* Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka,
567-0047, Japan; and Biosignal Research Center, Kobe University, Kobe, Hyogo, 657-8501, Japan
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Abstract |
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By the yeast two-hybrid screening of a rat
brain cDNA library with the regulatory domain of protein kinase C (PKC
) as a bait, we have cloned a gene
coding for a novel PKC
-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation
protein zeta-1) consisting of 393 amino acid residues
shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the
developmental stages of mouse embryo. By the yeast
two-hybrid assay with various deletion mutants of PKC,
FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKC
and weakly with that of
PKC
. In the COS-7 cells coexpressing FEZ1 and
PKC
, FEZ1 was present mainly in the plasma membrane, associating with PKC
and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKC
. When the constitutively active mutant
of PKC
was used, FEZ1 was found in the cytoplasm of
COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the
cytoplasm to the plasma membrane, suggesting that the
cytoplasmic translocation of FEZ1 is directly regulated
by the PKC
activity. Although expression of FEZ1
alone had no effect on PC12 cells, coexpression of
FEZ1 and constitutively active PKC
stimulated the
neuronal differentiation of PC12 cells. Combined with
the recent finding that a human FEZ1 protein is able to
complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1
plays a crucial role in the axon guidance machinery in
mammals by interacting with PKC
.
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Introduction |
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PROTEIN kinase C (PKC)1 was originally isolated as a
Ca2+- and phospholipid-dependent Ser/Thr protein
kinase, exerting a wide range of physiological functions (Nishizuka, 1995). The PKC family consists of at
least 10 isoforms and commonly possesses two functional domains, a regulatory domain in the NH2-terminal half
and a catalytic domain in the COOH-terminal half. The
PKC isoforms are widely distributed in many types of
mammalian cells. Besides the conventional (
,
I,
II, and
) and the novel (
,
,
, and
) isoforms, the PKC family
also comprises two atypical isoforms,
and
/
, that are
distinguished structurally from the former isoforms by the presence of only a single PKC zinc finger motif in their
regulatory domains (Nishizuka, 1995
). PKC
, one of the
atypical isoforms, has been shown to be involved in a wide
variety of important cellular functions: maturation of
Xenopus laevis oocytes (Dominguez et al., 1992
), enhancement of the NF
B-dependent promoter activity (Folgueira et al., 1996
), activation of the mitogen-activated protein kinase (MAPK) (Berra et al., 1995
), control of
apoptosis (Diaz-Meco et al., 1996
), regulation of neuronal
differentiation (Wooten et al., 1994
), and maintenance of
the long-term potentiation in nervous systems (Sacktor et al.,
1993
). For exhibiting these cellular functions, it is essential
that PKC
interacts specifically with respective cellular
substrates. Since all PKC isoforms show a subtle difference in the substrate specificity for synthetic peptides in
the in vitro phosphorylation studies (Kazanietz et al., 1993
;
Nishikawa et al., 1997
), it is conceivable that the regulatory domain of PKC isoforms contributes to the recognition of their own cellular substrates by protein-protein interaction. Recently, several proteins interacting with the
regulatory domain of PKC isoforms have emerged to be
the determinants for subcellular localization of PKC isoforms, cellular regulators for the activity of PKC isoforms,
or cellular substrates specific for various PKC isoforms
(Mochly-Rosen, 1995
; Diaz-Meco et al., 1996
; Faux and Scott, 1996
; Jaken, 1996
; Kuroda et al., 1996
; Puls et al.,
1997
). For elucidating novel signaling pathways involving
PKC
, it is thus a prerequisite to identify the proteins interacting with the regulatory domain of PKC
.
In this study, we have identified a novel PKC-interacting protein homologous to the nematode Caenorhabditis
elegans UNC-76 protein necessary for axonal outgrowth
(Bloom and Horvitz, 1997
). We here demonstrate that the
rat cDNA-derived protein, designated FEZ1 (fasciculation and elongation protein zeta-1) (Bloom and Horvitz,
1997
), is a cellular substrate of PKC
and is translocated from the plasma membrane to the cytoplasm by activation
of PKC
. We also show that FEZ1 mRNA is abundantly
expressed in adult rat brain and throughout the developmental stages of mouse embryo. A human FEZ1 protein
has been shown to rescue the defects caused by unc-76
mutations in the nematode (Bloom and Horvitz, 1997
), indicating that both UNC-76 and FEZ1 are conserved
evolutionarily on the functional and structural bases.
Therefore, it is predicted that FEZ1 is involved in the
axon guidance machinery in mammals by interacting with
PKC
.
