effective in inhibiting the catalytic activity of p85·PP1
Next we tested the effects of co-expression of this PIM50 motif on the
p85NT-induced morphological changes. As shown in Fig.
7, A and B, PIM50 expression is sufficient in reversing the effect of
p85NT-induced actin stress fiber losses. The
phosphorylation-deficient mutant PIM50AA was totally
inefficient (Fig. 7B). These inhibitory effects became less
pronounced when p85NT was co-expressed with PP1 Two candidate proteins from rat brain cytosolic extract were
identified in this study on the basis of their in vitro
phosphorylation by MRCK A central conserved motif that contains the sole phosphorylation site
for MRCK Here we have also demonstrated that p85 is functionally similar to
MBS130. First it is specifically associated with PP1 Similar conclusions can be derived from the morphological assays that
clearly reflect the biochemical interactions. HeLa cells expressing the
N terminus of MBS85 alone exhibits the greatest loss in actin stress
fibers, an indicator for activation of PP1 in vivo.
Interestingly, the two conserved motifs (PIM and It is therefore important to demonstrate a direct interaction of the
conserved central motif (PIM50) with PP1-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an
-helical C terminus with leucine repeats, and a centrally located
conserved motif with the MRCK
-kinase phosphorylation site. Like
MBS130, p85 is specifically associated with protein phosphatase 1
(PP1
), and this requires the N terminus, including the ankyrin
repeats. This association is required for the regulation of both the
catalytic activities and the assembly of actin cytoskeleton. The N
terminus, in association with PP1
, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The
C-terminal effects consist of two independent events that involved both
the conserved phosphorylation inhibitory motif and the
-helical
leucine repeats. The former was able to interact with PP1
only in
the phosphorylated state and result in inactivation of PP1
activity.
This provides further evidence that phosphorylation of a myosin binding
subunit protein by specific kinases confers conformational changes in a
highly conserved region that plays an essential role in the regulation
of its catalytic subunit activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and an unidentified mitotic kinase, but in contrast, such
phosphorylation resulted in activation of protein phosphatase activity
(18, 19). MBS130 therefore appears to serve as a scaffold for multiple
protein interactions as well as phosphorylation regulation. Indeed, a
recent report has indicated that a number of proteins involve in the
Rho signaling pathways including Ezrin, Radixin and Moesin family
proteins, adducin, and MBS130 can be found to colocalize at cell
periphery upon stimulation. It is possible that MBS130 may provide a
bridge for various Rho-dependent components to function in
a coordinated manner (20). Since the myotonic dystrophy kinase-related
Cdc42-binding kinases (MRCKs) also consist of similar catalytic domains
with differential cellular localization and cellular functions (7, 21),
it is of interest to investigate if these kinases could share similar
and different sets of substrates for their cellular activities.
kinase, we identified two potential substrate
proteins, one of which was MBS130; the other was a novel related protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix leucine zipper
(p85
LZ; residues 591-782) was derived from
PCR product using 5' primer 5'-CGGATCCCGAAGGCCCCGCGTC-3'. In-frame
deletions by Nar1 restriction enzyme digest, which removed a
central conserved region (p85
Nar; deleted
residues 521-610), and by BssHII
(p85
AR1), which deleted an ankyrin repeat
(residues 51-71), were likewise subcloned. Mutagenesis was carried out
as previously described (4) using primer pairs
5'-CTGCATGCAGCGGCACACTGG-3' and 5'-CTGCATGCAGGGCAGAGGCGCC-3' to
generate p85
AR2 (deleted residues 232-264)
and primer pairs 5'-GACGTCGACGGGCCGCACAGGGTGTGACTCTTAC3-' and
5'-GACGTCGACTCTGGCGCATGAGACGG-3' to generate
p85S559A/T560A (p85AA). PP1
was derived from
EST clone AI156840, which contains the whole of the coding sequence.
The nucleotide sequences encoding the 50 amino acids of
phosphorylation inhibitory motif
(PIM; residues 526-575) of the wild-type p85 and mutant
p85AA were obtained by PCR using primer pairs
5'-CAGGATCCCGGGACCGACGGAG-3' and pXJ40 reverse primer. The
156-base pair BamHI/PstI-digested PCR fragments
were subcloned into pXJ40-GST vector for mammalian cell transfection
(22) and into pGEX-4T2 for the production of GST-PIM50 and
GST-PIM50AA fusion proteins. The N terminus containing the
kinase domain of MRCK
(residues 1-473) was obtained by subcloning a
1.4-kilobase BamHI/HindIII fragment from
full-length MRCK
into pGEX4T2 to generate a vector producing
MRCK
-CAT. GST-MLC2 was prepared as previously described (7).
