(Received for publication, June 18, 1996, and in revised form, November 13, 1996)
From the West-Western screening of a cDNA expression
library using 32P-labeled, autophosphorylated protein
kinase C Protein kinase C (PKC)1 is a
serine/threonine kinase thought to act in diverse cellular processes
such as the secretion of hormones and neurotransmitters and the
regulation of cell proliferation and differentiation. So far, more than
10 PKC isozymes have been reported, and these can be divided into three
distinct classes based on differences in their structures and
biochemical properties, conventional PKC (cPKC) members ( PKC The stable interaction of PKC and its substrate has been demonstrated
by using MARCKS, a well characterized PKC substrate (10). Furthermore,
our kinetic analysis of the phosphorylation of MARCKS by PKC isozymes
revealed a very low Km value (10-20
nM), supporting the high affinity interaction between
MARCKS and PKC isozymes ( In the present study, we used West-Western screening to isolate
cDNA clones encoding PKC PKC Purified PKC Poly(A)+ RNA was isolated from NIH3T3 cells,
and randomly primed cDNA was synthesized using HindIII
primer adapter (Novagen). cDNA was ligated to Approximately 2 × 105 clones were plated at 10,000 phages per plate using Escherichia coli strain BL21 (DE3)
pLysE as host cells. After incubation for 6 h at 37 °C, the
plaques were covered with Hybond-C extra (Amersham Corp.) impregnated
with 10 mM
isopropyl-1-thio- PKC Overlay assay was performed based on the procedure of Wolf
and Sahyoun (23). PKC Protein-blotted PVDF
membrane was overlaid with 20 µg/ml
[14C]phosphatidylserine (specific activity = 1 µCi/15 µg, Amersham Corp.) diluted in 50 mM Tris-HCl,
pH 7.5, containing 0.5 M NaCl and 10 mg/ml bovine serum
albumin at room temperature for 1 h. Membrane was washed briefly
in phosphate-buffered saline and exposed for autoradiography.
cDNAs
encoding mouse PKC The protein-transferred PVDF membrane was incubated with 0.7 µM MBP-PKC An
E. coli expression vector for GST-SRBC was constructed using
expression vector pGEX-3X (Pharmacia) and cDNA encoding SRBC, and
the fusion protein was purified on glutathione-Sepharose 4B (Pharmacia). The reaction mixture contained 20 mM Tris-HCl,
pH 7.5, 5 mM Mg(OAc)2, 10 µg/ml leupeptin, 50 ng/ml TPA, 25 µg/ml phosphatidylserine, 3.74 ng of PKC An expression vector
for tag-SRBC NIH3T3 cells were routinely cultured in DMEM
supplemented with 10% fetal calf serum (FCS). For serum starvation,
the medium was changed to 0.5% FCS when the cells were still
subconfluent. For serum stimulation, fresh medium containing 20% FCS
was added to the starved cells. Cells were harvested at the desired
times for RNA isolation. Differentiation of P19 cells was performed following a standard procedure (24, 25).
Poly(A)+ RNA was isolated using a QuickPrep Micro mRNA
Purification Kit (Pharmacia), and Northern blot analysis was performed according to the standard protocol.
A mouse NIH3T3-
The original pEXlox clone,
clone 53, encodes only a C-terminal part of SRBC (156 to 263 amino
acids) as fusion proteins with the T7 gene 10 product, indicating that
the C-terminal part is sufficient for the interaction with PKC
To test whether full-length SRBC binds to PKC
Previous studies showed that most of the PKC-binding
proteins are phosphatidylserine-binding proteins (10, 13, 16, 23, 31).
Thus, phosphatidylserine may form a bridge between PKC and PKC-binding
proteins and stabilize the binding. Since binding of SRBC to PKC Since the
binding of PKC
As already shown in
Fig. 2, the C-terminal part of mouse SRBC, the clone 53 product, can be
phosphorylated by PKC
To monitor
the phosphorylation of SRBC in vivo, we designed an
epitope-tagged SRBC
Northern blot analysis showed that SRBC is ubiquitously
expressed in almost all tissues tested except liver, and higher levels of expression are observed in uterus and ovary (Fig.
7A). In cultured cell lines, SRBC mRNA
was detected in NIH3T3 cells and 3Y1 cells but not in COS1 cells or
mouse embryonal carcinoma cell lines including F9 and P19 (Fig.
7B).
It has been reported that the expression of sdr mRNA is
strongly induced by serum starvation in NIH3T3 cells and down-regulated within 6 h after the addition of serum to starved cells (29). The
considerable structural similarity between SRBC and Sdr allowed us to
test whether SRBC mRNA is also induced upon serum starvation. The
level of SRBC mRNA was relatively low in growing NIH3T3 cells (Fig.
7C, time 0), and was induced by serum deprivation
reaching a maximum level within 12 h. The induced mRNA
expression level was retained at least for 48 h (Fig.