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Materials and Methods |
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Yeast Two-Hybrid Screening and Sequence Analysis
The yeast two-hybrid screening (Chien et al., 1991) of a rat brain cDNA library () was conducted with a yeast strain CG-1945 [MATa ura3-52 his3-200 lys2-801 trp1-901 ade2-101 leu2-3,112 gal4-542 gal80-538
LYS2::GAL1-HIS3 cyhr2 URA3::(GAL4 17-mers)3-CYC1-lacZ], a derivative of HF7c (Feilotter et al., 1994
), by using the regulatory domain of rat
PKC
(residues 1-250) (Ono et al., 1988b
) fused with the yeast GAL4
DNA-binding domain as a bait. The cDNA fragment of a positive clone
obtained was sequenced with a DNA sequencer (model 373S; /Applied Biosystems). The full-length cDNA was obtained from a
rat brain Marathon-Ready cDNA library () by the method of
rapid amplification of cDNA ends (RACE) (Frohman et al., 1988
). Prediction for the coiled-coil structure (Lupas, 1996
) was performed by software available at the web site of the Swiss Institute for Experimental Cancer Research (http://ulrec3.unil.ch/software/COILS_form.html).
Plate Assay for -Galactosidase (
-Gal) Activity in
Yeast Cells
-Gal activity in yeast cells was measured by the plate assay method.
Yeast transformants (Leu+, Trp+, His+) were transferred onto nylon
membranes, permeabilized in liquid nitrogen, and placed on Whatman
3MM papers that had been soaked in Z buffer (60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM MgCl2, 50 mM 2-mercaptoethanol, pH 7.0) containing 1 mg/ml 5-bromo-4-chloro-3-indoryl-
-D-galactoside (X-Gal). After developing at 37°C for 30 min, the yeast cells forming dark blue colonies were
classified into a strongly positive group (+++). After developing at 37°C
for 10 h, the yeast cells forming either dark blue or blue colonies were
classified into a moderately positive (++) and a weakly positive group
(+), respectively. Those forming white colonies were classified into a negative group (
). All measurements were repeated at least four times.
Northern Blot Analysis
Northern blots containing poly(A)+ RNA (~2 µg/lane) from eight tissues
of adult rats and mouse embryos in four different developmental stages
were obtained from . The amount of poly(A)+ RNA in each lane
was calibrated using the rat -actin gene. The full-length FEZ1 cDNA
fragment labeled with [
-32P]dCTP (~110 TBq/mmol) by a Ready-To-Go
DNA labeling kit () was used as a probe. Hybridization
was carried out under highly stringent conditions. The blots were autoradiographed by using a BAS-2000 bioimage analyzing system (Fuji).
Expression of Epitope-tagged Proteins in COS-7 Cells
For expression of the NH2-terminally FLAG-tagged FEZ1 protein
(FEZ1-FLAG), a pTB701-FLAG-FEZ1 plasmid was constructed by placing in frame FEZ1 cDNA 3' downstream of the FLAG epitope sequence of pTB701-FLAG (Kuroda et al., 1996). Similarly, for expression of the
NH2-terminally HA-tagged PKC
(PKC
-HA), a pTB701-HA-PKC
plasmid was constructed from pTB701-HA (Kuroda et al., 1996
). An expression plasmid for a kinase-negative mutant protein of PKC
-HA (K281M
PKC
-HA), pTB701-HA-K281M PKC
, was prepared by replacing the
ATP-binding Lys-281 residue by Met with a Quick-Change site-directed
mutagenesis kit (Stratagene Cloning Systems). An expression plasmid for
a constitutively active mutant of PKC
-HA (caPKC
-HA), pTB701-HA-caPKC
, was prepared by deleting the pseudosubstrate region from
Arg-116 to Trp-122 (Schonwasser et al., 1998
). These plasmids were transferred into COS-7 cells by electroporation using a Gene Pulser II (Bio-Rad Laboratories).
Subcellular Fractionation of COS-7 Cells
COS-7 cells (~5.0 × 107 cells) expressing FEZ1-FLAG were suspended in 1 ml of PBS and sonicated on ice for 15 s. After centrifugation at 10,000 g at 4°C for 10 min, the supernatant was collected as a cytoplasmic fraction. The pellet was resuspended in 1 ml of PBS containing 1% (vol/vol) Triton X-100 and sonicated on ice for 15 s. After centrifugation at 10,000 g at 4°C for 10 min, the supernatant was collected as a membrane fraction. Samples (10 µl) derived from ~5.0 × 105 cells were subjected to SDS-PAGE (12.5%) and analyzed by Western blotting using an anti-FLAG mAb M2 ().
In Vitro Transcription and Translation
In vitro synthesis of FEZ1-FLAG was performed with a Single Tube Protein System 2 (Novagen). In brief, the cDNA for FEZ1-FLAG was placed 3' downstream of the T7 promoter and then was transcribed with T7 RNA polymerase in the presence of dNTPs at 30°C for 15 min. The synthesized mRNA was translated in the rabbit reticulocyte lysate containing 1.5 MBq of [35S]Met (~37 TBq/mmol) at 30°C for 60 min. Samples were analyzed by SDS-PAGE (12.5%) and subsequent autoradiography.
Immunoprecipitation and Phosphorylation Assay
COS-7 cells (~5.0 × 107 cells) coexpressing FEZ1-FLAG and either
PKC-HA or K281M PKC
-HA were suspended in 500 µl of the lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM
2-mercaptoethanol, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 1%
[vol/vol] Triton X-100, pH 7.5) containing 1 tablet of the complete protease inhibitor cocktail () per 50 ml of the buffer.