-glycerol phosphate, 5% glycerol, 0.1% Triton X-100
and 1× inhibitor mix (Roche Molecular Biochemicals)), separated
on a SDS-polyacrylamide gel, transferred to nitrocellulose, and probed
with anti-HA or anti-FLAG antibodies for expression.
-CAT fusion
protein, and 10 µCi/ml [
-32P]ATP for 30 min, and
washed with phosphate-buffered saline, 0.1% Triton and 6 M
guanidinium chloride before autoradiography. Control experiments were
also performed using GST-
-p21-activated kinase (22) and
protein kinase A (Sigma).
-CAT and 1 µM
[
-33P]ATP for 1 h at 30 °C. After an extensive
wash with GST purification buffer, the phosphorylated protein was
eluted with 5 mM reduced glutathione. Phosphatase assays
were carried out at 30 °C using 33P-GST-MLC2 as
substrate. To show phophorylation-mediated PP1 inhibition, the
immunoprecipitated FLAG-p85·HA-PP1
complex was first
incubated in 20 µl of kinase buffer with 0.15 mM ATP
S
in the presence or absence of 0.5 µg of GST-MRCK
-CAT for 30 min at
30 °C. Phosphatase assays were initiated by the addition of 5 µM 33P-GST-MLC2 in 30 mM
Tris-HCl, PH 7.5, 0.1 M KCl, 2 mM
MgCl2, and 0. 1 mg/ml bovine serum albumin. The reactions
were stopped by adding an equal volume of SDS sample buffer at each
time point indicated and boiling for 5 min before gel loading. To show
PP1 inhibition by phosphorylated GST-PIM50, 10 µg of the fusion
protein was first incubated in kinase buffer (as above using ATP
S)
with and without GST-MRCK
-CAT for 30 min at 30 °C. These
phosphorylated and nonphosphorylated GST-PIM50 proteins were then
separately mixed with the immunoprecipitated FLAG-p85·HA-PP1
complex and preincubated for 1 min before the start of phosphatase
assays. Phosphatase activities were quantified using the Molecular
Dynamics PhosphorImager System.
constructs were lysed in
buffer containing 25 mM HEPES, pH 7.3, 0.15 M
NaCl, 0.5 mM MgCl2, 0.2 mM EDTA, 20 mM
-glycerol phosphate, 1 mM sodium
vanadate, 5% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton, 1 µg/ml
each aprotinin, leupeptin, and pepstatin A, and 1× protease inhibitor
mixture and incubated with anti-FLAG-conjugated agarose beads (Sigma)
for 1 h at 4 °C. After an extensive wash, the
immunoprecipitates were resolved by SDS-polyacrylamide gel
electrophoresis and subjected to immunoblot analysis using anti-FLAG or
anti-HA antibodies as mentioned. To detect any interaction of these
complex with the phosphorylated GST-PIM50, an in vitro assay
containing either the unphosphorylated or phophorylated GST-PIM50 was
added to COS-7 cell lysates co-expressing FLAG-p85AA and
HA-PP1
and followed by immunoprecipitation using
anti-FLAG-conjugated agarose beads. Immunoblot analyses were carried
out as before.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CAT--
To identify and characterize potential substrates for
ROK
and MRCK
, we made GST fusion proteins of the catalytic
domains of both ROK
(1) and MRCK
(1). The yield and
catalytic activities of MRCK
-CAT were consistently higher than
ROK
-CAT, and subsequent experiments were thus carried out with
GST-MRCK
-CAT. Here we observed that renatured proteins separated on
SDS-polyacrylamide electrophoresis gels and transferred onto PVDF
membrane filters were readily phosphorylated by MRCK
-CAT.
-CAT (Fig. 1)
but not by
-p21-activated kinase or protein kinase A (data
not shown). These two proteins are not abundant in tissues such as
brain and testis but could easily be enriched by passing through an
affinity dye Reactive Brown 10-Sepharose column (Sigma), and this
constitutes a simple one-step enrichment of these proteins for further
purification (Fig. 1A). Further separation of these two
proteins was achieved with two-dimensional gel electrophoresis (Fig.