7C). The amount of SRBC mRNA in starved cells (Fig.
7D, time 0) decreased rapidly after the addition
of serum, reaching a minimum level after 3 h (Fig. 7D). When the amount of mRNA was normalized to that of
glyceraldehyde-3-phosphate dehydrogenase mRNA, the SRBC mRNA
level was ~eight times higher in serum-starved cells compared with
serum-stimulated or exponentially growing cells. The induction of SRBC
mRNA was also observed upon retinoic acid-induced differentiation
of mouse embryonal carcinoma P19 cells to neuron-like cells (Fig.
7B). Taken together, these results show that the level of
SRBC mRNA expression correlates with cell growth suppression.
We cloned a cDNA encoding a PKC-binding protein by
West-Western screening using 32P-labeled,
autophosphorylated PKC The Km value for the phosphorylation of SRBC by
PKC The binding of SRBC to PKC SRBC binds to PKC The primary structure of SRBC shows considerable similarity to Sdr and
one of the chicken expression sequence tags (CHKESTFLLE). sdr has been identified as a gene induced in NIH3T3 cells by
serum starvation but whose function remains unknown. Most of the
conserved amino acids in these proteins are clustered in the N-terminal region named SCR1, which includes a "leucine zipper"-like motif. Although this region is not required for the binding of SRBC to PKC The expression pattern of the srbc gene shares some common
features with that of sdr; both srbc and
sdr are induced by serum starvation of NIH3T3 cells and
down-regulated by the addition of serum. This suggests that
sdr and srbc form a family of genes that play a
role in cell-growth control. In addition, other genes induced by growth
arrest of cells have been identified in different systems (34, 35) but
show no structural homology with SRBC. Noteworthy, the N-terminal 185 amino acids of SRBC, including SCR1, have been found in the N-terminal
part of the oncogenic c-RAF-1 gene, replacing the N-terminal
regulatory domain of c-RAF-1 (30). The significance of the SRBC
sequence fused to c-RAF-1 is unclear since a variety of genes can
activate the c-RAF-1 gene by similar gene fusion (36).
However, since the SRBC-RAF fusion gene seems to be under the control
of the srbc promoter, the fusion gene is most likely
expressed at high levels in quiescent cells, and this could explain
some of the oncogenic properties of the fusion gene. The ubiquitous
expression of srbc in different tissues and its induction
upon retinoic acid-induced differentiation of mouse embryonal carcinoma
P19 cells suggest the involvement of SRBC in a variety of the cellular
events that accompany growth arrest.
A mutant PKC The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D85435[GenBank]. We thank Dr. Hiroshi Nojima (Osaka University)
for providing the rat 3Y1-
Department of Molecular Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
(PKC
) as a probe, led us to identify cDNA clones
encoding a PKC
-binding protein that contains a leucine zipper-like
motif in its N-terminal region and two PEST sequences in its C-terminal
region. This protein shows overall sequence similarity (43.3%) to the
serum deprivation response (sdr) gene product, and we
named it SRBC (
dr-
elated gene product that
inds to
-kinase).
PKC
binds to the C-terminal half of SRBC through the regulatory
domain and phosphorylates it in vitro. In COS1 cells, the
phosphorylation of over-expressed SRBC is stimulated by
12-O-tetradecanoylphorbol-13-acetate and further enhanced
by the over-expression of PKC
. The mRNA for SRBC is detected in
a wide variety of cultured cell lines and tissues and is strongly
induced by serum starvation. Furthermore, SRBC mRNA is induced
during retinoic acid-induced differentiation of P19 cells. These
results suggest that SRBC serves as a substrate and/or receptor for PKC
and might be involved in the control of cell growth mediated by
PKC.
,
I,
II, and
), novel PKC (nPKC) members (
,
,
,
, and
µ), and atypical PKC (aPKC) members (
and
/
). All PKC
members consist of an N-terminal regulatory domain and a C-terminal
catalytic domain; the co-factor binding site has been identified in the
regulatory domain (1-4).
is an nPKC member that is expressed in a variety of tissues and
cultured cell lines (5-8). We have previously shown that a
constitutively active mutant of PKC
acts as a potent inducer of
transcription factor activator protein 1/Jun and that a
kinase-deficient mutant of PKC
can inhibit the activity of the
mutant (9). This dominant-negative effect of kinase-deficient PKC
can be explained by the titration of an effector, substrate, or
receptor molecule(s) that binds stably to PKC
.
