From the Laboratory for Developmental Neurobiology,
Brain Science Institute, RIKEN (the Institute of Physical and Chemical
Research), 2-1 Hirosawa, Wako, Saitama 351-0198 and the ¶ Division
of Molecular Neurobiology, Department of Basic Medical Science, The
Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received for publication, March 8, 2001, and in revised form, April 16, 2001
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ABSTRACT |
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The C2 domain was originally
defined as a homologous domain to the C2 regulatory region of
Ca2+-dependent protein kinase C and has
been identified in more than 50 different signaling molecules. The
original C2 domain of protein kinase C The C2 domain is a Ca2+ binding motif that
consists of ~130 amino acids, and it has been identified in various
signaling molecules including protein kinases, lipid modification
enzymes, GTPase-activating proteins, ubiquitination enzymes, and
proteins involved in vesicular trafficking (reviewed in Refs. 1 and 2).
The C2 domain was originally defined as a homologous domain to the C2
regulatory region of mammalian Ca2+-dependent
protein kinase C isoforms The role of the C2 domain is not limited to the phospholipid membrane
interaction sites and has been shown to be a
Ca2+-dependent and -independent protein
interaction site. For instance, the synaptotagmin I (Syt
I)1 C2 domain, one of the
best characterized C2 domains essential for neurotransmitter release
(reviewed in Refs. 6-9), has been shown to interact with negatively
charged phospholipids (10-13), syntaxin (14), and Syt I itself in a
Ca2+-dependent manner (15-20). In addition,
the Syt I C2B domain binds inositol polyphosphates (21, 22), clathrin
assembly protein, AP-2 (23), SV2 (24), In this paper, we report the discovery of an unexpected function
of the C2A domain of a third isoform of double C2 protein that contains
a C2A domain and a C2B domain (Doc2 Plasmid Construction--
pEF-T7-Doc2 Site-directed Mutagenesis of Doc2 Cell Culture, Transfections, and
Immunocytochemistry--
Transfection of pEF-T7-Doc2 into PC12 cells
(0.5-1 × 105 cells, the day before
transfection/35-mm dish; MatTek Corp., Ashland, MA) or into
COS-7 cells (5 × 105 cells, the day before
transfection/10-cm dish) was performed as described previously (19, 35,
37). After washing twice with phosphate-buffered saline, the PC12 cells
were fixed, incubated with anti-T7 tag mouse monoclonal antibody
(1/5000 dilution; Novagen, Madison, WI) and anti-p300 rabbit polyclonal
antibody (1/500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA),
and then visualized with anti-mouse Alexa 488 and anti-rabbit Alexa 568 antibodies (1/5000 dilution; Molecular Probes, Eugene, OR),
respectively. In some cases, Vectashield mounting medium with DAPI
(Vector Laboratories, Burlingame, CA) was added after immunostaining
with anti-T7 tag antibody. Immunoreactivity was analyzed with a
fluorescence microscope (TE300; Nikon, Tokyo, Japan) attached to a
laser confocal scanner unit CSU 10 (Yokogawa Electric Corp., Tokyo,
Japan) and HiSCA CCD camera (C6790; Hamamatsu Photonics, Hamamatsu,
Japan). Images were pseudo-colored and superimposed with Adobe
PhotoShop software (Version 4.0).
Phospholipid Binding Assay--
Glutathione
S-transferase (GST) fusion proteins were expressed and
purified on glutathione-Sepharose (Amersham Pharmacia
Biotech) by the standard method (38). Preparation of
liposomes consisting of L- Nuclear Localization of Doc2 Mapping of the Domain Responsible for the Nuclear Localization of
Doc2 The Doc2
To determine whether the basic cluster of the Doc2 Conclusions--
This study revealed the novel function of the
Ca2+-independent type of the Doc2 functions as a
Ca2+ binding module, and the Ca2+ binding to
the C2 domain allows translocation of proteins to phospholipid
membranes. By contrast, however, some C2 domains do not exhibit
Ca2+ binding activity because of amino acid substitutions
at Ca2+-binding sites, and their physiological meanings
remain largely unknown. In this study, we discovered an unexpected
function of the Ca2+-independent C2A domain of double C2
protein
(Doc2
) in nuclear localization. Deletion and mutation
analyses revealed that the putative Ca2+ binding loop 3 of
Doc2
contains six Arg residues
(177RLRRRRR183) and that this basic
cluster is both necessary and sufficient for nuclear localization of
Doc2
. Because of the presence of the basic cluster, the C2A domain
of Doc2
did not show Ca2+-dependent
phospholipid binding activity. Our findings indicate that by changing
the nature of the putative Ca2+ binding loops the C2 domain
has more diversified function in cellular signaling than a simple
Ca2+ binding motif.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
, and
(reviewed in Ref. 3). The
C2 domains are composed of a common eight-stranded antiparallel
-sandwich consisting of four-stranded
-sheets, although their
structures have been classified into two groups based on their topology
(e.g. synaptotagmin I C2A domain with type I
topology and phospholipase C
1 C2 domain with type II topology) (2,
4, 5). Three flexible loops protrude from the tip of the
-sandwich
structure, and some of them are involved in Ca2+ binding
(4, 5). The Ca2+ binding allows interaction of the C2
domain with phospholipids to enable translocation of proteins to
phospholipid membranes (2).
