The C2A Domain of Doc2 Contains a Functional Nuclear

1

(Accelerated Publications)

The C2A Domain of Doc2 Contains a Functional Nuclear

Localization Signal*

Mitsunori Fukuda‡§, Chika Saegusa‡, Eiko Kanno‡, and Katsuhiko

Mikoshiba‡¶

From the ‡Laboratory for Developmental Neurobiology, Brain Science Institute,

RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako,

Saitama 351-0198, Japan, 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

(Running title: A Novel Function of the Doc2γ C2A Domain)

* This work was supported in part by grants from the Science and Technology Agency to

Japan (to K.M.) and Grants 11780571 and 12053274 from the Ministry of Education,

Science, and Culture of Japan (to M.F.).

§ To whom correspondence should be addressed: Laboratory for Developmental

Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-

0198, Japan. Tel.: +81-48-467-9745; Fax: +81-48-467-9744; E-mail:

[email protected].

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 22, 2001 as Manuscript C100119200 by guest on February 12, 2018

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SUMMARY

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 Cα

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 due to 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 do uble C2

protein γ (Doc2γ) in nuclear localization. Deletion and mutation analyses revealed that the

putative Ca2+-binding loop 3 of Doc2γ contains six Arg residues (177 RLRRRRR 183)

and that this basic cluster is both necessary and sufficient for nuclear localization of

Doc2γ. Because of the presence of the basic cluster, the C2A domain of Doc2γ 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.

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INTRODUCTION

The C2 domain is a Ca2+-binding motif that consists of approximately 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 α, β, and γ (reviewed in ref. 3). The C2

domains are composed of a common eight-stranded anti-parallel β-sandwich consisting of

four-stranded β-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 δ1 C2 domain with type II topology) (2, 4, 5). Three flexible loops

protrude from the tip of the β-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).

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), β-SNAP (25),

SNAP25 (26), Ca2+ channels (27), and SYNCRIP (28), irrespective of the presence of

Ca2+. Furthermore, some Syt isoforms fail to exhibit Ca2+-binding due to amino acid


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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 largely remains unknown.

In this paper, we report the discovery of an unexpected function of the C2A

domain of Doc2γ in nuclear localization (Doc2γ, a third isoform of do uble C2 protein that

contain a C2A domain and a C2B domain; see Fig. 1A) (30, 31). Unlike other members

of the Doc2 family, the C2A domain of Doc2γ lacks Ca2+-dependent phospholipid binding

activity, probably due to 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 Doc2γ C2A domain (see Fig. 3, #). Our

deletion and mutation analyses indicate that these basic residues are essential for nuclear

localization of Doc2γ instead of Ca2+-binding.


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EXPERIMENTAL PROCEDURES

Plasmid Construction

pEF-T7-Doc2γ, -Doc2γ∆C2AB (amino acid residues 1-80), -Doc2γ∆C2B (amino

acid residues 1-217), -Doc2γ-C2A (amino acid residues 80-217), Doc2γ-C2B (amino acid

residues 234-388), and T7-Doc2β (amino acid residues 1-418) (32) were constructed by

polymerase chain reaction using the following sets of primers with appropriate restriction

enzyme sites (underlined) and/or termination codons (bold letters), as described

previously (19, 35): 5’-C GGATCC ATGGCATGTGCAGGGCCAGCC-3’ (Met primer,

sense), 5’-GC ACTAG T CAGTCATCCGAGTCTCCTTC-3’ (∆C2AB primer,

antisense), 5’- GC GGATCC GACAGCACTGCCCTAGGCAC-3’ (C2A upper primer,

sense), 5’-GC ACTAG T CACCTCTTGGTCAGCTTCCGCT-3’ (C2A lower primer,

antisense), 5’-GC GGATCC GAGGTGGAGGCAGAGGTGTT-3’ (C2B upper primer,

sense), GCTG ACTAG T CACCAAGTT-3’ (C1 primer, antisense), 5’-

GC GGATCC ATGACCCTCCGGCGGCGCGGGGAGAAGGCGACCATCAGCA-3’

(Doc2β-Met primer, sense), and 5’-GC ACTAG T CAGTCGCTGAGTACAGC-3’

(Doc2β-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-mini preps (Promega; Madison,

WI, USA) or QIAGEN (Chatsworth, CA, USA) Maxi prep kits.