,
, and
) (11). Some other
PKC-binding proteins have been identified by the screening of a
cDNA expression library using a purified brain PKC mixture as a
probe. One such protein is RACK1, which is thought to serve as a
receptor for activated PKC (12). Others include F52/Mac-MARCKS and
35H/
-adducin, which is a known substrate for PKC in vivo
and in vitro (13, 14). A yeast two-hybrid system has also
been used to identify PKC
-binding protein PICK1 (15). In addition to
these proteins, several proteins are known to bind to a brain PKC
mixture in vitro. These include annexin (I, II, and VI),
vinculin, talin, and 72/53ORIG (16, 17). In addition, Bruton tyrosine
kinase binds to cPKC (
,
I, and
II), nPKC (
), and aPKC (
)
in vitro (18). Actin binds to PKC
in vitro
(19) and PKC
II in vitro and in vivo (20).
Recently, it has been shown that AKAP79, an A kinase anchoring protein,
can also bind to PKC
and PKC
II in vitro and in
vivo (21), and human immunodeficiency virus Nef protein can bind to PKC
in vitro and in vivo (22).
-binding proteins. Here we describe a
clone encoding as sdr-related gene product named SRBC, which serves as a substrate for PKC
and whose expression is induced by
serum starvation of NIH3T3 cells or differentiation of mouse embryonal
carcinoma P19 cells.
Purification and 32P Labeling of PKC
was purified from recombinant baculovirus-infected Sf21 cells as
described elsewhere (11). Briefly, 3 days after infection, Sf21 cells
were lysed, and PKC
was purified by a series of chromatographic steps on DEAE-cellulose (Tosoh), hydroxyapatite (Koken), and MonoQ (Pharmacia Biotech Inc.) columns. No additional proteins were obvious
in the final PKC
fraction as judged by SDS-PAGE, and the specific
activity was 380 units/mg. One unit of PKC
activity was defined as 1 nmol of 32P incorporated into a myelin basic protein
(MBP4-14) peptide per min in a reaction buffer
containing 20 mM Tris-HCl, pH 7.5, 5 mM
Mg(OAc)2, 10 µg/ml leupeptin, 25 µg/ml
phosphatidylserine, 50 ng/ml TPA, 20 µM ATP, and 0.5 µCi of [
-32P]ATP (Amersham Corp.).
(400 ng) was labeled with 32P by
autophosphorylation in 155 µl of kinase buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2,
10% glycerol, 0.6 mCi/ml [
-32P]ATP, 40 µg/ml
phosphatidylserine, and 400 ng/ml TPA at 30 °C for 2 h. After
the reaction, 32P-labeled PKC
was separated from
unreacted ATP by gel filtration on Sephadex G-50 equilibrated with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mM dithiothreitol, and 0.5% Triton X-100 and used as a
probe for screening a
EXlox cDNA expression
library.
-binding
Protein
EXlox
vector arm (Novagen) and packaged in phage particles using a Phage
Marker System (Novagen). This library contains approximately 0.95 × 106 independent clones.
-D-galactopyranoside and allowed to
grow for an additional 3.5 h at 37 °C. The filters were then
washed with TBS buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl), blocked with 5% skim milk at 4 °C overnight,
and incubated with 1 mM ATP solution (TBS buffer containing
1 mM ATP) at room temperature for 2 h to saturate any
auto-phosphorylation sites or ATP-binding sites of the proteins. The
filters were then incubated with 32P-labeled PKC
(106 cpm/ml) in 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 50 µg/ml phosphatidylserine, and 1% bovine serum
albumin for 5 h at room temperature. After washing in TBS buffer,
the filters were exposed for autoradiography. Positive phage clones
were converted to plasmids (pEXlox + cDNA insert) using
E. coli strain BM25.5 according to the manufacturer's instructions. A cDNA insert was used as a probe to screen a
3Y1-
ZAP II cDNA library to obtain cDNA clones encoding the
full SRBC coding sequence. Plaque hybridization with a
ZAP II
cDNA library and the isolation of positive phage clones were
performed according to standard procedures.
-binding proteins were subjected to SDS-PAGE,
blotted onto PVDF membranes, and treated with a 5% skim milk solution.
The phosphorylation reaction was carried out by incubating the PVDF membranes with phosphorylation buffer (83 ng/ml PKC
, 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2,
10% glycerol, 50 µg/ml phosphatidylserine, 400 ng/ml TPA, and 2.4 µCi/ml [
-32P]ATP) at room temperature for 2 h.
