ACCELERATED PUBLICATION
The C2A Domain of Double C2 Protein gamma  Contains a Functional Nuclear Localization Signal*

Mitsunori FukudaDagger §, Chika SaegusaDagger , Eiko KannoDagger , and Katsuhiko MikoshibaDagger

From the Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 Calpha 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 gamma  (Doc2gamma ) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2gamma contains six Arg residues (177RLRRRRR183) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2gamma . Because of the presence of the basic cluster, the C2A domain of Doc2gamma 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

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 alpha , beta , and gamma  (reviewed in Ref. 3). The C2 domains are composed of a common eight-stranded antiparallel beta -sandwich consisting of four-stranded beta -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 delta 1 C2 domain with type II topology) (2, 4, 5). Three flexible loops protrude from the tip of the beta -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), beta -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.

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 (Doc2gamma ; see Fig. 1A) in nuclear localization (30, 31). Unlike other members of the Doc2 family, the C2A domain of Doc2gamma 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 Doc2gamma C2A domain (see Fig. 3, #). Our deletion and mutation analyses indicate that these basic residues are essential for nuclear localization of Doc2gamma instead of Ca2+ binding.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Plasmid Construction-- pEF-T7-Doc2gamma , -Doc2gamma Delta C2AB (amino acid residues 1-80), -Doc2gamma Delta C2B (amino acid residues 1-217), -Doc2gamma -C2A (amino acid residues 80-217), Doc2gamma -C2B (amino acid residues 234-388), and T7-Doc2beta (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' (Delta 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' (Doc2beta -Met primer; sense), and 5'-GCACTAGTCAGTCGCTGAGTACAGC-3' (Doc2beta -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).

Site-directed Mutagenesis of Doc2beta and Doc2gamma -- A mutant Doc2gamma Delta 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' (Delta R-5' primer; antisense) (left half); and 5'-CGGGGGCCCCCCCTGGGGGAGCTA-3' (Delta R-3' primer; sense) and C1 primer (right half). Briefly, the right and left halves were separately amplified by using pGEM-T-Doc2gamma (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 Doc2gamma Delta 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 Doc2beta (R6) was similarly constructed by using the following mutagenic oligonucleotides: 5'-GGGCCCTCGCCGCCGGCGCCGCAGCCGTGACTCATCACACACGGAGAT-3' (Doc2beta (R6)-5' primer; antisense) and 5'-GGGCCCCCCATTGGAGAGACTCGGGTGCCC-3' (Doc2beta (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., 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-alpha -phosphatidylcholine (PC), dipalmitoyl, and L-alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Nuclear Localization of Doc2gamma Proteins in PC12 Cells-- The Doc2 family consists of three isoforms (alpha , beta , and gamma ) 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 Doc2alpha ) (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). Doc2alpha is specifically expressed in neuronal cells (34, 41), whereas Doc2beta and Doc2gamma are expressed ubiquitously (30, 32, 39). Both Doc2alpha and Doc2beta have been shown to be associated with synaptic vesicle fractions in the brain (34, 41), but the subcellular localization of Doc2gamma has yet to be determined. To address this we expressed T7-tagged Doc2gamma proteins in PC12 cells. To our surprise the Doc2gamma 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 Doc2gamma proteins seemed to be uniformly present throughout the nucleoplasm. By contrast, Doc2beta proteins are mainly present in the cytosol, the same as Doc2alpha proteins (Fig. 1B, bottom panels) (42).


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Fig. 1.   Differential expression of mouse Doc2beta and Doc2gamma in PC12 cells. A, schematic representation of mouse Doc2alpha , Doc2beta , and Doc2gamma . 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-Doc2gamma (top panels) and Doc2beta (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 Doc2gamma proteins were localized in nucleus, whereas the Doc2beta proteins were localized in the cytosol. Scale bar indicates 10 µm.