Site-directed Mutagenesis of Doc2 and Doc2


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A mutant Doc2γ∆R (deletion of amino acids 179-183 (four Arg residues) in the

C2A domain) was essentially produced by means of two-step polymerase chain reaction

techniques, as described previously (21), using the following pairs of oligonucleotides:

Met primer and 5’-GGG GGGCCC CCGCAGCCGTGAGTCCTC-3’ (∆R-5’ primer;

antisense) (left half); and 5’-CGG GGGCCC CCCCTGGGGGAGCTA-3’ (∆R-3’ primer;

sense) and C1 primer (right half). Briefly, the right and left halves were separately

amplified by using pGEM-T-Doc2γ (30), as a template, and the two resulting PCR

fragments were digested with ApaI (underlined and italics above), ligated to each other,

and reamplified with the Met and C1 primers. The PCR fragment obtained that encoded

the mutant Doc2γ∆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

Doc2β(R6) was similarly constructed by using the following mutagenic oligonucleotides:

5’- GGGCCC TCGCCGCCGGCGCCGCAGCCGTGACTCATCACACACGGAGAT-3’

(Doc2β(R6)-5’ primer; antisense) and 5’-

GGGCCC CCCATTGGAGAGACTCGGGTGCCC-3’ (Doc2β(R6)-3’ primer; antisense).

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., MA, USA) 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;


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Madison, WI, USA) and anti-p300 rabbit polyclonal antibody (1/500 dilution; Santa Cruz

Biotechnology, Santa Cruz, CA, USA), and then visualized with anti-mouse Alexa 488

and anti-rabbit Alexa 568 antibodies (1/5000 dilution; Molecular Probe, Eugene, OR,

USA), respectively. In some cases, Vectashield mounting medium with DAPI (Vector

Laboratories, Burlingame, CA, USA) 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 (Ver. 4.0).

Phospholipid Binding Assay

Glutathione S-transferase (GST) fusion proteins were expressed and purified on

glutathione Sepharose (Amersham Pharmacia Biotech, Buckinghamshire, UK) by the

standard method (38). Preparation of liposomes consisting of L-α-phosphatidylcholine

(PC), dipalmitoyl and L-α-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 Laboratories,

Hercules, CA, USA) by using bovine serum albumin for reference.


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RESULTS AND DISCUSSION

Nuclear Localization of Doc2 Proteins in PC12 Cells

The Doc2 family consists of three isoforms (α, β, and γ) 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 Doc2α) (40) and two C2 domains at the carboxyl

terminus (the C2A domain and the C2B domain) (see 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). Doc2α is specifically

expressed in neuronal cells (34, 41), whereas Doc2β and Doc2γ are expressed

ubiquitously (30, 32, 39). Both Doc2α and Doc2β have been shown to be associated

with synaptic vesicle fractions in the brain (34, 41), but the subcellular localization of

Doc2γ has yet to be determined. To address this we expressed T7-tagged Doc2γ proteins

in PC12 cells. To our surprise the Doc2γ 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 Doc2γ proteins seemed to be uniformly present

throughout the nucleoplasm. By contrast, Doc2β proteins are mainly present in the

cytosol, the same as Doc2α proteins (Fig. 1B, bottom panels) (42).

Mapping of the Domain Responsible for the Nuclear Localization of Doc2 Proteins


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To determine which domain is essential for the nuclear localization of Doc2γ, we

produced four deletion mutants, each of which involves a different domain of Doc2γ

(Doc2γ∆C2AB, Doc2γ∆C2B, Doc2γ-C2A and Doc2γ-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

Doc2γ∆C2B and Doc2γ-C2A proteins showed nuclear localization in PC12 cells, whereas

the amino terminal Mid domain was localized in the cytosol, and the Doc2γ-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.

The Doc2 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 Doc2γ-C2A domain

contains a cluster of basic residues (177 R L RRRRR 183) in the putative Ca2+-binding

loop 3, between the β6 and β7 strands (Fig. 3, #) (44). Interestingly, the loop 3 domain

of the Doc2γ 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

( s ynaptotagmin- l ike p rotein) (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


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have been no reports of tandem C2 proteins that specifically localized in nucleus. While

three Asp residues between the β6 and β7 strands in the C2A domain of Syt I (asterisks

in Fig. 3) are known to bind Ca2+ ions (48), the C2A domain of Doc2γ lacks two Asp

residues (Ser-176 and Pro-185), and because of these amino acid substitutions, the Doc2γ

C2A domain does not display any clear Ca2+-dependent phospholipid (PS/PC liposome)

binding activity (Fig. 4B) (30).

To determine whether the basic cluster of the Doc2γ C2A domain is the sole

nuclear localization signal of this protein, we produced a deletion mutant lacking four of

six Arg residues (named Doc2γ ∆R; see Fig. 4A). As expected, the Doc2γ∆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 Doc2γ is a

sufficient nuclear localization signal by producing chimera proteins between Doc2β and

Doc2γ in which the loop 3 domain of Doc2β was replaced by that of Doc2γ (named

Doc2β(R6); see Fig. 4A). As a result of this substitution, the Doc2β(R6) C2A domain

completely lost its Ca2+-dependent phospholipid binding activity (Fig. 4B), whereas the

Doc2β(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 Doc2γ is both necessary

and sufficient for nuclear localization of Doc2γ protein.