-SNAP (25), SNAP25
(26), Ca2+ channels (27), and SYNCRIP
(Syt-binding, cytoplasmic
RNA-interacting protein) (28),
irrespective of the presence of Ca2+. Furthermore, some Syt
isoforms fail to exhibit Ca2+ binding because of amino acid
substitutions (mutation of the Glu or Asp residue involved in
Ca2+ binding) in the putative Ca2+ binding
loops (14, 29). However, the function of the
Ca2+-independent type of C2 domains remains largely unknown.
; see Fig. 1A) in
nuclear localization (30, 31). Unlike other members of the Doc2
family, the C2A domain of Doc2
lacks
Ca2+-dependent phospholipid binding activity,
probably because of the amino acid substitutions of the key amino acids
(Glu or Asp) responsible for Ca2+ binding (30, 32-34).
Interestingly, six Arg residues are clustered at one of the putative
Ca2+ binding loops in the Doc2
C2A domain (see Fig. 3,
#). Our deletion and mutation analyses indicate that these basic
residues are essential for nuclear localization of Doc2
instead of
Ca2+ binding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, -Doc2
C2AB
(amino acid residues 1-80), -Doc2
C2B (amino acid residues
1-217), -Doc2
-C2A (amino acid residues 80-217), Doc2
-C2B (amino
acid residues 234-388), and T7-Doc2
(amino acid residues 1-418)
(32) were constructed by polymerase chain reaction (PCR) using the
following sets of primers with appropriate restriction enzyme
sites (underlined) and/or termination codons (bold letters), as
described previously (19, 35):
5'-CGGATCCATGGCATGTGCAGGGCCAGCC-3' (Met primer; sense),
5'-GCACTAGTCAGTCATCCGAGTCTCCTTC-3' (
C2AB primer; antisense),
5'-GCGGATCCGACAGCACTGCCCTAGGCAC-3' (C2A upper primer;
sense),
5'-GCACTAGTCACCTCTTGGTCAGCTTCCGCT-3' (C2A lower primer; antisense),
5'-GCGGATCCGAGGTGGAGGCAGAGGTGTT-3' (C2B upper primer;
sense),
5'-GCTGACTAGTCACCAAGTT-3' (C1
primer; antisense),
5'-GCGGATCCATGACCCTCCGGCGGCGCGGGGAGAAGGCGACCATCAGCA-3' (Doc2
-Met primer; sense), and
5'-GCACTAGTCAGTCGCTGAGTACAGC-3' (Doc2
-stop primer; antisense). Briefly, purified PCR products digested with BamHI and SpeI were subcloned into
the BamHI/SpeI site of a modified pEF-BOS vector
with a T7 tag (19, 35, 36) and verified by DNA sequencing with a
Hitachi SQ-5500 DNA sequencer. Plasmid DNA was prepared by using
Wizard® minipreps (Promega, Madison, WI) or Maxi prep kits (Qiagen,
Chatsworth, CA).
