A Protein Kinase Cdelta -binding Protein SRBC Whose Expression Is Induced by Serum Starvation*

(Received for publication, June 18, 1996, and in revised form, November 13, 1996)

Yasushi Izumi Dagger §, Syu-ichi Hirai Dagger , Yoko Tamai Dagger , Ariko Fujise-Matsuoka Dagger , Yoshifumi Nishimura and Shigeo Ohno Dagger par

From the Dagger  Department of Molecular Biology, Yokohama City University School of Medicine, 3-9, Fuku-ura, Kanazawa-ku, Yokohama 236 and the  Graduate School of Integrated Science, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama 236, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

West-Western screening of a cDNA expression library using 32P-labeled, autophosphorylated protein kinase Cdelta (PKCdelta ) as a probe, led us to identify cDNA clones encoding a PKCdelta -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 (<UNL><IT>s</IT></UNL>dr-<UNL>r</UNL>elated gene product that <UNL>b</UNL>inds to <UNL>c</UNL>-kinase). PKCdelta 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 PKCdelta . 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.


INTRODUCTION

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 (alpha , beta I, beta II, and gamma ), novel PKC (nPKC) members (delta , epsilon , eta , theta , and µ), and atypical PKC (aPKC) members (zeta  and iota /lambda ). 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).

PKCdelta 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 PKCdelta acts as a potent inducer of transcription factor activator protein 1/Jun and that a kinase-deficient mutant of PKCdelta can inhibit the activity of the mutant (9). This dominant-negative effect of kinase-deficient PKCdelta can be explained by the titration of an effector, substrate, or receptor molecule(s) that binds stably to PKCdelta .

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 (alpha , delta , and epsilon ) (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/gamma -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 PKCalpha -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 (alpha , beta I, and beta II), nPKC (epsilon ), and aPKC (zeta ) in vitro (18). Actin binds to PKCepsilon in vitro (19) and PKCbeta II in vitro and in vivo (20). Recently, it has been shown that AKAP79, an A kinase anchoring protein, can also bind to PKCalpha and PKCbeta II in vitro and in vivo (21), and human immunodeficiency virus Nef protein can bind to PKCtheta in vitro and in vivo (22).

In the present study, we used West-Western screening to isolate cDNA clones encoding PKCdelta -binding proteins. Here we describe a clone encoding as sdr-related gene product named SRBC, which serves as a substrate for PKCdelta and whose expression is induced by serum starvation of NIH3T3 cells or differentiation of mouse embryonal carcinoma P19 cells.


EXPERIMENTAL PROCEDURES

Purification and 32P Labeling of PKCdelta

PKCdelta was purified from recombinant baculovirus-infected Sf21 cells as described elsewhere (11). Briefly, 3 days after infection, Sf21 cells were lysed, and PKCdelta 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 PKCdelta fraction as judged by SDS-PAGE, and the specific activity was 380 units/mg. One unit of PKCdelta 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 [gamma -32P]ATP (Amersham Corp.).

Purified PKCdelta (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 [gamma -32P]ATP, 40 µg/ml phosphatidylserine, and 400 ng/ml TPA at 30 °C for 2 h. After the reaction, 32P-labeled PKCdelta 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 lambda EXlox cDNA expression library.

Isolation of a cDNA Clone Encoding a PKCdelta -binding Protein

Poly(A)+ RNA was isolated from NIH3T3 cells, and randomly primed cDNA was synthesized using HindIII primer adapter (Novagen). cDNA was ligated to lambda EXlox vector arm (Novagen) and packaged in phage particles using a Phage Marker System (Novagen). This library contains approximately 0.95 × 106 independent clones.

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-beta -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 PKCdelta (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-lambda ZAP II cDNA library to obtain cDNA clones encoding the full SRBC coding sequence. Plaque hybridization with a lambda ZAP II cDNA library and the isolation of positive phage clones were performed according to standard procedures.

Phosphorylation of Proteins Fixed on PVDF Membranes

PKCdelta -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 PKCdelta , 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 [gamma -32P]ATP) at room temperature for 2 h. After washing with TBS containing 1% SDS buffer, the membranes were exposed for autoradiography.

Overlay Assay Using 32P-Labeled PKCdelta as a Probe

Overlay assay was performed based on the procedure of Wolf and Sahyoun (23). PKCdelta -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 PKCdelta (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.

Phosphatidylserine Overlay Assay

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.

Overlay Assay Using MBP Fusion Proteins as Probes

cDNAs encoding mouse PKCdelta 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-PKCdelta , MCD299, MCD209, and MCD111). The proteins were purified on amylose resin (New England BioLabs) and used as probes for the overlay assay.

The protein-transferred PVDF membrane was incubated with 0.7 µM MBP-PKCdelta 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).

In Vitro Phosphorylation of GST-SRBC Protein by PKCdelta

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 PKCdelta , 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 [gamma -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).

Phosphorylation of SRBC in COS1 Cells

An expression vector for tag-SRBCDelta 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 PKCdelta 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).

Cell Culture Conditions for the Analysis of SRBC mRNA

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.


RESULTS

Cloning of SRBC

A mouse NIH3T3-lambda EXlox expression library was screened for proteins that bind to PKCdelta in the presence of phosphatidylserine. Using 32P-labeled PKCdelta 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-lambda 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-lambda EXlox expression library using PKCdelta 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.
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PKCdelta Binds to SRBC

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 PKCdelta . The overlay assay showed that PKCdelta 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 PKCdelta 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. PKCdelta 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 PKCdelta 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.
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To test whether full-length SRBC binds to PKCdelta , SRBC was expressed as a GST-fusion protein in E. coli and tested for PKCdelta binding by overlay assay. MARCKS, a known binding protein/substrate for PKC, and GST alone were also tested for PKCdelta 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). PKCdelta 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 PKCalpha instead of PKCdelta was used as a probe, essentially the same results were obtained, whereas the signal was barely detectable when PKCzeta was used as the probe (data not shown).