1B), and the Coomassie Blue-stained spots corresponding to
the phosphorylated proteins were excised for peptide sequencing.
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Fig. 1.
Identification of p85 and p130 as major
phosphorylated polypeptides from rat brain. Soluble rat brain
extract was loaded onto Reactive Brown 10-Sepharose column (Sigma;
0.5-ml bed volume). After extensive washes, the bound fraction was
eluted with sample buffer and separated either on a one- (A)
or two-dimensional gel (B) for Coomassie Blue staining
(top panel) or transferred onto PVDF membrane filters for
phosphorylation reactions with GST-MRCK -CAT and
[
-32P]ATP. In A, each lane was
loaded with 100 µg each of soluble extract (1), flow-through (2),
wash (3), and eluents (4, 5). Arrows indicate
equivalent positions of p85 and p130 on filters. Mr,
molecular mass marker.
-helical structure with leucine zippers at the C-terminal
end (
LZ). This structure can form homodimers or heterodimers
(e.g. with M20, which also contains this structural motif
(26)). Of most striking similarity (87% identity) is a central motif,
which contains the sole phosphorylation site for MRCK
-CAT (refer to
Fig. 3).
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Fig. 2.
p85 amino acid sequence comparison, genomic
organization, and expression analysis. A, the amino
acid sequence was derived from the first exon of human genomic DNA and
a human EST clone AI825921. The conserved ankyrin repeats
(AR), PIM with threonine 560 indicated by an
asterisk, and -helical leucine zipper (
LZ) are in
bold. The putative consensus sequence for PP1 binding is
boxed. Peptide sequences matched with the derived amino acid
sequence were underlined. B, a diagrammatic
comparison of p85 and MBS130. C, the genomic organization of
human p85. The genomic sequences were derived from two overlapping
genomic sequences (S51329 and AC005782) from chromosome 19. The 22 exons corresponding to the cDNA sequence were boxed, and
larger intron sequences were indicated by double parallel
lines. Arrows indicate the adenovirus-associated virus
preinsertion sites (AAVS1). D, junctional sequences of
hotspots of adenovirus-associated virus integration locus (AAVS1) on
exon 1 of chromosome 19q13.3-qter. Bold letters highlight
some of the junctional recipient sites reported. E, Northern
blot analysis of p85. mRNA blot was purchased from
CLONTECH and probed with
[32P]-labeled full-length p85. Ht, heart;
Br, brain; Pa, pancreas; Lu, lung;
Li, liver; Sm, smooth muscle; Ki,
kidney; Pl, placenta; kb, kilobases.
F, cellular localization of p85. Full-length p85 in
pXJ40-FLAG vector was co-transfected with pXJ40-HA-tagged PP1
in
HeLa cells. Cells were serum-starved for 4-6 h (a) and
treated with lysophosphatidic acid (b) or phorbol myristic
acetate (c). Cells were stained with anti-FLAG antibody for
detecting the expressed p85. Arrows indicated the
tranlocated p85 in peripheral membranes.
-CAT--
To confirm the nature of phosphorylation of p85,
we expressed the FLAG-tagged wild-type and deletion mutants of p85 in
COS-7 cells (Fig. 3A). The
immunoprecipitated proteins were phosphorylated with MRCK
-CAT to map
the phosphorylation site(s). It is clear that the major site(s) is
within the central conserved region, as mutants deleted of this region
were not phosphorylated (Fig. 3B, lanes 2 and
4). To confirm this, we expressed GST fusion protein containing the wild-type conserved PIM motif (PIM50; also refer to Fig.
4) and a mutant S559A/T560A
(PIM50AA) and showed that the mutant was not phosphorylated
(Fig. 3B, lane 8), unlike the wild-type
control (lane 7). This in vitro phosphorylation
site of p85 on threonine 560 by MRCK
-CAT was further confirmed by
mass spectrometry on the tryptic phosphopeptides derived from p85
phosphorylation.
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Fig. 3.