After washing with TBS containing 1% SDS buffer, the membranes were
exposed for autoradiography.
as a
Probe
-binding proteins were subjected to SDS-PAGE and blotted onto a PVDF membrane. After treatment with a 5% skim milk
solution, the PVDF membrane was incubated with 32P-labeled
PKC
(106 cpm/ml) diluted in 50 mM Tris-HCl,
pH 7.5, 0.5 M NaCl, 50 µg/ml phosphatidylserine, and 1%
bovine serum albumin at room temperature for 5 h. Excess ligand
was removed by washing the PVDF membrane with TBS buffer, and the
membrane was exposed for autoradiography.
and its deletion mutant (lacking amino acid
residues 299-654, 209-654, and 111-654) were subcloned into E. coli expression vector pMAL-c2 (New England BioLabs) for expression of these proteins as fusion proteins with maltose-binding protein (MBP-PKC
, MCD299, MCD209, and MCD111). The proteins were purified on amylose resin (New England BioLabs) and used as probes for
the overlay assay.
or 1 µM MCD299, MCD209, or
MCD111 in 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl,
50 µg/ml phosphatidylserine, and 1% bovine serum albumin for 3 h at room temperature. After washing the membrane with TBS buffer,
bound protein was fixed by incubation with 0.5% formaldehyde. Excess
formaldehyde was removed by washing with 2% glycine, and MBP fusion
proteins were detected using anti-MBP antibody (New England BioLabs),
alkaline phosphatase-conjugated second antibody (TAGO, Inc.), and
artificial substrate for alkaline phosphatase (VECTOR).
, and
various concentrations of GST-SRBC proteins in a total volume of 20 µl. The reaction was started by the addition of 20 µM
ATP and 0.5 µCi of [
-32 P]ATP and incubated at
30 °C. After 5 min of incubation, the reaction was stopped by the
addition of 5 µl of 5 × Laemmli's SDS-sample buffer. Proteins
were then separated on SDS-PAGE, and the 32P incorporated
in the GST-SRBC protein was quantified using a Bio-Image analyzer (BAS
2000 FUJI).
15 (SRHis-SRBC) encodes a SRBC protein whose N-terminal
15 amino acids are replaced by six histidine residues and a 12-amino
acid sequence from the T7 gene 10 leader sequence derived from pBLUE
Bac (Invitrogen). The PKC
expression plasmid (M241) was described
previously (6). COS1 cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS)
and seeded 24 h before transfection at a concentration of 3 × 105 cells/6-cm dish. Transfection by calcium phosphate
co-precipitation was carried out for 6 h using the appropriate
expression vectors, as described in the figure legends. The culture
medium was then changed to serum-deprived DMEM to starve the cells.
After 48 h, the medium was changed to phosphate-free DMEM, and the
cells were further incubated for 2 h. The cells were then
incubated in medium containing [32P]orthophosphate (125 µCi/ml) for 4 h prior to stimulation. After treatment for 10 min
with TPA (50 ng/ml) or vehicle (dimethyl sulfoxide) alone, the cells
(in 6-cm dishes) were harvested and suspended in 100 µl of lysis
buffer containing 20 mM Hepes, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 50 mM NaF, 1 mM
Na3VO4, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 1.8 µg/ml aprotinin, 1% Triton X-100,
0.1% deoxycholate, and 0.1% SDS. After 30 min incubation on ice, the
lysate was clarified by centrifugation at 14,000 rpm for 30 min and
incubated with anti-T7-tag antibodies (Novagen) preabsorbed with
Protein G-Sepharose (Pharmacia), for 1 h at 4 °C. The
immunocomplexes on Sepharose were washed 5 times with lysis buffer and
2 times with final wash buffer containing 20 mM Hepes, pH
7.5, 1 mM EDTA, 50 mM NaF, 1 mM
Na3VO4, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 1.8 µg/ml aprotinin, and 0.1% Triton
X-100. The proteins were then separated by SDS-PAGE, transferred to a
PVDF membrane, and probed with anti-T7 antibody to estimate the amount
of tag-SRBC precipitated. The amount of 32P incorporated
into tag-SRBC was measured by a Bio-Image analyzer (BAS 2000 FUJI).
Cloning of SRBC
EXlox expression
library was screened for proteins that bind to PKC
in the presence
of phosphatidylserine. Using 32P-labeled PKC
as a probe,
two clones, clone 53 and 91, that appeared to originate from the same
mRNA were isolated. To isolate full-length cDNAs, a rat
3Y1-
ZAPII cDNA library was screened using clone 53 as a probe.
The nucleotide sequence of a rat homologue of clone 53/91, and the
structural features of the protein are shown in Fig.
1A. The ATG at position 1 is most likely an
initiation codon because 1) the surrounding nucleotide sequence
fulfills Kozak's criteria (26), and 2) there is an in-frame TAG codon
in the 5
non-coding sequence. The open reading frame starting with
this initiation codon encodes a protein with 263 amino acid residues. The calculated molecular mass of the protein, named SRBC, is 27,879. Mouse clone 53 encodes 105 amino acid residues corresponding to the
C-terminal part of rat SRBC (Fig. 1A, lower panel), and the amino acid sequence identity in this region is 86%. SRBC contains a
leucine zipper-like motif in the N-terminal part and two PEST regions,
which are commonly found in short-lived proteins (27, 28), in the
C-terminal part (Fig. 1A). A comparison of the SRBC nucleotide sequence with the GenBank data base revealed high homologies to mouse sdr (29) and a chicken mRNA for the expressed
sequence tag (CHKESTFLLE) (Fig. 1B).The overall amino acid
sequence identities for SRBC to Sdr and CHKESTFLLE are 43.3 and 25.9%,
respectively. Amino acid residues conserved among the proteins are
clustered in two regions, SRBC conserved region 1 (SCR1) and SRBC
conserved region 2 (SCR2). SCR1 contains the leucine zipper-like motif
in its N-terminal part, and SCR2 contains one or two PKC
phosphorylation sites (Fig. 1B). Amino acid identity in SCR1
is 43.8% between SRBC and Sdr and 29.0% between SRBC and CHKESTFLLE.