After centrifugation at 10,000 g at 4°C for 10 min, the lysates (500 µl) were
incubated on ice for 1 h with 2 µg of either an anti-FLAG or anti-HA
12CA5 () mAb and then mixed with 20 µl of protein G-Sepharose 4 fast flow beads (50% slurry; ). After shaking at 4°C for 1 h, the beads were washed four times with the lysis
buffer. For Western blotting, the beads were subjected to SDS-PAGE
(12.5%). Tagged proteins were detected with either an anti-FLAG or
anti-HA mAb as a primary antibody and an alkaline phosphatase-conjugated anti-mouse IgG () as a secondary antibody. For phosphorylation assay, the beads were mixed with 25 µl of the reaction mixture containing 20 mM Tris, 10 mM MgCl2, 20 µM ATP, pH 7.5 (without
PKC activators). After addition of 3.7 KBq of [
-32P]ATP (~220 TBq/
mmol), the beads were incubated at 30°C for 30 min. Samples were analyzed by SDS-PAGE (12.5%) and subsequent autoradiography.
In Vitro Phosphorylation Assay
The glutathione-S-transferase (GST)-fused FEZ1 protein was synthesized
in Escherichia coli BL21 cells by using a pGEX6P-1 vector () and purified by a glutathione-Sepharose 4B column () according to the supplier's protocol. The phosphorylation reaction mixture (see above) (25 µl) and 50 ng of the conventional PKC isoforms (mixture of ,
I,
II, and
) purified from the rat brain (Kikkawa
et al., 1986
) were mixed with 5 µg of the purified GST-fused FEZ1 protein
and the reaction was started by addition of 3.7 KBq of [
-32P]ATP (~220
TBq/mmol). The mixture was incubated at 30°C until incorporation of
phosphate was saturated (~30 min). Samples were analyzed by SDS-PAGE (12.5%) and autoradiography, followed by measurement of radioactivities with a BAS-2000 image analyzer. Under the same conditions,
~1.8 mol of phosphate was incorporated into each mole of H1 histone, a
commonly used substrate of PKC (Kikkawa et al., 1986
; Kuroda et al.,
1996
).
Deletion Analysis
Essential regions in the PKC isoforms for interaction with FEZ1 were investigated by a yeast two-hybrid assay using the following 12 PKC-deletion mutants: -R, residues 1-336 of rat PKC
(Ono et al., 1988a
);
-R,
residues 1-340 of rat PKC
I (Ono et al., 1986
);
-R, residues 1-349 of rat
PKC
(Ono et al., 1988a
);
-R, residues 1-345 of rat PKC
(Ono et al.,
1988b
);
-R, residues 1-406 of rat PKC
(similarly,
-V1, residues 1-133;
-V1/C1, residues 1-297; and
-C1/V3, residues 134-406) (Ono et al.,
1988b
); and
-R, residues 1-250 of rat PKC
(similarly,
-V1, residues
1-113;
-V1/C1, residues 1-180; and
-C1/V3, residues 114-250).
Immunocytochemical Observation
COS-7 cells coexpressing FEZ1-FLAG and either PKC-HA, K281M
PKC
-HA, or caPKC
-HA were seeded in a 3.5-cm glass-bottom plate (MetTek Co.) at a concentration of ~5.0 × 104 cells/plate. For experiments involving the treatment with a PKC inhibitor, cells were treated
with 0.1 µM staurosporin (Wako Pure Chemical Ind., Ltd.) (Tamaoki et al.,
1986
) at 37°C for 2 h. Cells were fixed in 4% (wt/vol) paraformaldehyde at
room temperature for 30 min, and then permeabilized and blocked in the
mixture of 0.25% (vol/vol) Triton X-100, 5% (vol/vol) normal goat serum, and 5% (wt/vol) skim milk at room temperature for 30 min. The cells were
incubated at room temperature for 2 h with 1 µg/ml of either an anti-FLAG or anti-HA mAb in PBS containing 0.03% (vol/vol) Triton X-100.
Subsequently, the cells were incubated at room temperature for 30 min
with 1 µg/ml of an FITC-conjugated anti-mouse IgG (.). The fluorescence was visualized under a LSM410 confocal laser scanning microscope (). Parental COS-7 cells
showed no fluorescence with either an anti-FLAG or anti-HA mAb.
Transient Expression Assay for Neuronal Differentiation of PC12 Cells
PC12 cells (~5.0 × 105 cells) were seeded in a 10-cm plate and cultured
for 24 h in DME supplemented with 10% (vol/vol) horse serum () and 5% (vol/vol) FCS. Cells were transfected with 9 µg of pTB701-FLAG-FEZ1, 3 µg of a pTB701-HA-PKC derivative, and 1 µg of a reporter plasmid, pRc-CMV-
-Gal (Higuchi et al., 1997
) by the liposome
method (SuperFect; QIAGEN GmbH). After 72 h, cells were washed
with PBS and fixed with 1% (vol/vol) glutaraldehyde at 4°C for 5 min, followed by washing twice with PBS containing 5 mM MgCl2. Cells were
stained by incubation at 37°C for 3 h in PBS containing 20 mM
K3Fe(CN)6, 20 mM K4Fe(CN)6, 1 mM MgCl2, and 1 mg/ml X-Gal. The
-Gal-positive blue cells were scored by phase-contrast microscopy. Morphologically altered cells were judged from neurite outgrowth with a flattened shape and increased body mass, as typified previously (Higuchi et al.,
1997
).