Fig. 3. SRBC binds to PKCdelta 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 PKCdelta 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).
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Phosphatidylserine Binds to SRBC and Clone 53 Product

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 PKCdelta 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 PKCdelta . These results suggest that the binding of SRBC and PKCdelta depends on phosphatidylserine-bridging as suggested for other PKC-binding proteins.

The Regulatory Domain of PKCdelta Binds to SRBC

Since the binding of PKCdelta to SRBC depends on the presence of phosphatidylserine, the co-factor binding site of PKCdelta 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 PKCdelta 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 PKCdelta was detected by anti-MBP antibodies. As shown in Fig. 4B, MBP-PKCdelta bound to GST-SRBC or MARCKS similarly to intact PKCdelta (Fig. 4B, lanes 1 and 2). Furthermore, MBP fused to the regulatory domain of PKCdelta (MCD299) also bound to GST-SRBC and MARCKS to the same extent (Fig. 4B, lanes 4 and 5). The regulatory domain of PKCdelta 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 PKCdelta , 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 PKCdelta raises the possibility that GST-SRBC modulates the activity of PKCdelta . However, we could not detect any effect of GST-SRBC on the myelin basic protein kinase activity of PKCdelta in vitro (data not shown).


Fig. 4. PKCdelta binds to SRBC in its regulatory domain. A, schematic drawing and Coomassie Brilliant Blue staining of purified maltose-binding protein (MBP) fused to PKCdelta (MBP-PKCdelta ) and its regulatory domain (MCD299, MCD209, and MCD111). Amino acids 299-654, 209-654, and 111-654 of PKCdelta are deleted in MCD299, MCD209, and MCD111, respectively. B, overlay assays were performed using MBP-PKCdelta (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.
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SRBC Is a Substrate for PKCdelta in Vitro

As already shown in Fig. 2, the C-terminal part of mouse SRBC, the clone 53 product, can be phosphorylated by PKCdelta in vitro. Full-length rat SRBC fused to GST is also phosphorylated by PKCdelta , whereas GST alone is barely phosphorylated (Fig. 5A). We next examined the kinetics for SRBC phosphorylation by PKCdelta in vitro. GST-SRBC (5-150 nM) was incubated with PKCdelta (0.7 nM), in the presence of phosphatidylserine, TPA, and [gamma -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 PKCalpha , with Km and Vmax values similar to those for PKCdelta , but GST-SRBC is a poor substrate for PKCzeta with a Vmax value one-eighth that for PKCdelta (data not shown). The maximum incorporation of phosphate into SRBC by PKCdelta was 0.78 mol per 1 mol of GST-SRBC.


Fig. 5. Phosphorylation of GST-SRBC by PKCdelta in vitro. A, GST-SRBC and GST, 150 nM each, were incubated in vitro with purified PKCdelta , cofactors, and [gamma -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 PKCdelta are indicated by arrowheads. Additional bands around 54 kDa are thought to represent degradation products of SRBC, which are also phosphorylated by PKCdelta .
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SRBC Is Phosphorylated Upon PKC Activation in Vivo

To monitor the phosphorylation of SRBC in vivo, we designed an epitope-tagged SRBCDelta 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 SRBCDelta 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 PKCdelta in the presence or absence of TPA. No significant effect of PKCdelta 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 PKCdelta 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 SRBCDelta 15 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 SRBCDelta 15 (5 µg) alone or together with the PKCdelta -encoded expression vector (2 µg). Cells were labeled with 32P and exposed to TPA (50 ng/ml) for 10 min before harvest. SRBCDelta 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 SRBCDelta 15 protein. DMSO, dimethyl sulfoxide.
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The Expression of SRBC mRNA Is Induced by Cell Growth Arrest

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).


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.
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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.


DISCUSSION

We cloned a cDNA encoding a PKC-binding protein by West-Western screening using 32P-labeled, autophosphorylated PKCdelta as a probe. This protein, called SRBC, binds to and is phosphorylated by PKCdelta in vitro. In COS1 cells, the phosphorylation of over-expressed SRBC is stimulated by TPA and is further enhanced by the over-expression of PKCdelta .

The Km value for the phosphorylation of SRBC by PKCdelta 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 PKCdelta . 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 PKCdelta . These features of the PKC-SRBC interaction are very similar to those for the interactions between PKC isozymes (alpha , delta , and epsilon ) and MARCKS (11).

The binding of SRBC to PKCdelta depends on the presence of phosphatidylserine; therefore, the active conformation of PKCdelta 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 PKCdelta through its bridging. Interaction with SRBC was also observed for MCD209, a deletion mutant of PKCdelta 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 PKCdelta and SRBC or not. We need further experiments to determine the SRBC binding region on the PKCdelta .

SRBC binds to PKCalpha , a cPKC family member, to the same extent as PKCdelta . And it barely binds to PKCzeta , an aPKC family member (data not shown). Thus, the binding of PKC to SRBC does not clearly show isotype specificity. We identified several PKCdelta -binding proteins in addition to SRBC by subsequent screening of the cDNA expression library using PKCdelta 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 PKCdelta -specific binding protein.3

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 PKCdelta , 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 PKCdelta . 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 PKCdelta (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.

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 PKCdelta 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 PKCdelta -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.


FOOTNOTES

*   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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D85435[GenBank].


§   Research Fellow of the Japan Society for the Promotion of Science for Young Scientists.
par    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.

Acknowledgment

We thank Dr. Hiroshi Nojima (Osaka University) for providing the rat 3Y1-lambda ZAPll cDNA library.


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