and Doc2
--
A mutant
Doc2
R (deletion of amino acids 180-183 (four Arg residues) in
the C2A domain) was essentially produced by means of two-step PCR
techniques, as described previously (21), using the following pairs of
oligonucleotides: Met primer and
5'-GGGGGGCCCCCGCAGCCGTGAGTCCTC-3' (
R-5' primer;
antisense) (left half); and 5'-CGGGGGCCCCCCCTGGGGGAGCTA-3' (
R-3' primer; sense) and C1 primer (right half). Briefly, the right
and left halves were separately amplified by using pGEM-T-Doc2
(30)
as a template, and the two resulting PCR fragments were digested with
ApaI (underlined above), ligated to each other, and
reamplified with the Met and C1 primers. The PCR fragment obtained that
encoded the mutant Doc2
R was digested with
BamHI/SpeI, inserted into the
BamHI/SpeI site of the pEF-T7 tag vector (19, 35), and verified by DNA sequencing. A mutant Doc2
(R6) was similarly constructed by using the following mutagenic
oligonucleotides: 5'-GGGCCCTCGCCGCCGGCGCCGCAGCCGTGACTCATCACACACGGAGAT-3'
(Doc2
(R6)-5' primer; antisense) and
5'-GGGCCCCCCATTGGAGAGACTCGGGTGCCC-3' (Doc2
(R6)-3' primer; antisense).
-phosphatidylcholine (PC),
dipalmitoyl, and L-
-phosphatidylserine (PS), dioleoyl
(1:1 w/w), and a phospholipid binding assay were performed as described
previously (13, 33). Proteins bound to the PS/PC liposomes were
analyzed by 10% SDS-polyacrylamide gel electrophoresis and then
stained with Coomassie Brilliant Blue R-250. The protein concentrations
were determined with a Bio-Rad protein assay kit (Bio-Rad) by using
bovine serum albumin for reference.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Proteins in PC12 Cells--
The
Doc2 family consists of three isoforms (
,
, and
) in rats and
mice (30, 32, 34, 39) and shares a highly conserved amino-terminal Munc13-1 interacting
domain (Mid domain; amino acid residues 13-37 of Doc2
) (40) and two
C2 domains at the carboxyl terminus (the C2A domain and the C2B domain)
(Fig. 1A). Although this
carboxyl-terminal tandem C2 domain structure is also found in the
synaptotagmin family and rabphilin-3A, the Doc2 family is distinguished
from other tandem C2 protein families in possessing a Mid domain at
their amino terminus (6, 40). Doc2
is specifically expressed in
neuronal cells (34, 41), whereas Doc2
and Doc2
are expressed
ubiquitously (30, 32, 39). Both Doc2
and Doc2
have been shown to
be associated with synaptic vesicle fractions in the brain (34, 41),
but the subcellular localization of Doc2
has yet to be determined.
To address this we expressed T7-tagged Doc2
proteins in PC12 cells. To our surprise the Doc2
proteins were almost exclusively localized in the nucleus and overlapped well with p300 transcription factor and
DAPI (Fig. 1B, top panels, and data not shown).
The Doc2
proteins seemed to be uniformly present throughout the
nucleoplasm. By contrast, Doc2
proteins are mainly present in the
cytosol, the same as Doc2
proteins (Fig. 1B, bottom
panels) (42).
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Fig. 1.
Differential expression of mouse
Doc2 and Doc2
in PC12
cells. A, schematic representation of mouse
Doc2
, Doc2
, and Doc2
. The amino acid identity of each domain
of the Doc2 family are indicated by percentages. The Mid domains and
two C2 domains are represented by hatched and shaded
boxes, respectively. Amino acid numbers are given on
both sides. B, subcellular localization of
T7-Doc2
(top panels) and Doc2
(bottom
panels) in PC12 cells. PC12 cells expressing T7-Doc2 proteins were
fixed, permeabilized, and co-stained with anti-T7 tag antibody
(green in left panels) and anti-p300
(red in middle panels), as described under
"Experimental Procedures." The right panels represent
an overlay (in yellow) of left and
middle panels. Note that Doc2
proteins were localized in
nucleus, whereas the Doc2
proteins were localized in the cytosol.
Scale bar indicates 10 µm.
Proteins--
To determine which domain is essential for the
nuclear localization of Doc2
, we produced four deletion mutants,
each of which involves a different domain of Doc2
(Doc2
C2AB,
Doc2
C2B, Doc2
-C2A, and Doc2
-C2B; see Fig.
2A). First, we checked the
size of the mutants by immunoblotting and confirmed that they were
expressed correctly, with no degradation (Fig. 2B). Each
deletion mutant was then expressed in PC12 cells, and its subcellular
localization was determined by immunocytochemistry, as described above
(Fig. 2C). Interestingly, both the Doc2
C2B and
Doc2
-C2A proteins showed nuclear localization in PC12 cells, whereas
the amino-terminal Mid domain was localized in the cytosol, and the
Doc2
-C2B protein was localized in both the nucleus and the cytosol.
