Noc2, a Putative Zinc Finger Protein Involved in Exocytosis in Endocrine Cells*

We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton.

gested to be involved in the recruitment of synaptic vesicles for exocytosis by Rab3A (7,8). Although, like synaptotagmins, which are a candidate for calcium sensor in neurotransmitter release (9), rabphilin-3A has two repeats of the C2 domain (regulatory domain of protein kinase C that confers Ca 2ϩ and phospholipid binding) in the C-terminal half, rabphilin-3A is unique in that it has a Rab3A-binding domain in the N-terminal region (6). The recently identified Doc2␣ and Doc2␤ also have two repeats of the C2 domain, but lack the Rab3A-binding domain (10,11). It has been shown that both rabphilin-3A and Doc2 also influence regulated exocytosis in neurons (12,13). Previously, we identified synaptotagmin III, which is expressed at high levels in endocrine tissues (14), and suggested that synaptotagmin III is a candidate for the Ca 2ϩ sensor in insulin release from pancreatic ␤-cells (15). However, the molecular basis of the exocytosis-associated proteins in endocrine cells is not well known. Although Rab3A is expressed in neuronal cells and endocrine cells, rabphilin-3A is expressed exclusively in neurons and neuron-like cells and is absent or present at very low levels in endocrine cells (16). Reasoning that a putative target for Rab3A might be expressed specifically in endocrine cells, we screened a rat pancreatic islet and the insulin-secreting cell line RINm5F cDNA libraries using a DNA fragment encoding the Rab3A-binding domain of rabphilin-3A as a probe. We identified a novel protein that has a high similarity to the Rab3A-binding domain of rabphilin-3A but lacks the C2 domain. Our data also suggest that Noc2 is involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton.

EXPERIMENTAL PROCEDURES
cDNA Cloning of Noc2-700,000 plaques of a rat islet cDNA library were hybridized under low stringency conditions (17) using a mouse rabphilin-3A cDNA (16) as a probe. Five clones encoded a partial rabphilin-3A-related protein (designated Noc2). A full-length Noc2 clone was isolated from a RINm5F cDNA library. Both strands of DNA were sequenced.
RNA Blot Analysis-RNA blot analysis was performed under standard stringent hybridization conditions with 32 P-labeled 580-base pair Noc2 cDNA. Membranes were washed with 0.1 ϫ SSC, 0.1% SDS at 50°C for 1 h and were exposed to x-ray film with intensifying screen at Ϫ80°C for 36 h.
Subcellular Fractionation-Discontinuous sucrose gradient fractionation of the insulin-secreting cell line MIN6 was performed according to the method described by Wendland and Scheller (18) with slight modifications (15). Each postnuclear fraction containing 2-20 g of protein was precipitated with 15% of trichloroacetate, separated on 10% SDSpolyacrylamide gel, and subjected to immunoblot analysis.
Immonocytochemstry by Confocal Laser Microscopy-MIN6 cells grown on chamber slides were incubated in HEPES balanced Krebs-Ringer buffer, fixed with 4% of paraformaldehyde, and permeabilized with 0.1% Triton X-100. Immunoreactivity of Noc2 was detected with anti-Noc2 antibody and rhodamine-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Immunoreactivity of insulin was detected with mouse monoclonal anti-rat insulin antibody (Advanced Immuno Chemical, Long Beach, CA) and fluores-* This work was supported by scientific research grants from the Ministry of Education, Science, Sports and Culture and from the Ministry of Health and Welfare, Japan, funds from the Uehara Memorial Foundation, a grant for studies on pathophysiology and complications of diabetes from Tsumura, a grant from Yamanouchi Foundation for Research on Metabolic Disorders, and a grant from the Juvenile Diabetes Foundation International. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF022774.