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Results |
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cDNA Cloning of PKC-interacting Protein
Using the regulatory domain of rat PKC (residues 1-250)
fused with the yeast GAL4 DNA-binding domain as a
bait, we screened a rat brain cDNA library by the two-hybrid method in yeast. Among ~1.0 × 107 yeast transformants (Leu+, Trp+) expressing rat cDNA-derived proteins fused with the GAL4 activation domain, one positive
clone that exhibited both
-Gal activity and His+ phenotype only in the presence of the bait plasmid was obtained. Since the clone was found to harbor a 5'-terminal truncated form of a cDNA fragment upon nucleotide sequencing (data not shown), RACE was performed with another
rat brain cDNA library to obtain a full-length cDNA.
Finally, a cDNA consisting of 1662 bp and encoding a
polypeptide of 393 amino acid residues was isolated and
named temporarily as zeta-1. By computer search of the
protein sequences registered in the GenBank/EBI/DDBJ
data bank, we have come across the C. elegans UNC-76
protein reported recently (Bloom and Horvitz, 1997
),
showing significant sequence homology with the rat zeta-1
protein (identity, 31%; similarity, 63%) (Fig. 1). The nematode gene unc-76 (unc, uncoordinated) is necessary for
normal axonal bundling and elongation within fascicles in the nematode (Hedgecock et al., 1985
; Desai et al., 1988
;
McIntire et al., 1992
). A human gene coding for a protein
homologous to the nematode UNC-76 protein has also
been cloned and shown to rescue locomotory defects
caused by unc-76 mutations in the nematode (Bloom and
Horvitz, 1997
). The rat zeta-1 protein shows a 96% sequence identity with the reported human homologue of
UNC-76. Hence, these mammalian homologues of the
nematode UNC-76 protein (human and rat zeta-1 proteins) have been designated FEZ1 on the basis of the presumed roles in axonal fasciculation and elongation in
mammals (Bloom and Horvitz, 1997
).
|
Rat FEZ1 is rich in acidic residues Asp and Glu (24% of
the total residues), especially in the NH2-terminal half. Although the NH2-terminal half of UNC-76 (residues 13-
186) has been demonstrated to play as a signal for axonal
localization in the nematode (Bloom and Horvitz, 1997), it
is unknown whether the NH2-terminal half of FEZ1 possesses the similar signaling function. A computer analysis of the deduced amino acid sequence of FEZ1 has predicted that four regions (Lys-57 to Ala-85, Ala-165 to Glu-192, Ser-231 to Glu-266, and Gln-279 to Lys-307) have potentials to form amphipathic helices, which can mediate
intra- and intermolecular interactions by constituting the
coiled-coil structure (Lupas, 1996
). Alignment of rat FEZ1
with the nematode UNC-76 (Fig. 1) reveals the presence of five well conserved regions (1-5), and three (1-3) of
them approximately coincide with the regions predicted
for forming amphipathic helical structures. In addition,
FEZ1 has 4 potential sites for N-glycosylation (Asn-Xaa-Thr/Ser, where Xaa represents any amino acid except for
Pro) and 13 putative sites for phosphorylation by PKC
(Pearson and Kemp, 1991
) (Fig. 1).
Expression of FEZ1 mRNA
Northern blot analysis of eight tissues from adult rat has
shown that FEZ1 mRNA with a size of ~1700 nucleotides
(nt) is expressed abundantly and exclusively in the brain,
although faint expression of mRNA with a size of ~4000
nt is also observed in the liver (Fig. 2 A). It is interesting to
note that PKC mRNAs are also highly expressed in the
brain (Ono et al., 1988b
). The nematode UNC-76 is detected throughout the nervous system of the animals at all
developmental stages from embryos (before outgrowth
of the first axons) through adult worms (Bloom and Horvitz, 1997
). Therefore, we investigated further the FEZ1
mRNA expression during development of mouse embryo
with rat FEZ1 cDNA as a probe; the mouse FEZ1 gene
deposited in the GenBank EST (Expressed Sequence Tag)
database shows a very high sequence identity (>92%) with rat FEZ1. As shown in Fig. 2 B, a 6000-nt mRNA is expressed abundantly in the early to mid stage of development (from 7 d postcoitum [dpc] to 15 dpc). In the late
stage of development (17 dpc), the 6000-nt mRNA disappears and instead a 1700-nt mRNA is expressed abundantly. Furthermore, a 2000-nt mRNA is expressed constantly, though slightly, in all developmental stages. It has
been known that development of the nervous system in
mouse embryo begins soon after 7 dpc with the neural
plate formation and ends before 17 dpc. By cDNA cloning, all mRNAs observed here (1700-, 2000-, 4000-, and
6000-nt mRNAs) were confirmed to contain a 5'-untranslated sequence (~100 nt), the same rat/mouse FEZ1 gene
(~1200 nt), a 3'-untranslated sequence of various lengths
(from 340 to 4600 nt), and a poly (A)+ sequence (<100 nt).