In SCR2, it is 60.9% between SRBC and Sdr and 52.2% between SRBC and
CHKESTFLLE. In addition, all of these proteins contain PEST regions
even though their primary structures are not conserved. It is
noteworthy that the sequence from 1 to 185 amino acids of SRBC shows
90% identity with the sequence from 1 to 185 amino acids of the human
7N-1 clone, identified as a gene fused to c-RAF-1 that
results in the constitutive activation of the c-RAF-1 gene
(30). In this clone, the N-terminal regulatory domain of c-RAF-1 is
replaced by the human SRBC homologue.
Fig. 1.
Primary structure of SRBC. A,
nucleotide sequence of rat SRBC and the deduced amino acid sequence.
Nucleotides are numbered at the left beginning with the
"A" of the predicted translation initiation codon; amino acid
residues are numbered at the right. Polyadenylation signals (AATAAA) are double-underlined.
Amino acid residues included in the leucine zipper-like motif are
marked by a black background; the PEST regions are
underlined. The asterisks indicate potential PKC
phosphorylation sites. The lower panels show schematic
drawings of the protein structures of SRBC and the clone 53 product.
Clone 53 encodes the C-terminal part of mouse SRBC, which was obtained
from the NIH3T3-EXlox expression library using PKC
as
a probe. B, amino acid alignment of rat SRBC, mouse Sdr, and
the CHKESTFLLE protein encoded by a chicken mRNA for the expressed
sequence tag. Identical residues are shaded and SRBC
conserved region 1 (SCR1) and SRBC conserved region 2 (SCR2) are
indicated by double-underlining. Asterisks
indicate amino acid residues included in the leucine
zipper-motif.
[View Larger Versions of these Images (33 + 62K GIF file)]
Binds to SRBC
. The
overlay assay showed that PKC
binds to the product of the T7 gene 10 fused clone 53 product in the presence of phosphatidylserine, a common
activator of PKCs (Fig. 2, lanes 3 and
5). On the other hand, no such interaction was observed with
the T7 gene 10 product itself (Fig. 2, lanes 3 and
4). Furthermore, the fusion protein but not the T7 gene 10 product itself can be phosphorylated by PKC
on the membrane (Fig. 2,
lanes 7 and 8), in agreement with the presence of
putative PKC-phosphorylation sites in the clone 53 product (Fig.
1A).
Fig. 2.
PKC binds to and phosphorylates the clone
53 product. E. coli lysates containing the T7 gene 10 product and the fusion protein of clone 53 product were separated by
SDS-PAGE and transferred onto a PVDF membrane. Proteins were detected
by Western blot analysis using anti-T7 tag antibody (lanes 1 and 2), and the binding of 32P-labeled PKC
was tested by overlay assay in the presence (lanes 3 and
4) or absence (lanes 5 and 6) of
phosphatidylserine. The phosphorylation of proteins on the PVDF
membrane is also shown (lanes 7 and 8). The
upper arrowheads indicate the position of the T7 gene
10-fused clone 53 product, and the lower arrowheads indicate
the position of the T7 gene 10 product.
[View Larger Version of this Image (42K GIF file)]
, SRBC was expressed as
a GST-fusion protein in E. coli and tested for PKC
binding by overlay assay. MARCKS, a known binding protein/substrate for
PKC, and GST alone were also tested for PKC
binding. GST-SRBC appeared as a 65.4-kDa band on SDS-PAGE, and MARCKS and GST appeared as
80- and 26-kDa bands, respectively (Fig. 3A, lanes
1-3). The positions of GST and GST-SRBC were confirmed by Western
blot analysis using anti-GST antibody (Fig. 3A, lanes 4 and
5). PKC
bound to GST-SRBC to the same extent as to MARCKS
(Fig. 3A, lanes 6 and 7) but did not bind to GST
(Fig. 3A, lane 8). Again, the binding depends entirely on
the presence of phosphatidylserine (Fig. 3A, lanes 9 and
10). When PKC
instead of PKC
was used as a probe, essentially the same results were obtained, whereas the signal was
barely detectable when PKC
was used as the probe (data not shown).
Fig. 3.