Mapping of phosphorylation sites of p85 by
MRCK -CAT. A, a schematic
diagram of the various p85 constructs. AR, ankyrin repeats;
LZ; PIM; mutations in PIM50AA (S559A/T560A)
were indicated by ××. B, immunoprecipitated wild-type
FLAG-tagged p85 (lane 1) and the various truncated mutants
(p85
Nar1 (lane 2),
p85CT (lane 3),
p85CT
Nar1 (lane 4),
p85NT (lane 5), and
p85
LZ (lane 6)) and 5 µg of
GST-PIM50 (lane 7) and mutant PIM50AA
(lane 8) were phosphorylated with MRCK
-CAT and
[32P]ATP. Proteins were resolved on a 10% polyacrylamide
gel electrophoresis-SDS gel for Coomassie Blue staining
(top) and autoradiography (bottom).
Arrowheads indicate the positions of IgG light and heavy
chains; the position for the kinase autophosphorylation was marked by
arrows. C, FLAG-tagged p85 was expressed alone or
together with either HA-tagged MRCK
-CAT or ROK
-CAT, and the
proteins transferred to PVDF filters were detected separately with
anti-FLAG, anti-p85-pT, and anti-HA to detect expression of the
kinases. FLAG-tagged immunoprecipitated p85 before and after
phosphorylation in vitro by GST-MRCK
-CAT was also shown
as controls for comparison (right panel). WB,
Western blot.
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Fig. 4.
p85 is associated with
PP1 /MLC-2 and is regulated by
phosphorylation. A, the N terminus of p85 is required
for association with PP1
. FLAG-tagged wild-type p85
(lane1), and the various mutant proteins (p85NT
(lane 2), p85
Nar1 (lane
3), p85AA (lane 4),
p85
AR2 (lane 5),
p85CT (lane 6), and
p85
LZ (lane 7)) were co-expressed
with HA-tagged PP1
in COS-7 cells. Proteins immunoprecipitated
(IP) with mouse anti-FLAG beads were separated on 10%
SDS-polyacrylamide electrophoresis gels, transferred to PVDF membranes,
and detected with a rabbit anti-FLAG antibody for p85 and a rabbit
anti-HA antibody for associated PP1
. Overexpressed PP1
present in
the cell lysate was also shown for comparison. WB, Western
blot. B, the N terminus of p85 binds MLC-2. FLAG-tagged p85
NT (lane 1) or p85LZ (lane
2) was co-expressed with HA-tagged MLC-2. Immunoprecipitated
proteins with anti-FLAG antibody transferred onto PVDF membrane were
detected with anti-FLAG or anti-HA as described in A. C, phosphorylation of p85 by GST-MRCK
-CAT inhibits
associated PP1
activities. Immunoprecipitated wild-type p85
(p85WT) or a phosphorylation-deficient mutant,
p85S559A/T560A (p85AA), coexpressed with PP1
were phosphorylated with GST-MRCK
-CAT in the presence of 0.1 mM ATP
S. The nonphosphorylated and phosphorylated
proteins were used to initiate the dephosphorylation activities of the
associate PP1
toward 33P-MLC2 at different time
intervals.
-CAT or
ROK
-CAT with an antibody that specifically recognizes phosphorylated threonine 560 of p85. Clearly, threonine 560 of p85 can be
phosphorylated by both MRCK
-CAT and ROK
-CAT in vivo
(Fig. 3C).
and Its Substrate MLC2,
and the Phosphorylation by MRCK
Regulates the Phosphatase
Activity--
To see if p85 can associate with PP1, we co-transfected
FLAG-tagged p85 and HA-tagged PP1
, -
, and -
isoforms. Only
PP1
isoform was immunoprecipitated with p85, indicating a specific interaction between p85 and PP1
. This was also evident from the co-
immunoprecipitation of the endogenous PP1
with the overexpressed p85
(data not shown). Constructs with an intact N terminus, including the
ankyrin repeats, could effectively interact with PP1
(Fig. 4A, lanes 1-4), whereas a deletion mutant
of a single ankyrin repeat (lane 5) can dramatically reduce
such interaction. Deletion mutants devoid of N terminus are totally
ineffective (Fig. 4A, lanes 6 and
7).
can form a tight complex with p85 and substrate MLC2 through its N terminus.
-CAT
can regulate PP1 activity, we measured the time course of
dephosphorylation toward 33P-MLC2. As shown in Fig.