|
Expression of FEZ1 in COS-7 Cells
The lysate of COS-7 cells expressing FEZ1-FLAG was separated into the cytoplasmic and membrane fractions. Western blotting with an anti-FLAG mAb indicated that FEZ1-FLAG (~55 kD) was equally present in both the cytoplasmic and membrane fractions (Fig. 3 A). The molecular mass of FEZ1-FLAG produced in COS-7 cells was ~10 kD larger than that of the protein synthesized by in vitro transcription and translation (Fig. 3 B), which agreed well with the value calculated from the deduced amino acid sequence of FEZ1 (45,207). As described above, FEZ1 contains four potential sites for N-glycosylation. In addition, FEZ1-FLAG was found to be phosphorylated by in vivo labeling of COS-7 cells with [32P]H3PO4 (data not shown), which could also be a cause for the retarded migration on SDS-PAGE. These results strongly suggest that FEZ1 undergoes posttranslational modification (N-glycosylation and/or phosphorylation) in the mammalian cells.
|
Association of FEZ1 and PKC in COS-7 Cells
To examine in vivo association of FEZ1 and PKC, the
lysates of COS-7 cells coexpressing FEZ1-FLAG and
PKC
-HA were analyzed by the immunoprecipitation assay. By Western blotting with the anti-HA mAb, PKC
-HA with an approximate Mr of 72,000 was detected in the
anti-FLAG immunoprecipitates (Fig. 4 A), indicating coprecipitation of PKC
-HA with FEZ1-FLAG. Similarly,
by Western blotting with an anti-FLAG mAb, FEZ1-FLAG with an approximate Mr of 55,000 was also detected in the anti-HA immunoprecipitates (Fig. 4 B), indicating coprecipitation of FEZ1-FLAG with PKC
-HA.
These results clearly show that FEZ1-FLAG associates
with PKC
-HA in vivo. Under the same conditions, FEZ1-FLAG did not associate with other HA-conjugated conventional and novel PKC isoforms (
,
I,
,
, and
) (data not shown). We then analyzed the anti-HA immunoprecipitates by the phosphorylation assay as described in Materials and Methods and found that FEZ1 in the FEZ1-FLAG/PKC
-HA complex could be phosphorylated (Fig.
4 C). When a kinase-negative mutant enzyme of PKC
-HA (K281M PKC
-HA) was coexpressed with FEZ1-FLAG in COS-7 cells, FEZ1-FLAG was found to be associated with K281M PKC
-HA (Fig. 4, A and B) but not
phosphorylated (Fig. 4 C). Collectively, these results indicate that FEZ1 is a novel cellular substrate of PKC
and
its association with PKC
is independent of the activity of
PKC
. On the other hand, when GST-FEZ1 expressed in
bacterial cells was incubated with the conventional PKC
isoforms (mixture of
,
I,
II, and
) purified from the
rat brain in the presence of [
-32P]ATP, ~0.4 mol of phosphate was incorporated per mole of GST-FEZ1. The GST
protein alone could not be phosphorylated under the same conditions (Kuroda et al., 1996
). This result shows that
FEZ1 can also be phosphorylated in vitro by the conventional PKC isoforms. The discrepancy between in vivo and
in vitro phosphorylation assays is discussed later (see Discussion).
|
Mapping of Regions Involved in the
FEZ1/PKC Association
To investigate the specificity of FEZ1 for PKC isoforms
and also to identify the region(s) in the PKC molecules involved in the association with FEZ1, the regulatory domains of various PKC isoforms were used as a bait in the
yeast two-hybrid assay. As shown in Fig. 5, FEZ1 interacted strongly with the regulatory domain of PKC (
-R,
residues 1-250) and moderately with that of PKC
(
-R,
residues 1-406) but not with those of other PKC isoforms.
Based on the sequence comparison of PKC isoforms, the regulatory domains of PKC
and PKC
are further divided
into a conserved region (C1) and two variable regions (V1
and V3) with a considerable sequence diversity (Nishizuka, 1988
). Therefore, various deletion mutants containing a part(s) of these regions of PKC
and PKC
were then
constructed to map the region interacting with FEZ1. The
yeast two-hybrid assays indicated that the V1 regions of
PKC
and PKC
were essential for interaction with FEZ1
(Fig. 5). However, in the case of full-length PKC isoforms,
only PKC
interacted with FEZ1 in the two-hybrid assay
(data not shown), indicating that PKC
but not PKC
could form a stable complex with FEZ1.
|
Cytoplasmic Translocation of FEZ1 in Response to the
PKC Activity
A confocal laser scanning microscope was used to observe
the intracellular localization of FEZ1 expressed in COS-7
cells. When the cells coexpressing FEZ1-FLAG and
PKC-HA were examined, the plasma membrane was
stained strongly with an anti-FLAG mAb and its cytoplasmic peripheries were also stained weakly (Fig. 6, panel 1).
