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Involvement of Epidermal Growth Factor-like Domain of NELL Proteins in the Novel Protein–Protein Interaction with Protein Kinase C
Biochemical and Biophysical Research Communications 265, 752–757 (1999) Article ID bbrc.1999.1753, available online at http://www.idealibrary.com on
Involvement of Epidermal Growth Factor-like Domain of NELL Proteins in the Novel Protein–Protein Interaction with Protein Kinase C 1 Shun’ichi Kuroda 2 and Katsuyuki Tanizawa Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan
Received October 16, 1999
NELL proteins are the thrombospondin-1-like proteins that are strongly expressed in neural tissues, containing six epidermal growth factor (EGF)-like domains. By radiolabeling of the NELL proteinexpressing COS-7 cells with [ 32P]orthophosphate, here we demonstrate that NELL proteins are synthesized as phosphoproteins by interacting with protein kinases in the cells. By immunoprecipitation and in vitro phosphorylation assays, we have also found that NELL proteins expressed in COS-7 cells are associated with and phosphorylated by protein kinase C bI (PKCbI). Further analysis using various deletion mutants of NELL proteins by the yeast two-hybrid assay has revealed their EGF-like domains to be involved in the isoform-specific interaction with PKC. Conversely, the NH 2-terminal variable region of PKC isoforms has been found essential for the interaction with NELL proteins. Because NELL proteins are expressed mainly in the cytoplasm of neuronal cells, unlike most EGF-like domain-containing extracellular proteins, the novel protein–protein interaction identified here between the EGF-like domains of NELL proteins and PKC suggests that EGF-like domains of intracellular proteins can be a target of PKC that mediates various signaling pathways. © 1999 Academic Press
Abbreviations: PKC, protein kinase C; EGF, epidermal growth factor; TSP-1, thrombospondin-1; vWF, von Willebrand factor; ER, endoplasmic reticulum; NELL-FLAG, COOH-terminally FLAGtagged NELL protein; PKCbI-HA, NH 2-terminally HA-tagged PKCbI; GAL4 AD, GAL4 activating domain; GAL4 DBD, GAL4 DNA-binding domain. 1 This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (#09780576, #11780446, #11139241), research grants from KatoMemorial Bioscience Foundation, Association for the Progress of New Chemistry (ASPRONC), Takeda Science Foundation, and TERUMO Life Science Foundation (to S.K.). 2 To whom correspondence should be addressed at Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 5670047, Japan. Fax: 181-6-6879-8464. E-mail: [email protected]. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Protein kinase C (PKC) is a Ca 21/phospholipiddependent Ser/Thr protein kinase and is classified into at least 10 isoforms (1, 2), each of which bears a specific physiological function in various cellular activities, such as cell growth, differentiation, oncogenesis, and apoptosis, by interacting with their own target proteins (3– 6). Recently, we have identified new PKCinteracting proteins, NELL1 and NELL2, which are homologous to the NELL proteins that are expressed abundantly in neural tissues and contain six epidermal growth factor (EGF)-like domains (7–9), by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of PKCbI as a bait (10). The rat NELL proteins show about 55% identities with each other and possess, in addition to the EGF-like domains (11, 12), protein motifs similar to thrombospondin-1 (TSP-1); an NH 2-terminal TSP-1-like module (13) and five von Willebrand factor (vWF) C domains (14, 15) (see Fig. 3). In chick embryo, the NELL2 mRNAs are ubiquitously expressed in all tissues, but after hatching the expression is restricted only in neural tissues (7). We have also shown that the expression of NELL1 and NELL2 mRNAs in mouse embryo is rigorously regulated during embryogenesis (10). These findings have