SRBC binds to PKC in a
phosphatidylserine-dependent manner. A, GST and
GST-SRBC were produced in E. coli, and MARCKS was produced
in Sf21 cells using baculovirus vector (11). These proteins were
subjected to SDS-PAGE and/or transferred to a PVDF membrane for the
overlay assay. The proteins were detected on the gel by silver staining
(lanes 1-3). GST-SRBC and GST were also detected by Western
blot analysis using anti-GST antibody (lanes 4 and
5). Overlay assay was performed in the presence (lanes 6-8) or absence (lanes 9-11) of phosphatidylserine
using 32P-labeled PKC
as a probe. Arrowheads
indicate the position of each protein. B,
protein-transferred membrane was overlaid with [14C]phosphatidylserine. Phosphatidylserine-binding
proteins were detected by autoradiography. Samples used are purified
GST-SRBC (same as A) and crude extract of E. coli
containing clone 53 products (same as Fig. 2).
[View Larger Version of this Image (26K GIF file)]
also depends on phosphatidylserine, we next tested whether SRBC binds
to phosphatidylserine by using [14C]phosphatidylserine
overlay assay. As shown in Fig. 3B, GST-SRBC binds to
phosphatidylserine, and clone 53 encoding only C-terminal part of SRBC
also binds to phosphatidylserine. Note that the C-terminal part of SRBC
was sufficient for the binding to PKC
. These results suggest that
the binding of SRBC and PKC
depends on phosphatidylserine-bridging as suggested for other PKC-binding proteins.
Binds to SRBC
to SRBC depends on the presence of
phosphatidylserine, the co-factor binding site of PKC
located in its
N-terminal regulatory domain might be involved in the interaction with
SRBC. It has been reported that the pseudosubstrate region, in the
regulatory domain, directly mediated
phosphatidylserine-dependent PKC binding to some PKC-binding
proteins (16, 31). To test whether the N-terminal regulatory domain is
sufficient for this interaction, whole PKC
and its regulatory domain
were expressed as MBP (maltose-binding protein) fusion proteins in
E. coli (Fig. 4A) and used as
probes for the overlay assay. In these experiments, bound PKC
was
detected by anti-MBP antibodies. As shown in Fig. 4B,
MBP-PKC
bound to GST-SRBC or MARCKS similarly to intact PKC
(Fig.
4B, lanes 1 and 2). Furthermore, MBP fused to the
regulatory domain of PKC
(MCD299) also bound to GST-SRBC and MARCKS
to the same extent (Fig. 4B, lanes 4 and 5). The
regulatory domain of PKC
includes a cysteine-rich domain conserved
among all cPKC and nPKC members. When the cysteine-rich domain was
totally deleted (MCD111), no binding to SRBC or MARCKS was observed
(Fig. 4B, lanes 10 and 11), whereas the
C-terminal half of the cysteine-rich domain was dispensable for binding
(MCD209 in Fig. 4B, lanes 7 and 8). These results
suggest that amino acid residues 111-209 on PKC
, a region that
includes a pseudosubstrate and half of the cysteine-rich domain, are
essential for the binding to GST-SRBC and MARCKS. The binding of SRBC
to the regulatory domain of PKC
raises the possibility that GST-SRBC
modulates the activity of PKC
. However, we could not detect any
effect of GST-SRBC on the myelin basic protein kinase activity of
PKC
in vitro (data not shown).
Fig. 4.
PKC binds to SRBC in its regulatory
domain. A, schematic drawing and Coomassie Brilliant Blue
staining of purified maltose-binding protein (MBP) fused to PKC
(MBP-PKC
) and its regulatory domain (MCD299, MCD209, and MCD111).
Amino acids 299-654, 209-654, and 111-654 of PKC
are deleted in
MCD299, MCD209, and MCD111, respectively. B, overlay assays
were performed using MBP-PKC
(lanes 1-3), MCD299
(lanes 4-6), MCD209 (lanes 7-9), and MCD111 (lanes 10-12) as probes. Binding of each MBP fusion protein
was detected by anti-MBP antibody and visualized by color development after incubating with alkaline phosphatase-conjugated second antibody. Arrowheads indicate the position of each protein as
indicated at right.
[View Larger Version of this Image (44K GIF file)]
in Vitro
in vitro. Full-length rat SRBC
fused to GST is also phosphorylated by PKC
, whereas GST alone is
barely phosphorylated (Fig. 5A). We next examined the kinetics for SRBC phosphorylation by PKC
in
vitro. GST-SRBC (5-150 nM) was incubated with PKC
(0.7 nM), in the presence of phosphatidylserine, TPA, and
[
-32P] ATP, and the phosphorylated proteins were
analyzed by SDS-PAGE followed by quantitative autoradiography (Fig.