Transfection and Growth Hormone Secretion Assay-A full-length cDNA of Noc2 or ␤-galactosidase was inserted into the mammalian expression vector pSR␣. PC12 cells, a rat catecholamine-secreting cell line, were plated at a density of 3 ϫ 10 5 /35-mm dish and cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 5% horse serum. The cells were cotransfected with GH 1 expression vector pXGH5 (Nichols Institute, San Juan Capistrano, CA) (1 g) and pSR␣Noc2 (1 g) using LipofectAMINE (Life Technologies, Inc.), according to the manufacturer's instructions. As controls, cells were cotransfected with pXGH5 (1 g) and pSR␣␤gal (1 g). 3 days after transfection, PC12 cells were washed with a PSS (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 1.2 mM KH 2 PO 4 , 20 mM HEPES, pH 7.4, and 11 mM glucose) and incubated for 2.5, 5, or 10 min with a high K ϩ solution (PSS containing 60 mM KCl and 85 mM NaCl) or a low K ϩ solution (PSS containing 4.7 mM KCl and 140 mM NaCl). GH was measured by enzyme immunoassay kit (Picoia, Sumitomo, Osaka, Japan). Secretion was expressed as a percentage of the GH amounts released into the medium relative to the total cellular GH amounts (19).

RESULTS
Structure of Noc2-The sequence of the longest insert (1934 base pairs) of clones contains an open reading frame encoding a 302-amino acid protein (M r ϭ 33431.2) having 40.7% identity with and 77.9% similarity to the amino acid sequence of the N-terminal region (amino acids 1-304) of rat rabphilin-3A (Fig.  1). However, unlike rabphilin-3A, it lacks the C2 domains and was therefore designated Noc2 (no C2 domain). Interestingly, Noc2 has a putative metal ion-binding domain containing a cluster of cysteine residues that is also conserved in rabphilin-3A. There are a potential protein kinase A-dependent phosphorylation site and three potential protein kinase C-dependent phosphorylation sites in Noc2.
Tissue Expression of Noc2-RNA blot analysis revealed that a single 2.2-or 2.6-kb transcript of Noc2 is expressed at very high levels in pancreatic islets; at moderate to high levels in ovary, adrenal, pituitary, and PC12 cells and the insulin secreting cell lines RINm5F (rat), MIN6 (mouse), and HIT-T15 (hamster); and at low levels in testis, the rat GH-secreting cell line GH3, and the mouse adrenocorticotropic hormone-secreting cell line AtT20 (Fig. 2). Noc2 mRNA is detected at only very low levels in brain after a longer exposure (data not shown). Noc2 protein is detected as a 38-kDa protein in total proteins prepared from MIN6 cells and PC12 cells (data not shown).
Subcellular Localization- Fig. 3A shows the immunoblot analysis of subcellular fractions prepared from MIN6 cells probed with anti-Noc2 and anti-synaptotagmin III antibodies. Noc2 was detected as a 38-kDa protein in fractions 2-8, peaking at fraction 3. In contrast, synaptotagmin III, which is present in large dense core vesicle in MIN6 cells (15), was detected in fraction at 9 and 10 (Fig. 3A). Fig. 3B shows the representative of double-immunostaining of MIN6 cell for Noc2 and insulin. Noc2 was detected diffusely and partially dotted in the cytoplasm of MIN6 cells.
Enhancement of GH Secretion from PC12 Cells Overexpressing Noc2-Total amounts of GH produced in PC12 cells transfected with human GH ranged from 3 to 10 ng/35-mm dish. The basal secretion of GH (expressed as a percentage of GH amounts released into the medium containing 4.7 mM K ϩ relative to amounts of total cellular GH) is not significantly different between Noc2-transfected (8.5 Ϯ 0.87%) and control cells (8.7 Ϯ 0.99%). Upon high K ϩ (60 mM) stimulation, GH secretion from PC12 cells cotransfected with GH and Noc2 was significantly higher than that seen in controls (Fig. 4).