This indicates that FEZ1-FLAG is present predominantly in the plasma membrane of COS-7 cells, in which the expressed PKC
is usually in an inactive form. On the other
hand, in the cells expressing caPKC
-HA (constitutively
active mutant) (Schonwasser et al., 1998
) instead of PKC
-HA, FEZ1-FLAG was detected uniformly in the cytoplasm (Fig. 6, panel 3). In the cells expressing K281M PKC
-HA (kinase-negative mutant), however, FEZ1-FLAG was again localized mostly in the plasma membrane (Fig. 6, panel 5). When these cells were treated with
staurosporin, a PKC inhibitor common to all isoforms
(Tamaoki et al., 1986
), FEZ1-FLAG was present in the
plasma membrane (Fig. 6, panels 2, 4, and 6). Particularly, it should be noted that FEZ1-FLAG present in the cytoplasm of the caPKC
-HA-expressing cells was dynamically translocated into the plasma membrane by the
staurosporin treatment (Fig. 6, panel 4). These results
demonstrate that the translocation of FEZ1 from the
plasma membrane, where it is normally localized, to the
cytoplasm is regulated directly by the PKC
activity.
|
Expression of FEZ1 Protein Stimulates the
Constitutively Active PKC-induced Neuronal
Differentiation of PC12 Cells
PC12 cells are known to differentiate into neuron-like
cells in response to NGF or ectopic expression of an activated form of either Ras or other signaling molecules
(Raf, MAPK kinase, MAPK) (Marshall, 1995). Because
PKC
is involved in the MAPK activation (Berra et al.,
1995
) and activation of PKC
is required for NGF-induced neuronal differentiation of PC12 cells (Wooten et al.,
1994
; Zhou et al., 1997
), we first examined the effects of
PKC
on morphology of PC12 cells by the transient expression assays. When PC12 cells were transfected with
pTB701-HA-caPKC
and the reporter plasmid pRc-CMV-
-Gal in a ratio of 9:1 (wt/wt), morphological changes (including extension of neurites, enlargement of cell mass,
and flattened shapes) were induced in ~18% of
-Gal-
positive cells (Table I). No apparent morphological
changes were observed in PC12 cells transfected with
pTB701-HA-PKC
and pRc-CMV-
-Gal. Under the same
conditions, expression of the oncogenically activated form
of Ras (RasG12V) significantly stimulated neuronal differentiation in ~71% of transfected PC12 cells. These
results suggest that caPKC
can induce neuronal differentiation of PC12 cells, though weakly, presumably by activation of MAPK. On the other hand, expression of FEZ1
alone had no effect on PC12 cells.
|
Since no FEZ1 protein was detected in PC12 cells by
Western blotting using a polyclonal anti-FEZ1 mAb (data
not shown), we next asked whether expression of FEZ1
would affect caPKC-induced neuronal differentiation of
PC12 cells. Cells were cotransfected with pTB701-FLAG-FEZ1, pTB701-HA-caPKC
, and pRc-CMV-
-Gal in a
ratio of 9:3:1 (wt/wt/wt) and the number of morphologically changed cells in
-Gal-positive cells was scored. The
percentage of differentiated cells was increased (~48%),
as compared with that (~18%) observed in the case of
expression of caPKC
alone (Table I). When PC12 cells
were transfected with pTB701-FLAG-FEZ1, pTB701-HA-PKC
, and pRc-CMV-
-Gal, there were no apparent
morphological changes. These results strongly suggest that
FEZ1 is located downstream of PKC
and stimulates the
caPKC
-induced neuronal differentiation of PC12 cells.
![]() |
Discussion |
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Predicted Role of UNC-76 in the C. elegans Axon Guidance Machinery
In the developing nervous systems, axons outgrow along
other axonal cell surfaces to reach their targets, and then
most axons associate with other axons in specific fascicles.
This axonal association in fascicles is crucial for the assembly of nervous systems (Jessell, 1988; Grenningloh and
Goodman, 1992
). By genetic analysis of the C. elegans unc
mutants with locomotory defects, the axonal elongation in
fascicles has been shown to require at least two groups of
genes (Hedgecock et al., 1985
; Desai et al., 1988
; McIntire
et al., 1992
). One group of genes (unc-14, unc-33, unc-44,
unc-51, and unc-73) is required for the axonal elongation along nonneuronal as well as neuronal cell surfaces, and is
likely to encode molecules essentially required for the axonal elongation. The other group of genes (unc-34, unc-71,
and unc-76) is necessary for the axonal elongation in fascicles but not for the axonal elongation along nonneuronal
cell surfaces, suggesting that the products of these genes
are involved in the interaction of axons with the neuronal
cell surfaces. The C. elegans unc-76 mutants show two
types of axonal defects; the axons in fascicles often do not
reach their full lengths and fail to bundle tightly together. Nonetheless, the axons around the body wall elongate normally, which are not accompanied by other axons, showing
that UNC-76 is required only for the recognition of adjacent neuronal cells (Hedgecock et al., 1985
; Desai et al.,
1988
; McIntire et al., 1992
; Bloom and Horvitz, 1997
).
Many molecules related to the axon guidance are conserved among mammals and the nematode in both the
structural and functional levels: netrin (Serafini et al.,
1994), a mammalian homologue of UNC-6; CRMP-62
(Goshima et al., 1995
), that of UNC-33; and transforming
growth factor-
(Colavita et al., 1998
), that of UNC-129.