4C, nonphosphorylated wild-type p85 (p85WT) or
the phosphorylation-defective p85 mutant (p85AA) were
equally active in MLC2 dephosphorylation. Wild-type p85 but not the
mutant p85AA, when phosphorylated in vitro with
MRCK
-CAT, showed significant reduction in the rate of MLC2
dephosphorylation. These results confirm a similar observation with
MBS130 where phosphorylation of a conserved threonine 695 within a
highly conserved motif was essential for the inhibition of phosphatase
catalytic activity (11). Based on the biochemical and functional
similarities between p85 and MBS130, we therefore designate p85 as MBS85.
may be
correlated with morphological effects in cultured cells. Furthermore
adherent cells mainly exhibit active Rho phenotype in serum medium, and
the interference of endogenous PP1
would be expected to affect this
actin structure. As shown in Fig.
5A, expression of wild-type
p85 alone did not give any obvious morphological consequence. However,
truncation of the C terminus led to various degrees of disassembly of
actin stress fibers, with the most striking effects when both the PIM and
LZ motifs were totally removed (p85NT; Fig. 5,
A and B). Deletion of the PIM motif
(p85
Nar1) or mutation of the phosphorylation
site (p85AA) was significantly effective in inducing
similar morphological effects, although this appears to depend on the
levels of expression (Fig. 5A, bottom panel).
Both the PIM and
LZ motifs at the C terminus appear to exert
opposing effects on the N-terminal function. Truncation of the N
terminus resulted in general increases in actin polymerization
(p85CT in Fig. 5A) that was resistant to C3
treatment (data not shown). As
LZ alone was also effective, although
to a lesser extent (data not shown), it is likely that PIM and
LZ
domains have independent activities toward actin assembly. Similar
trends were also observed when the various constructs were co-expressed
with PP1
(Fig. 5B). In this case, the morphological
effects were more pronounced in the presence of exogenous PP1
. As
expected, both ankyrin repeat mutants p85
AR1
and p85
AR2 that were defective in binding to
PP1
were totally ineffective in inducing morphological changes (Fig.
5B and data not shown). Hence the phenotypic effects of the
various MBS85 variants on actin cytoskeleton correlate well with the
biochemical data described earlier.
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Fig. 5.
Morphological effects of MBS85 in HeLa
cells. Full-length wild-type p85, p85NT (1),
p85CT (346), p85 Nar1,
p85AA, p85
LZ, and
p85
AR2 in pXJ40-FLAG vector were singly
(A) or doubly transfected with pXJ40-HA-PP1
construct.
Cells were immunostained with anti-FLAG or anti-HA for the expressed
protein and tetramethylrhodamine B isothiocyanate-phalloidin for actin
filaments. B, a statistical analysis of the scores of stress
fiber loss with the various single (white bar) and double
tranfections in A (black bar). The values represent
means and S.D. obtained from 3-4 independent experiments.
When
Phosphorylated by MRCK
-CAT and Exerted an Inhibitory Effect on
PP1
Activity--
It is known that phosphorylation of threonine 695 of MBS (which is equivalent to the threonine 560 of p85) is critical
for the inhibitory effects on PP1 catalytic function (11). It is likely
that a similar mechanism may operate for MBS85. To test this
possibility, we derived an assay to examine the effect of phosphorylation on the interaction of the highly conserved GST-PIM with
p85/PP1
complex. The phosphorylation-deficient mutant
p85AA was used to eliminate possible competition for
binding. As shown in Fig. 6A,
phosphorylated GST-PIM, but not the nonphosphorylated form, was
detected in the p85/PP1
immuno-complex. Similarly only glutathione
beads with the phosphorylated GST-PIM, but not the nonphosphorylated or
phosphorylation-deficient mutant GST-PIM50AA, were able to
pull down the expressed PP1
or p85/PP1
complex (Fig.
6B), clearly indicating that phosphorylation of threonine 560 of MBS85 is essential for its interaction with PP1
.
View larger version (21K):
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Fig. 6.
Phosphorylated PIM50 binds and inhibits
PP1 activity. A,
phosphorylated GST-PIM50 by MRCK
-CAT and ATP
S or
nonphosphorylated GST-PIM50 control was preincubated with COS-7 cell
extract expressing FLAG-tagged p85AA and HA-tagged PP1
before immunoprecipitation (IP) with anti-FLAG beads.