5B). The Km and
Vmax values estimated from the saturation curve
are 60 nM and 0.69 nmol of ATP/min/nmol, respectively. The
Km value is roughly comparable with that for MARCKS
(20.7 nM) obtained by similar experiments (11), whereas the
Vmax is considerably lower than MARCKS (5.2 nmol
of ATP/min/nmol). GST-SRBC is also phosphorylated by PKC
, with
Km and Vmax values similar to
those for PKC
, but GST-SRBC is a poor substrate for PKC
with a
Vmax value one-eighth that for PKC
(data not
shown). The maximum incorporation of phosphate into SRBC by PKC
was
0.78 mol per 1 mol of GST-SRBC.
Fig. 5.
Phosphorylation of GST-SRBC by PKC
in vitro. A, GST-SRBC and GST, 150 nM each, were incubated in vitro with purified PKC
, cofactors, and [
-32P]ATP as described under
"Experimental Procedures." Phosphorylated proteins were analyzed by
SDS-PAGE followed by autoradiography. Arrowheads indicate
the position of each protein. B, different concentrations of
GST-SRBC were used for the phosphorylation reaction to obtain kinetic
constants. The Km and Vmax
values obtained from a saturation curve are 60 nM and 0.69 nmol of ATP/min/nmol, respectively. The position of phosphorylated
GST-SRBC and autophosphorylated PKC
are indicated by
arrowheads. Additional bands around 54 kDa are thought to
represent degradation products of SRBC, which are also phosphorylated
by PKC
.
[View Larger Version of this Image (33K GIF file)]
15 expression vector. In this construction, the
N-terminal 15 amino acids of SRBC are replaced by a T7 gene 10 epitope
tag. The apparent molecular mass of this protein is 43 kDa on SDS-PAGE.
The SRBC
15 was spontaneously phosphorylated in serum-starved COS1
cells, and TPA stimulation caused a ~2-fold increase in the level of
phosphorylation (Fig. 6). We next examined the effect of
the co-expression of PKC
in the presence or absence of TPA. No
significant effect of PKC
over-expression was observed in the
absence of TPA. But a more enhanced induction of phosphorylation by TPA
(2.5-3-fold) was observed when PKC
was over-expressed. These
results are consistent with the idea that SRBC is phosphorylated in vivo as a consequence of PKC activation.
Fig. 6.
Phosphorylation of SRBC15 in response to
PKC activation in vivo. COS1 cells (3 × 105 cells/6-cm dish) were transiently transfected with the
expression vector encoding epitope-tagged SRBC
15 (5 µg) alone or
together with the PKC
-encoded expression vector (2 µg). Cells were
labeled with 32P and exposed to TPA (50 ng/ml) for 10 min
before harvest. SRBC
15 was immunoprecipitated from cell extracts
using anti-T7 tag antibody as described under "Experimental
Procedures." Values represent arbitrary units normalized to the
amount of immunoprecipitated SRBC
15 protein. DMSO,
dimethyl sulfoxide.
[View Larger Version of this Image (23K GIF file)]
Fig. 7.
Northern blot analysis of SRBC mRNA.
A, expression of SRBC mRNA in mouse tissues. Total RNA
was extracted from mouse organs and analyzed on Northern blot (10 µg)
using 32P-labeled SRBC cDNA as a probe. The positions
of ribosomal RNAs (18 S and 28 S) are indicated. The same blot was
sequentially probed with SRBC (upper panel) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(lower panel) cDNAs. B, expression of SRBC mRNA in various cultured cell lines. Poly(A)+ RNA from
NIH3T3 cells, 3Y1 cells, and F9 cells (0.4 µg
each), total RNA from COS1 cells (10 µg), and
poly(A)+ RNA (1.6 µg) from undifferentiated P19 cells
(P19 cells) and differentiated, retinoic acid-treated, P19
cells (P19 cells-RA) were applied. C, SRBC
mRNA level after serum starvation was analyzed by Northern blot
using equal amounts of poly(A)+ RNA (0.5 µg) isolated
from growing NIH3T3 cells (time 0) and cells at the
indicated times after the start of starvation with 0.5% FCS/DMEM.
D, analysis of SRBC mRNA level during G0 and
G1 transition. NIH3T3 cells were starved for 48 h in
0.5% FCS and stimulated to reenter the cell cycle by the addition of
20% FCS. At the indicated times, RNA was isolated and 0.5 µg of
poly(A)+ RNA was analyzed by Northern blot with SRBC and
glyceraldehyde-3-phosphate dehydrogenase probes.