Mapping of the Domain Responsible for the Nuclear Localization of Doc2gamma Proteins-- To determine which domain is essential for the nuclear localization of Doc2gamma , we produced four deletion mutants, each of which involves a different domain of Doc2gamma (Doc2gamma Delta C2AB, Doc2gamma Delta C2B, Doc2gamma -C2A, and Doc2gamma -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 Doc2gamma Delta C2B and Doc2gamma -C2A proteins showed nuclear localization in PC12 cells, whereas the amino-terminal Mid domain was localized in the cytosol, and the Doc2gamma -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 Doc2gamma . A, schematic representation of deletion mutants of Doc2gamma . 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 Doc2gamma -C2B proteins were localized both in nucleus and cytoplasm. Amino acid numbers are given on both sides. B, expression of T7-Doc2gamma deletion mutants. Total homogenates of COS-7 cells expressing T7-Doc2gamma 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-Doc2gamma ; lane 2, Doc2gamma Delta C2AB; lane 3, Doc2gamma Delta C2B; lane 4, Doc2gamma -C2A; lane 5, Doc2gamma -C2B; and lane 6, Doc2gamma Delta R. C, subcellular localization of T7-Doc2gamma deletion mutants. PC12 cells expressing pEF-T7-Doc2gamma deletion mutants were fixed, permeabilized, and stained with anti-T7 tag antibody. Note that the C2A domain of Doc2gamma alone is sufficient for nuclear localization. Scale bar indicates 10 µm.

The Doc2gamma 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 Doc2gamma -C2A domain contains a cluster of basic residues (177RLRRRRR183) in the putative Ca2+ binding loop 3, between the beta 6 and beta 7 strands (Fig. 3, #) (44). Interestingly, the loop 3 domain of the Doc2gamma 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 beta 6 and beta 7 strands in the C2A domain of Syt I (asterisks in Fig. 3) are known to bind Ca2+ ions (48), the C2A domain of Doc2gamma lacks two Asp residues (Ser-176 and Pro-185), and because of these amino acid substitutions, the Doc2gamma 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 Doc2gamma . The location of the beta -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), Doc2alpha (53), Doc2beta (32), Doc2gamma (30), Slp1-3 (45), and Syt B/K and Syt XII.2


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Fig. 4.   A basic cluster in the Doc2gamma C2A domain is essential for nuclear localization signal. A, schematic representation of Doc2gamma deletion mutant (Doc2gamma Delta R) and chimera between Doc2beta and Doc2gamma (Doc2beta (R6)). Doc2gamma Delta R lacks four Arg residues (dashes) between the beta 6 and beta 7 strands of the C2A domain. Doc2beta (R6) contains basic residues of Doc2gamma (SRLRRRRRGPP; underlined) between the beta 6 and beta 7 strands of the C2A domain. B, Ca2+-dependent phospholipid binding properties of the Doc2gamma -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-Doc2beta (R6)-C2A completely lost phospholipid binding activity. The results shown are representative of three independent experiments. C, subcellular localization of T7-Doc2gamma Delta R and T7-Doc2beta (R6). PC12 cells expressing pEF-T7-Doc2gamma and Doc2beta 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 Doc2beta is sufficient for nuclear localization. Scale bar indicates 10 µm.

To determine whether the basic cluster of the Doc2gamma C2A domain is the sole nuclear localization signal of this protein, we produced a deletion mutant lacking four of six Arg residues (named Doc2gamma Delta R; see Fig. 4A). As expected, the Doc2gamma Delta 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 Doc2gamma is a sufficient nuclear localization signal by producing chimera proteins between Doc2beta and Doc2gamma in which the loop 3 domain of Doc2beta was replaced by that of Doc2gamma (named Doc2beta (R6); see Fig. 4A). As a result of this substitution, the Doc2beta (R6) C2A domain completely lost its Ca2+-dependent phospholipid binding activity (Fig. 4B), whereas the Doc2beta (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 Doc2gamma is both necessary and sufficient for nuclear localization of Doc2gamma protein.

Conclusions-- This study revealed the novel function of the Ca2+-independent type of the Doc2gamma 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 beta -sandwich structure of the Doc2gamma 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 Doc2gamma in the nucleus remains unclear, but because Doc2alpha 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), Doc2gamma might be involved in nuclear envelope assembly. As far as we know, Doc2gamma 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 Doc2gamma regulates nuclear envelope assembly.

    ACKNOWLEDGEMENTS

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

    FOOTNOTES

* 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.

    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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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