The C2A Domain of Double C2 Protein γ Contains a Functional Nuclear Localization Signal*

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 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 γ (Doc2γ) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2γ contains six Arg residues (177RLRRRRR183) 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.

The C2 domain is a Ca 2ϩ binding motif that consists of ϳ130 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 Ca 2ϩ -dependent protein kinase C isoforms ␣, ␤, 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. synap-totagmin 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 Ca 2ϩ binding (4,5). The Ca 2ϩ binding allows interaction of the C2 domain with phospholipids to enable translocation of proteins to phospholipid membranes (2).
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␥; see Fig. 1A) in nuclear localization (30,31). Unlike other members of the Doc2 family, the C2A domain of Doc2␥ lacks Ca 2ϩ -dependent phospholipid binding activity, probably because of the amino acid substitutions of the key amino acids (Glu or Asp) responsible for Ca 2ϩ binding (30,(32)(33)(34). Interestingly, six Arg residues are clustered at one of the putative Ca 2ϩ 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 Ca 2ϩ binding.
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-␣-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% SDSpolyacrylamide 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
Nuclear Localization of Doc2␥ 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-  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. Ϯ 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). 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).
Mapping of the Domain Responsible for the Nuclear Localization of Doc2␥ 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 2 M. F., unpublished data.

FIG. 3. Alignment of the putative Ca 2؉ 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 Ca 2ϩ 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   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␥ (SRLR-RRRRGPP; underlined) between the ␤6 and ␤7 strands of the C2A domain. B, Ca 2ϩ -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 Ca 2ϩ for 15 min at room temperature. After centrifugation at 12,000 ϫ 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 m.
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.
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 Doc2␥-C2A domain contains a cluster of basic residues ( 177 RLRRRRR 183 ) in the putative Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ -dependent phospholipid (PS/PC liposome) binding activity (Fig. 4B) (30).
To determine whether the basic cluster of the Doc2␥ 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 Ca 2ϩ -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.
Conclusions-This study revealed the novel function of the Ca 2ϩ -independent type of the Doc2␥ C2A domain in nuclear localization. It is noteworthy that the basic cluster (RLRRRRR) is present in the putative Ca 2ϩ 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 Ca 2ϩ -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.