We therefore concluded that only the C2A domain contains a functional
nuclear localization signal.
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Fig. 2.
Mapping of the domain responsible for the
nuclear localization of Doc2 .
A, schematic representation of deletion mutants of Doc2
.
The T7 tag, Mid domain, and two C2 domains are represented by
black, hatched, and shaded boxes,
respectively. Systematic deletions were made from the amino or carboxyl
terminus. The nuclear localization of each mutant is indicated after
its name and was determined on the basis of the results shown in
C. ± means that the Doc2
-C2B proteins were localized
both in nucleus and cytoplasm. Amino acid numbers are given
on both sides. B, expression of T7-Doc2
deletion mutants. Total homogenates of COS-7 cells expressing
T7-Doc2
proteins were loaded on a 12.5% SDS-polyacrylamide gel, and
immunoblotting with horseradish peroxidase-conjugated anti-T7 tag
antibody was performed as described previously (35). The positions of
the molecular weight markers (× 10
3) are shown on
the left. Lane 1, T7-Doc2
; lane 2,
Doc2
C2AB; lane 3, Doc2
C2B; lane 4,
Doc2
-C2A; lane 5, Doc2
-C2B; and lane 6,
Doc2
R. C, subcellular localization of T7-Doc2
deletion mutants. PC12 cells expressing pEF-T7-Doc2
deletion mutants
were fixed, permeabilized, and stained with anti-T7 tag antibody. Note
that the C2A domain of Doc2
alone is sufficient for nuclear
localization. Scale bar indicates 10 µm.
C2A Domain Contains a Functional Nuclear Localization
Signal--
Various nuclear localization signals have been determined
in many proteins localized in nucleus, and they have often consisted of
clusters of basic residues (Arg and Lys; reviewed in Ref. 43). Consistent with this, we found that the Doc2
-C2A domain contains a
cluster of basic residues
(177RLRRRRR183) in the putative
Ca2+ binding loop 3, between the
6 and
7 strands
(Fig. 3, #) (44). Interestingly, the loop
3 domain of the Doc2
C2A domain is three amino acids longer than in
other carboxyl-terminal type (C-type) tandem C2 protein families,
including Syts I-XIII (9, 29), Slp1-3
(synaptotagmin-like protein) (45),
granuphilin-a (46), rabphilin-3A (47), and other members of the Doc2
family (31). It is also noteworthy that other C-type tandem C2 domains
do not contain an Arg cluster at this position (Fig. 3). Consistent
with this, there have been no reports of tandem C2 proteins that
specifically localized in nucleus. Although three Asp residues between
the
6 and
7 strands in the C2A domain of Syt I
(asterisks in Fig. 3) are known to bind Ca2+
ions (48), the C2A domain of Doc2
lacks two Asp residues (Ser-176 and Pro-185), and because of these amino acid substitutions, the Doc2
C2A domain does not display any clear
Ca2+-dependent phospholipid (PS/PC liposome)
binding activity (Fig. 4B)
(30).
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Fig. 3.
Alignment of the putative Ca2+
binding loop 3 of the two C2 domains of the mouse C-type tandem C2
protein family. Residues, half of whose sequences were conserved
or were similar, are shown on a black background and
shaded background, respectively. Asterisks
indicate the conserved Asp or Glu residues, which may be crucial for
Ca2+ binding by analogy with the Syt I-C2A domain (44, 48).
The number signs (#) indicate the basic (six Arg) residues that are
only conserved in the C2A domain of Doc2 . The location of the
-strands is indicated by arrows (44, 48). Amino acid
numbers are indicated on the right. The amino
acid sequences of the mouse C-type tandem C2 proteins were from Syt I,
Syt II, and rabphilin-3A (12), Syts III and IV (21), Syts V-XI (35),
Syt XIII (29), granuphilin-a (46), Doc2
(53), Doc2
(32), Doc2
(30), Slp1-3 (45), and Syt B/K and Syt
XII.2
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Fig. 4.