[View Larger Version of this Image (30K GIF file)]
as a probe. This protein, called SRBC, binds
to and is phosphorylated by PKC
in vitro. In COS1 cells,
the phosphorylation of over-expressed SRBC is stimulated by TPA and is
further enhanced by the over-expression of PKC
.
is quite low (60 nM), which may reflect a strong
interaction between the two molecules. On the other hand, the
Vmax value is quite low indicating that SRBC is
not a good substrate for PKC
. However, 1 mol of SRBC incorporates
nearly 1 mol of phosphate after prolonged phosphorylation reaction
(data not shown). Therefore, the low Vmax value
could reflect the low exchange rate of the substrate, SRBC, as a result
of stable binding to PKC
. These features of the PKC-SRBC interaction
are very similar to those for the interactions between PKC isozymes
(
,
, and
) and MARCKS (11).
depends on the presence of
phosphatidylserine; therefore, the active conformation of PKC
might be necessary for the interaction. In addition, the fact that SRBC binds
to phosphatidylserine suggests that phosphatidylserine mediates or
stabilizes the interaction between SRBC and PKC
through its bridging. Interaction with SRBC was also observed for MCD209, a
deletion mutant of PKC
lacking the kinase domain and the C-terminal half of the cysteine-rich domain that is responsible for the binding to
phorbol ester or diacylglycerol (32, 33). This indicates that these
functional domains are dispensable for the interaction with SRBC. Since
MCD111 cannot bind to SRBC, the N-terminal half of the cysteine-rich
domain and the pseudosubstrate region may be essential for binding.
Previous studies demonstrated that the pseudosubstrate region is one of
several motifs that mediate PKC binding (16, 31). However, from our
results, we cannot conclude whether the pseudosubstrate region is
important to the binding to PKC
and SRBC or not. We need further
experiments to determine the SRBC binding region on the PKC
.
, a cPKC family member, to the same extent as
PKC
. And it barely binds to PKC
, an aPKC family member (data not
shown). Thus, the binding of PKC to SRBC does not clearly show isotype
specificity. We identified several PKC
-binding proteins in addition
to SRBC by subsequent screening of the cDNA expression library
using PKC
as a probe. These include MARCKS and several other
proteins that do not include any of previously reported PKC-binding
proteins.2 Among these novel PKC-binding
proteins, we could find a PKC
-specific binding
protein.3
,
it might be involved in the formation of complexes with themselves or
other proteins. The rest of the conserved amino acids are clustered in
a small region called SCR2. This region in SRBC includes two putative
PKC phosphorylation sites, one of which is conserved among the three
proteins. Since only the C-terminal half of SRBC was encoded by the
cDNA clones first identified in the expression library, SCR1 must
be dispensable for the binding to PKC
. No striking sequence homology
is found among known PKC-binding proteins, AKAP79 (21), MARCKS (10),
and the C-terminal part of SRBC including SCR2. However, it is
noteworthy that SCR2 as well as the PKC-binding region in AKAP79 and
MARCKS show a high content of basic amino acids and both SRBC and
MARCKS show similar binding patterns to PKC
(Figs. 3 and 4).
Therefore, SCR2 could be responsible for the binding to PKC in the same
way as the basic amino acid region of MARCKS.
lacking kinase activity shows a dominant-negative
effect on the TPA-induced activation of the TRE-tk-CAT reporter gene in
NIH3T3 cells (9). Furthermore, the same dominant negative mutant
suppresses TRE-tk-CAT expression caused by the ectopic expression of a
PKC
-active mutant (9). Thus, we examined the effect of SRBC on
TRE-tk-CAT expression in NIH3T3 cells. However, we failed to detect any
significant effect (data not shown). Another series of preliminary
experiments to examine the effect of SRBC overexpression on the growth
of NIH3T3 cells demonstrated the inhibition of DNA synthesis and colony
formation,4 supporting the idea that SRBC
is involved in the regulation of cell growth. We cannot conclude that
PKC is involved in these events; however, the further characterization
of SRBC might provide a clue to understanding the signaling pathway by
which PKC controls cell growth and differentiation.
*
This work was supported in part by research grants from the
Ministry of Education, Science, Sports and Culture of Japan, The Cell
Science Research Foundation, and the Uehara Memorial Foundation.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.
§
Research Fellow of the Japan Society for the Promotion of Science
for Young Scientists.
To whom correspondence should be addressed: Dept. of Molecular
Biology, Yokohama City University School of Medicine, 3-9, Fuku-ura,
Kanazawa-ku, Yokohama 236, Japan. Tel.: 81-45-787-2596; Fax:
81-45-785-4140; E-mail: ohnos{at}med.yokohama-cu.ac.jp.
1
The abbreviations used are: PKC, protein kinase
C; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; MARCKS,
myristoylated alanine-rich protein kinase C substrate; sdr, serum
deprivation response; MBP, maltose-binding protein; GST, glutathione
S-transferase; TPA,
12-O-tetradecanoylphorbol-13-acetate; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; PVDF, polyvinylidene difluoride.
2
Y. Tamai, Y. Izumi, S. Hirai, and S. Ohno,
manuscript in preparation.
3
Y. Izumi, Y. Tamai, S. Hirai, and S. Ohno,
manuscript in preparation.
4
Y. Izumi, S. Hirai, and S. Ohno, manuscript in
preparation.
ZAPll cDNA library.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.