led to a suggestion that NELL proteins may play important roles in the development of neural tissues. The EGF-like domain is a polypeptide segment with 40 –50 amino acid residues including 6 conserved Cys residues and has so far been found in about 100 eukaryotic proteins related to cell proliferation, growth inhibition, and differentiation (11). These EGF-like domain-containing proteins mainly occur in the extracellular fractions, as membrane-associated or secreted proteins. However, some intracellular proteins are also known to contain EGF-like domains, e.g., NELL proteins occurring mainly in the cytoplasm (10), amphiregulin present in the nucleus (16), and prostaglandin endoperoxide H synthases existing in the endoplasmic reticulum (ER) and nuclear envelopes (17). Some groups of EGF-like domains have been demonstrated
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to participate in the Ca 21-dependent protein-protein interactions, although their interacting proteins have been unidentified (12). To identify proteins interacting with EGF-like domains, particularly with those contained in the intracellular proteins, we have dissected the NELL proteins newly isolated as PKC-interacting proteins by the yeast two-hybrid screening. We here report that the EGF-like domains of NELL proteins interact with PKC in an isoform-specific manner and suggest that those domains in the intracellular proteins may be a target of PKC that mediates various intracellular signaling pathways. MATERIALS AND METHODS Expression of epitope-tagged proteins in COS-7 cells. For expression of the COOH-terminally FLAG-tagged NELL proteins (NELL1FLAG, NELL2-FLAG), the plasmids pTB701-FLC-NELL1 and pTB701-FLC-NELL2 were used (10). For expression of the NH 2terminally HA-tagged form of PKCbI (PKCbI-HA) and kinasenegative PKCbI (K371M PKCbI-HA), plasmids pTB701-HA-PKCbI and pTB701-HA-K371M PKCbI (4) were used, respectively. These plasmids were transferred into COS-7 cells by electroporation using a Gene Pulser II (Bio-Rad Laboratories). COS-7 cells were cultured for 60 –72 h in a Dulbecco’s modified MEM (DMEM) medium supplemented with 10% (v/v) fetal bovine serum, and then used for expression assays. In vivo protein labeling with [ 32P]orthophosphate. About 5 3 10 7 COS-7 cells expressing either NELL1-FLAG or NELL2-FLAG were incubated for 5– 6 h in 5 ml of a phosphate-free DMEM medium (Life Technologies) supplemented with 10% (v/v) dialyzed FBS (Life Technologies) and 0.5 mCi/ml of [ 32P]orthophosphate (;9000 Ci/mmol, NEN). The NELL-FLAG proteins recovered in the cytoplasmic fractions were immunoprecipitated with 2 mg of an anti-FLAG monoclonal antibody (M2, Eastman Kodak) as described below, subjected to SDS-PAGE, and then autoradiographed. Immunoprecipitation. COS-7 cells from a 10-cm plate (about 5 3 10 7 cells) expressing either NELL1-FLAG or NELL2-FLAG were suspended in 500 ml of lysis buffer (4). Cleared lysates (500 ml) were incubated on ice for 1 h with 2 mg of an anti-FLAG or anti-HA (12CA5, Boehringer Mannheim) monoclonal antibody, and then mixed with 20 ml of protein G-Sepharose 4 fast flow beads (50% slurry, Pharmacia). After incubation at 4°C for 1 h with shaking, the beads were washed four times with lysis buffer, subjected to SDSPAGE, and then analyzed by Western blotting. In vitro phosphorylation assay. Cleared lysates (500 ml) prepared from the COS-7 cells (about 5 3 10 7 cells) coexpressing a NELLFLAG protein and PKCbI-HA were subjected to the immunoprecipitation using 2 mg of an anti-HA antibody and 20 ml of protein G-Sepharose 4 fast flow beads (50% slurry). The beads were mixed with 25 ml of the reaction mixture (20 mM Tris, pH 7.5, 10 mM MgCl 2, 1 mM CaCl 2, 20 mM ATP, 8 mg/ml phosphatidylserine, 0.8 mg/ml diolein) and 1 ml of [g- 32P]ATP (10 mCi/ml, .7000 Ci/mmol, Amersham). The reaction mixture was incubated at 30°C for 30 min, subjected to