Conclusions


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This study revealed the novel function of the Ca2+-independent type of the Doc2γ

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 β-sandwich

structure of the Doc2γ 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 Doc2γ in the nucleus remains

unclear, but since Doc2α isoform is involved in secretory vesicle exocytosis (42, 49, 50)

and vesicle traffic is thought to be regulated by 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), Doc2γ might be involved in nuclear

envelope assembly. As far as we know, Doc2γ 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 Doc2γ regulates nuclear envelope assembly.


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FOOTNOTES

1The abbreviations used are: C-type, carboxyl-terminal type; Doc2, double C2 protein;

GST, Glutathione S-transferase; Mid, Munc13-1 interacting domain; PC,

phosphatidylcholine; PS, phosphatidylserine; Syt, synaptotagmin(s).

Acknowledgements- We thank Dr. Shigekazu Nagata for the expression vector (pEF-

BOS).


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FIGURE LEGENDS

FIG. 1. Differential expression of mouse Doc2 and Doc2 in PC12 cel ls .

A, schematic representation of mouse Doc2α, Doc2β, and Doc2γ. 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-Doc2γ

(top panels) and Doc2β (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 in “Experimental Procedures”.

The right panels are overlay (in yellow) between left and middle panels. Note that Doc2γ

proteins were localized in nucleus, whereas the Doc2β proteins were localized in the

cytosol. Scale bar indicates 10 µm.

FIG. 2. Mapping of the domain responsible for the nuclear localization of

Doc2 . A, schematic representation of deletion mutants of Doc2γ. 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 Doc2γ-C2B proteins were localized

both in nucleus and cytoplasm. Amino acid numbers are given on both sides. B,

expression of T7-Doc2γ deletion mutants. Total homogenates of COS-7 cells expressing


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T7-Doc2γ 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-Doc2γ; lane 2, Doc2γ∆C2AB; lane 3,

Doc2γ∆C2B; lane 4, Doc2γ-C2A; lane 5, Doc2γ-C2B; and lane 6, Doc2γ∆R. C,

subcellular localization of T7-Doc2γ deletion mutants. PC12 cells expressing pEF-T7-

Doc2γ deletion mutants were fixed, permeabilized, and stained with anti-T7 tag antibody.

Note that the C2A domain of Doc2γ alone is sufficient for nuclear localization. Scale bar

indicates 10 µm.

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 Doc2γ. The location of the β-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 ref. 12 (Syts I, II and rabphilin-3A), ref. 21 (Syts

III and IV), ref. 35 (Syts V-XI) ref. 29 (Syt XIII), ref. 46 (granuphilin-a), ref. 53

(Doc2α), ref. 32 (Doc2β), ref. 30 (Doc2γ), ref. 45 (Slp1-3), and M.F. unpublished data

(Syt B/K and Syt XII).


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19

FIG. 4. A basic cluster in the Doc2 C2A domain is essential for nuclear

localization signal. A, schematic representation of Doc2γ deletion mutant (Doc2γ∆R)

and chimera between Doc2β and Doc2γ (Doc2β(R6)). Doc2γ∆R lacks four Arg residues

(dashes) between the β6 and β7 strands of the C2A domain. Doc2β(R6) contains basic

residues of Doc2γ (SRLRRRRRGPP, underlined) between the β6 and β7 strands of the

C2A domain. B, Ca2+-dependent phospholipid binding properties of the Doc2γ-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-PAGE and then stained with Coomassie Brilliant Blue R-250.

Note that GST-Doc2β(R6)-C2A completely lost phospholipid binding activity. The

results shown are representative of three independent experiments. C, subcellular

localization of T7-Doc2γ∆R and T7-Doc2β(R6). PC12 cells expressing pEF-T7-Doc2γ

and Doc2β 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 in “Experimental

Procedures”. The right panels are overlay (in yellow) between left and middle panels.

Note that the insertion of SRLRRRRRGPP sequence into Doc2β is sufficient for nuclear

localization. Scale bar indicates 10 µm.


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Mitsunori Fukuda, Chika Saegusa, Eiko Kanno and Katsuhiko MikoshibaThe C2A domain of Doc2g contains a functional nuclear localization signal

published online May 22, 2001J. Biol. Chem.

10.1074/jbc.C100119200Access the most updated version of this article at doi:

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The C2A Domain of Doc2 Contains a Functional Nuclear