FEZ1 protein identified here as a PKC
-interacting protein was shown recently to rescue the locomotory defects
caused by unc-76 mutations (Bloom and Horvitz, 1997
),
indicating that both FEZ1 and UNC-76 are also conserved
evolutionarily. In the nematode, the axon guidance machinery involves several signaling molecules, for instance,
UNC-33 protein in the heterotrimeric GTP-binding protein cascade (Goshima et al., 1995
), Dock protein in the
Tyr kinase cascade (Garrity et al., 1996
), and UNC-51 and
UNC-14 proteins in the Ser/Thr kinase cascade (Ogura et al.,
1994
, 1997
). In addition, various PKC isoforms similar to
the mammalian PKC isoforms have been identified in the
nematode (Land et al., 1994a
,b; Sano et al., 1995
; Islas-Trejo et al., 1997
). Thus, it is likely that both mammals and
the nematode share a common machinery for axon guidance using similar signaling molecules. To date, however, there has been no report describing the relationship between the nematode PKCs and the molecules involved in
axon guidance such as UNC-76.
FEZ1 Protein as a Cellular Substrate for PKC
The findings presented herein demonstrate that FEZ1
protein, a mammalian homologue of the nematode UNC-76 protein, is a novel cellular substrate for PKC. Northern blot analysis has shown that FEZ1 mRNA is abundantly expressed in rat brain (see Fig. 2 A). PKC
mRNA
is also highly expressed in rat brain (Ono et al., 1988b
). In
mammalian cells including the neuronal cells, endogenous
PKC
is localized evenly in the cytoplasm and the plasma membrane (Wooten et al., 1994
; Goodnight et al., 1995
;
Parrow et al., 1995
). Therefore, it is likely that FEZ1 and
PKC
coexist in the cytoplasmic periphery of the plasma
membrane and the activation of PKC
induces the cytoplasmic translocation of FEZ1 in the mammalian neuronal
cells (Fig. 6). Various FEZ1 mRNAs of different sizes are
expressed throughout the developmental stages of mouse
embryo (Fig. 2 B), whereas all mRNAs observed encode
the same FEZ1 gene. Thus, we assume that these FEZ1
mRNAs may be expressed in different cell types and/or
may possess distinct in vivo stabilities. The 6000-nt FEZ1
mRNA coordinately expressed in embryos during 7-15 dpc is suggested to play an important role in the development of the mouse nervous system, whereas the 1700-nt
FEZ1 mRNA likely bears a continual role in the neuronal
tissues already developed. However, further studies by in
situ hybridization analysis of the mouse embryos and construction of the FEZ1 gene-deficient mice are needed to
clarify the role of FEZ1 protein during development.
A mixture of conventional PKC isoforms (PKC,
I,
II, and
) could phosphorylate FEZ1 in vitro, although
these PKC isoforms did not form a stable complex with
FEZ1 in vivo (Fig. 4). Recently, many proteins interacting
with the regulatory domain of PKC isoforms were shown
to regulate the in vivo PKC functions (Mochly-Rosen, 1995
; Diaz-Meco et al., 1996
; Faux and Scott, 1996
; Jaken,
1996
; Kuroda et al., 1996
; Puls et al., 1997
). Therefore, it is
suggested that the absence of an unidentified cellular protein(s) determining the specificity of PKC isoforms to
FEZ1 may cause the in vitro phosphorylation of FEZ1 by
conventional PKC isoforms. Attempts to identify the cellular factor(s) interacting with the PKC
/FEZ1 complex
are underway. Alternatively, in the phosphorylation by PKC, the substrate protein may not necessarily form a stable complex with PKC. Indeed, H1 histone and myelin basic protein, both of which are good substrates for PKC and
are often used in the in vitro phosphorylation assay, could
not form a stable complex with PKC in vivo (data not shown).
By the yeast two-hybrid assay, the NH2-terminal variable region (V1) of PKC was shown to interact with
FEZ1 protein (Fig. 5). Since the V1 region of all PKC isoforms shows a considerable sequence diversity (Nishizuka,
1988
), involvement of this region in the association with
FEZ1 appears reasonable for displaying specific protein-
protein interactions and is consistent with our recent finding that several PKC-interacting proteins containing the
LIM domain recognize the similar region of PKC isoforms
(Kuroda et al., 1996
). As for the region(s) in FEZ1 protein
involved in the association with PKC
, the COOH-terminal half of FEZ1 (residues 185-393) likely participates in
the association with the regulatory domain of PKC
, because the clone containing a 5'-terminal truncated form of
cDNA has been isolated in the first two-hybrid screening. Furthermore, the sequence homology between the COOH-
terminal halves of FEZ1 and nematode UNC-76 proteins
is higher than that between their NH2-terminal halves (see
Fig. 1). Also, the COOH-terminal truncated forms of
UNC-76 were observed in most of the nematode mutant
alleles that failed to complement unc-76 (Bloom and Horvitz, 1997
). Although further analyses with various deletion mutants of FEZ1 are needed to map the PKC
-interacting region in more detail, it is suggested that interaction
of the COOH-terminal half of FEZ1 with the regulatory
domain of PKC
is essential for the predicted cellular
function(s) of FEZ1, as discussed below.