Immunoprecipitated proteins on Western blots (WB) were
detected with various anti-tag antibodies. GST-PIM50 in total lysate is
shown in the bottom panel for comparison. B,
GST-PIM50 or GST-PIM50AA on glutathione beads was
phosphorylated with MRCK
-CAT and ATP
S. Nonphosphorylated
GST-PIM50 was used as the control. A pull-down assay was performed by
incubating these beads with extracts expressing PP1
alone
(left lanes) or PP1
together with p85AA
(right lanes). Immunoblots of the bound proteins were
detected with the anti-tag antibodies. A nonspecific band recognized by
the HA in total extract was marked by an asterisk.
C, time course of dephosphorylation of
33P-MLC2 by p85AA and PP1
immunoprecipitates in the presence of phosphorylated and
nonphosphorylated GST-PIM50 was assayed as described under Fig.
4B.
complex (Fig. 6C). This provides further evidence that the interaction is functional.
,
suggesting that PIM50 may well be competing with the catalytic subunit
in regulating actin dynamics. We therefore conclude that from both
biochemical and morphological data that the central conserved motif of
MBS85 can be regulated by phosphorylation, resulting in conformational
changes that affect the associated PP1
catalytic property and
subsequently effects on actin morphology, probably through the eventual
effects on myosin phosphorylation.
View larger version (44K):
[in a new window]
Fig. 7.
Co-transfection of PIM50 counteracts MBS85
N-terminal-induced morphological effects. A, HeLa cells
were either transfected with pXJ40-FLAG-p85NT or
co-transfected with pXJ40-HA-PP1 . The additional effects of
expressing pXJ40-GST-PIM50 construct on top were compared by
co-staining of the various expressed tagged proteins with different
antibodies and actin filament with phalloidin. B,
statistical analysis of the effects of the expression of GST-PIM50 and
GST-PIM50AA on p85NT-induced stress
fiber loss. The bar represents mean and S.D. from 3-4
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CAT. The p130 protein was confirmed to be
MBS130 by peptide sequencing and immunoreactive toward specific
antibodies. The smaller protein p85 is a novel protein that is
structurally related to MBS130. Overall p85 shares low similarity to
MBS130 (<40%). The N terminus of p85 contains six ankyrin repeats
that are known to be involved in protein-protein interactions. This
motif shares a 48% identity to MBS130, which has 7-8 repeats (23,
24). Preceding these repeats is a short stretch (RTVRF) that resembles PP1 binding consensus sequence (25). The C terminus of p85 is also
conserved and consists of an
-helical structure with four leucine
heptad repeats at the C-terminal end. This motif is known to be
involved in dimerization and interaction with other proteins. M20, a
small subunit protein of PP1, is found to be an integral part of the
heterotrimeric complex (28), and it contains a similar helical
structure. Indeed, it has been reported that this M20 may be a spliced
product from skeletal muscle isoform of MBS130 (30). More recently, it
has also been reported that other proteins can interact with the C
terminus of MBS130. This includes RhoA and cGMP-dependent
protein kinase 1
(9, 18). The former links the PP1 complex to
Rho-dependent regulatory events, and the kinase
phosphorylates and activates PP1 activity. The presence of a similar
motif in p85 at the C terminus suggests that it may serve similar
functional roles. Indeed p85 can readily translocate to peripheral
membrane upon treatment with lysophosphatidic acid and phorbol ester,
factors that are known to have prominent effects on Rho GTPases and
actin cytoskeleton (2).
and ROK
exhibits a more striking similarity. Threonine
560 of p85 is equivalent to threonine 695 of MBS130 and is located
within a ~50-amino acid conservative phosphorylation inhibitory motif
(PIM50). Phosphorylation of the threonine 695 by ROK
has been shown
to inhibit associated PP1 activity (11, 30). A second phosphorylation
site (serine 854) by ROK
has also been described in MBS130, but this
is absent in p85 (Fig. 2A and Ref. 10).
, and this
depends on the N terminus including the whole of the ankyrin repeats.