Interaction of Noc2 with Zyxin-A putative target of Noc2 was isolated from a mouse embryonic cDNA library by the yeast two-hybrid system with the full-length Noc2 used as a bait. We isolated four clones, one encoding a partial amino acid sequence of zyxin (amino acids 353-509). FLAG-zyxin was specifically coimmunoprecipitated by glutathione-Sepharose 4B (Fig. 5). In addition, GST-Noc2 was also coimmunoprecipitated with zyxin by Anti-FLAG M2 Affinity Gel (Fig. 5). DISCUSSION In the present study we have identified a novel protein designated Noc2. Noc2 has structural features characterized by 1) a high degree of similarity to the N-terminal region of rabphilin-3A, 2) no C2 domain, and 3) no putative transmembrane region. Interestingly, Noc2 has a putative zinc finger motif composed of a cysteine-rich sequence that is conserved among rabphilin-3A and a recently identified putative Rab3 effector, RIM (21). Because rabphilin-3A has been shown to bind Rab3A via this domain in a zinc-dependent manner (22), we assumed that Noc2 might also interact with Rab3 members. However, FIG. 1. Comparison of amino acid sequences of rat Noc2 and rat rabphilin-3A. Amino acids are indicated in single-letter codes. Identical and chemically similar amino acid residues are indicated by double dots and single dots, respectively. The N-terminal region (amino acid residues 1-304) of rat rabphilin-3A (Rabp) is shown. Potential protein kinase A-dependent phosphorylation site and protein kinase C-dependent phosphorylation sites of Noc2 are indicated by * and #, respectively. Cysteine residues that constitute a putative zinc finger domain are boxed.
Noc2 did not bind Rab3A, Rab3B, or Rab3C under the conditions in which rabphilin-3A binds Rab3A, 2 although we cannot exclude the possibility that Noc2 might bind other members of the Rab3 subfamily or their related proteins.
To search for a molecule that interacts with Noc2, we screened a mouse embryonic cDNA library using the yeast two-hybrid system (20) and identified zyxin as a candidate. Coimmunoprecipitation experiments confirmed that Noc2 interacts with zyxin. Zyxin is a cytoskeletal element that exhibits an unusual proline-rich N terminus followed by three tandemly arrayed LIM domains (23). The LIM domain contains the cysteine-rich consensus sequence CX 2 CX 16 -23 HX 2 CX 4 CX 2 CX 16 -21CX 2-3 (C/H/D) (24). This domain participates in protein-protein interaction in a zinc-dependent manner (24). It is possible, therefore, that the zinc finger domain of Noc2 interacts with one of the LIM domains in zyxin. In fact, we have found by the yeast two-hybrid system that the zinc finger containing Nterminal fragment (amino acids 1-200) of Noc2 interacts with the fragment between the first and a part of the third LIM domain of zyxin (data not shown).
Rabphilin-3A has been shown to interact with ␣-actinin, a component of the cytoskeleton, and possibly to be involved in the predocking and docking process of synaptic vesicles by linking Rab3A to the cytoskeleton (25). This Rab3A-rabphilin-3A system is suggested to regulate reorganization of actin filaments (25). Interestingly, a proline-rich domain in the Nterminal region of zyxin has been shown to interact with ␣actinin (26). Accordingly, Noc2 may participate in the reorganization of actin filaments by interacting with zyxin.
Although rabphilin-3A is expressed predominantly in brain, Noc2 is expressed predominantly in endocrine cells and is poorly expressed in brain. Overexpression of rabphilin-3A in PC12 cells enhances high K ϩ -induced GH secretion from the cells transfected with GH (27). We have found that overexpression of Noc2 in PC12 cells also enhances high K ϩ -induced secretion, further suggesting involvement of Noc2 in Ca 2ϩtriggered secretion. Because Noc2 lacks the C2 domain and is not present in large dense core vesicle in MIN6 cells, it remains to be determined whether Noc2 interacts directly or indirectly with vesicle-associated proteins having the C2 domain. Considering these findings together, Noc2 participates in regulated exocytosis in endocrine cells probably by interacting with the cytoskeleton.