Possible Function of FEZ1 in the Neuronal Differentiation of PC12 Cells
In this study, we have shown that FEZ1 interacts with
PKC in vivo and its intracellular localization is regulated
by the PKC
activity. By the transient expression assay,
FEZ1 protein was found to stimulate the caPKC
-induced
neuronal differentiation of PC12 cells. On the other hand,
in the NGF-induced neuronal differentiation of PC12
cells, a PKC
substrate "nucleolin" was proposed recently
to play a pivotal role in the connection between cell surface signaling and nucleus in PC12 cells (Zhou et al., 1997
). Although it has been shown that the intracellular
localization of nucleolin is changed by the activation of
PKC
in the NGF-induced neuronal differentiating PC12
cells, there is no structural similarity between nucleolin
and FEZ1. Furthermore, no direct evidence is available
showing that nucleolin enhances the caPKC
-induced neuronal differentiation of PC12 cells. Thus, it is unclear
whether or not there is any functional relationship between nucleolin and FEZ1.
Rho family GTP-binding proteins, which work downstream of the PKC activation (Tominaga et al., 1993), have
also been shown to regulate the axon guidance in mammals and the nematode (Kozma et al., 1997
; Luo et al.,
1997
; Zipkin et al., 1997
). To examine the effects of Rho
family proteins on the intracellular localization of FEZ1
protein (Fig. 6), we have coexpressed each of the constitutively active Rho family mutants (RhoA G14V, Rac1
G12V, and Cdc42Hs G12V/Q61L) with PKC
-HA and
FEZ1-FLAG in COS-7 cells. In all the cells examined, the
localization of FEZ1 protein in the plasma membrane was
unaffected by the ectopic expression of these Rho family
mutants (data not shown), strongly suggesting that FEZ1
protein is not located downstream of the Rho family proteins but may be rather located upstream. As shown in Fig.
1, most of the FEZ1 protein is predicted to form coiled-
coil structures (at least four amphipathic helices). These helices are considered to form a four-helix bundle, where
the four helices are packed together by intramolecular interactions (Kohn et al., 1997
), and the amphipathic property of these helices may contribute to the membrane localization of FEZ1 protein. More importantly, it has been
demonstrated that the amphipathic helices (coiled-coil
structures) often participate in the association with RhoA
proteins, e.g., p116Rip (Gebbink et al., 1997
), citron (Madaule et al., 1995
), p160 ROCK (Ishizaki et al., 1996
), and
Rho-kinase (Leung et al., 1995
). Thus, it is likely that the
amphipathic helices of FEZ1 protein may interact with
RhoA family proteins.
Concluding Remarks
Taken together, we propose that FEZ1 transduces the
signals from unidentified receptors for neuronal cells to
the axon guidance machinery by interacting with PKC
through the following presumed pathway. First, cell-surface receptors receive the signals from adjacent neuronal
cells. Second, receptors evoke an unknown molecule(s) that activates PKC
. Third, FEZ1 proteins phosphorylated
by PKC
are translocated from the plasma membrane to
the cytoplasm. Fourth, FEZ1 proteins in the cytoplasm
transduce the signals to the intracellular machinery involving the Rho family GTP-binding proteins, which will finally induce axonal growth in fascicles. We expect that the
FEZ1-PKC
interaction demonstrated herein will be an
important clue for elucidation of the signaling pathway
and molecules involved in the axon guidance machinery in mammals.
![]() |
Footnotes |
---|
Address correspondence to S. Kuroda, Department of Structural Molecular Biology, Institute for Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan. Tel.: 81-6-6879-8462. Fax: 81-6-6879-8464. E-mail: skuroda{at}sanken.osaka-u.ac.jp
Received for publication 24 September 1998 and in revised form 4 January 1999.
We thank Dr. K. Mizuno for providing the reporter plasmid pRc-CMV--Gal. We also thank Drs. L. Bloom and H.R. Horvitz for sharing unpublished data. We are also grateful to Drs. U. Kikkawa and N. Saito for encouragement and helpful advice.
This study was supported in part by a Grant-in-Aid for Scientific Research (No. 09780576) from the Ministry of Education, Science, Sports, and Culture of Japan.
![]() |
Abbreviations used in this paper |
---|
-Gal,
-galactosidase;
caPKC
-HA, a
constitutively active mutant of PKC
-HA;
dpc, days postcoitum;
FEZ1, fasciculation and elongation protein zeta-1;
FEZ1-FLAG, NH2-terminally
FLAG-tagged FEZ1 protein;
GST, glutathione-S-transferase;
K281M
PKC
-HA, a kinase-negative mutant protein of PKC
-HA;
MAPK, mitogen-activated protein kinase;
nt, nucleotides;
PKC, protein kinase C;
PKC
-HA, NH2-terminally HA-tagged PKC
;
RACE, rapid amplification
of cDNA ends;
X-Gal, 5-bromo-4-chloro-3-indoryl-
-D-galactoside.
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