N-terminal as well as ankyrin-repeat deletion mutants are ineffective
in binding PP1
. In agreement with a previous report for MBS130 (32),
we also detected binding of the substrate MLC2 within the N-terminal
region. In this respect, the N terminus of p85 alone can therefore act
as scaffold for PP1 and myosin and confers specific phosphatase
activity on MLC2 substrate (30). p85 (designated here as MBS85) is
therefore a genuine myosin binding subunit of PP1
, similar to
MBS130. Furthermore, the phosphorylation of threonine 560 of the
central motif of MBS85 resulted in inhibition of PP1
activity, also
suggesting a conservation of phosphorylation mechanism in regulating
the catalytic event.
LZ) appear to act
independently to counteract this N-terminal function as deletion of
either motif attenuates the stress fiber losses. Overexpression of
C-terminal
LZ motif alone can induce actin stress fibers that are
resistant to C3 treatment, suggesting that this activates an event
downstream of Rho. The exact mechanism of how this
LZ motif works is
currently not clear. One possibility is that this motif may directly
compete with binding proteins such as cGMP-dependent kinase
1
, which is known to activate PP1 (18). Furthermore, the
LZ motif
is known to be able to form homodimers or heterodimers with the small
subunit M20, although the biological functions of these complexes are
currently not known (data not shown; Refs. 26 and 30). We conclude from both biochemical and morphological analyses that different domains of
the MBS85 have opposing activities in regulating actin polymerization and that the phosphorylation of threonine 560 in a central motif appears to fine tune these events.
. Such an interaction occurred only when the threonine 560 was phosphorylated. Moreover, not
only did such a motif play a role in intramolecular interaction, it is
also equally effective both in vitro and in vivo
in promoting intermolecular interactions when introduced separately.
This leads to the conclusion that phosphorylation of MBS85 by myotonic
dystrophy kinase-related kinases such as MRCK
and ROK
induces a
conformational change at the central conserved motif that results in
higher affinity toward PP1
. Such an interaction may change the
orientation or catalytic properties of PP1
toward the associated
myosin (through MLC2; Fig. 6C), resulting in myosin
phosphorylation and subsequent cytoskeletal changes. This is depicted
as a working model in Fig. 8. It remains
unclear as to which GTPase(s) and downstream kinase(s) are regulating
this event, as endogenous levels of p85 in cultured cells are much
lower than p130MBS, and the specific antibody that recognizes the
phosphorylated peptide was unable to detect the endogenous
phosphoprotein. Further experiments are therefore required to clarify
this issue.
View larger version (18K):
[in a new window]
Fig. 8.
A model for the phosphorylation regulation of
threonine 560 of p85MBS on PP1 activity.
When threonine 560 (T) MBS85 is not phosphorylated, PP1
assumes an orientation in contact with its substrate MLC2, resulting in
an active conformation for the dephosphorylation of MLC2 and subsequent
actin-myosin disassembly. Upon phosphorylation (T with an
asterisk), it presents a conformation that has a higher
affinity to PP1
and disrupts its accessibility or catalytic activity
toward MLC2, resulting in a shutdown of MLC2 dephosphorylation that
favors myosin phosphorylation and actin-myosin assembly. AR,
ankyrin repeats.
In summary, we have isolated a novel myosin binding subunit that is
ubiquitously expressed. Compared with MBS130, the smaller size and
simpler arrangement of the regulatory domains of this novel MBS85 allow
an easier analysis of structure and function relationships. The
identification of an increasing number of these myosin binding
subunits, which share a similar regulatory mechanism, should help to
understand how each of these are regulated by various diverse signaling
pathways in the control of the actin cytoskeleton.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Ito for the antibody to phosphorylated threonine 695 of MBS130 and Dr. Robert Qi for peptide sequence analysis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Glaxo-Singapore Research Fund.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF312028.
¶ To whom correspondence should be addressed. Tel.: 65-874 6167; Fax: 65-774 0742; E-mail: mcbthoml@imcb.nus.edu.sg.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M102615200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MRCK, myotonic
dystrophy kinase-related Cdc42 binding kinase;
ROK, RhoA binding
kinase;
MLC2, myosin light chain 2;
MBS, myosin binding subunit;
PP1, protein phosphatase 1;
PIM, phosphorylation inhibitory motif;
LZ,
-helical leucine zipper;
CAT, catalytic domain;
PCR, polymerase
chain reaction;
HA, hemagglutinin;
PVDF, polyvinylidene
difluoride;
GST, glutathione S-transferase;
ATP
S, adenosine 5'-O-(thiotriphosphate);
EST, expressed sequence
tag.
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