A basic cluster in the
Doc2 C2A domain is essential for nuclear
localization signal. A, schematic representation
of Doc2
deletion mutant (Doc2
R) and chimera between Doc2
and Doc2
(Doc2
(R6)). Doc2
R lacks four Arg residues
(dashes) between the
6 and
7 strands of the C2A
domain. Doc2
(R6) contains basic residues of Doc2
(SRLRRRRRGPP;
underlined) between the
6 and
7 strands of the C2A
domain. B, Ca2+-dependent
phospholipid binding properties of the Doc2
-C2A domain. PS/PC
liposomes and GST fusion proteins were incubated in 50 mM
HEPES-KOH, pH 7.2, in the presence of 2 mM EGTA or 1 mM Ca2+ for 15 min at room temperature. After
centrifugation at 12,000 × g for 10 min, the
supernatants (S; non-binding fraction) and pellets
(P; phospholipid binding fraction) were separated as
described previously (13, 33). Equal proportions of the supernatants
and pellets were subjected to 10% SDS-polyacrylamide gel
electrophoresis and then stained with Coomassie Brilliant Blue R-250.
Note that GST-Doc2
(R6)-C2A completely lost phospholipid binding
activity. The results shown are representative of three independent
experiments. C, subcellular localization of T7-Doc2
R
and T7-Doc2
(R6). PC12 cells expressing pEF-T7-Doc2
and Doc2
mutants were fixed, permeabilized, and co-stained with anti-T7 tag
antibody (green in left panels) and anti-p300
(red in middle panels) as described under
"Experimental Procedures." The right panels
represent an overlay (in yellow) of left and
middle panels. Note that the insertion of SRLRRRRRGPP
sequence into Doc2
is sufficient for nuclear localization.
Scale bar indicates 10 µm.
C2A domain is the
sole nuclear localization signal of this protein, we produced a
deletion mutant lacking four of six Arg residues (named Doc2
R;
see Fig. 4A). As expected, the Doc2
R proteins were mainly localized in the cytosol of PC12 cells and mostly absent in the
nucleus (Fig. 4C, top panels). Finally, we
investigated whether the basic cluster alone of Doc2
is a sufficient
nuclear localization signal by producing chimera proteins between
Doc2
and Doc2
in which the loop 3 domain of Doc2
was replaced
by that of Doc2
(named Doc2
(R6); see Fig. 4A). As a
result of this substitution, the Doc2
(R6) C2A domain completely lost
its Ca2+-dependent phospholipid binding
activity (Fig. 4B), whereas the Doc2
(R6) proteins
acquired the ability to localize in the nucleus of PC12 cells (Fig.
4C, bottom panels). These findings indicate that
the basic cluster of Doc2
is both necessary and sufficient for
nuclear localization of Doc2
protein.
C2A domain in nuclear
localization. It is noteworthy that the basic cluster (RLRRRRR) is
present in the putative Ca2+ binding loop 3, which is
located at the apex of
-sandwich structure of the Doc2
C2A domain
(i.e. loop 3 functions as a nuclear localization signal rather than a Ca2+-binding site). Thus, the function
of the loop domains of the C2 domain is more diversified than we
expected. The function of Doc2
in the nucleus remains unclear, but
because Doc2
isoform is involved in secretory vesicle exocytosis
(42, 49, 50), and vesicle traffic is thought to be regulated by a
conserved protein family, such as SNARE (soluble
N-ethylmaleimide-sensitive factor
attachment protein receptor) proteins,
C-type tandem C2 protein families, and rab family (51, 52), Doc2
might be involved in nuclear envelope assembly. As far as we know,
Doc2
is the only isoform of the C-type tandem C2 protein family that is localized in the nucleus. Further work is necessary to elucidate whether Doc2
regulates nuclear envelope assembly.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Shigekazu Nagata for the expression vector (pEF-BOS).
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FOOTNOTES |
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* This work was supported in part by grants from the Science and Technology Agency of Japan (to K. M.) and by Grants 11780571 and 12053274 from the Ministry of Education, Science, and Culture of Japan (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail: mnfukuda@brain.riken.go.jp.
Published, JBC Papers in Press, May 22, 2001, DOI 10.1074/jbc.C100119200
2 M. F., unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: Syt, synaptotagmin(s); C-type, carboxyl-terminal type; Doc2, double C2 protein; GST, glutathione S-transferase; Mid domain, Munc13-1 interacting domain; PC, phosphatidylcholine; PS, phosphatidylserine; DAPI, 4,6-diamidino-2-phenylindole; PCR, polymerase chain reaction.
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