SDS-PAGE, and then autoradiographed. Yeast two-hybrid assay. Protein-protein interaction was analyzed by the yeast two-hybrid assay (18) using both of the NELL proteins fused with the GAL4 activating domain (GAL4 AD) and the regulatory domain of PKC fused with the GAL4 DNA-binding domain (GAL4 DBD) in the yeast strain CG-1945 (Clontech). Deletion mutants of PKC isoforms used for the yeast two-hybrid assay were as follows (4, 6): a-R, residues 1–336 of rat PKCa; b-R, residues 1–340 of rat PKCbI; b-V1, residues 1–19 of rat PKCbI; b-V1/C1, residues 1–159 of rat PKCbI; b-C1, residues 20 –159 of rat PKCbI; b-V2/V3,
residues 160 –340 of rat PKCbI; g-R, residues 1–349 of rat PKCg; d-R, residues 1–345 of rat PKCd; «-R, residues 1– 406 of rat PKC«; «-V1, residues 1–135 of rat PKC«; «-V1/C1, residues 1–297 of rat PKC«; «-C1/V3, residues 134 – 406 of rat PKC«; z-R, residues 1–250 of rat PKCz; z-V1, residues 1–113 of rat PKCz; z-V1/C1, residues 1–180 of rat PKCz; and z-C1/V3, residues 114 –250 of rat PKCz. Deletion mutants of NELL proteins used were as follows: NELL2, residues 1– 819 (full length) of rat NELL2; NELL2DN1, residues 443– 819 of rat NELL2; NELL2DN2, residues 663– 819 of rat NELL2; NELL1, residues 1– 810 (full length) of rat NELL1; NELL1DN1, residues 434 – 810 of rat NELL1; and NELL1DN2, residues 654 – 810 of rat NELL1. The activity of b-galactosidase (b-Gal) in yeast cells was detected by the plate assay method (18). Briefly, yeast transformants were transferred onto nylon membranes, permeabilized in liquid nitrogen, and placed on Whatman 3MM papers that had been soaked in Z buffer (4) containing 1 mg/ml 5-bromo-4-chloro-3-indolylb-D-galactoside (X-gal). After developing at 37°C for 10 h, the yeast cells forming either dark blue or blue colonies were classified into a positive (11) and a weakly positive group (1), respectively. The yeast cells forming white colonies were classified into a negative group (2). All measurements were repeated at least four times. Immunocytochemical observation of COS-7 cells expressing NELLFLAG proteins. COS-7 cells (about 5 3 10 5 cells) expressing either NELL1-FLAG or NELL2-FLAG were grown on cover glasses (1-cm diameter), washed with phosphate-buffered saline (PBS), and fixed with 4% (w/v) paraformaldehyde in PBS at room temperature for 30 min. The cells were washed again with PBS and permeabilized by incubating in PBS containing 0.25% (v/v) Triton X-100, 5% (v/v) normal goat serum, and 5% (w/v) skim milk at room temperature for 30 min. The anti-FLAG antibody was applied at a concentration of 0.4 mg/ml at 4°C and allowed to react overnight. A fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Amersham) was added at a concentration of 1 mg/ml at room temperature, and after 30 min the fluorescence was observed under a Zeiss LSM410 confocal laser scan microscope (Carl Zeiss).
RESULTS In vivo phosphorylation of NELL proteins expressed in COS-7 cells. When the plasmids carrying the COOH-terminally FLAG-tagged NELL genes were introduced into COS-7 cells, two types of FLAG-tagged proteins with molecular sizes of about 140-kDa and .400-kDa were found to be expressed in the cytoplasm by Western blotting (10). The .400-kDa protein was thought to be a homotrimeric form of the 140-kDa protein. In this study, COS-7 cells transiently expressing the NELL-FLAG proteins were labeled with [ 32P]orthophosphate for 5– 6 h. The cleared lysates obtained were mixed with an anti-FLAG antibody and the immunoprecipitates were subjected to SDS-PAGE, followed by autoradiography. As shown in Fig. 1, both of the 140- and .400-kDa proteins were found to be phosphorylated. The .400-kDa protein were converted to 140-kDa protein by prolonged denaturation before applying to SDS-PAGE (data not shown). This result indicates that the NELL proteins are phosphorylated in vivo by interacting with intracellular protein kinases. The NELL-FLAG proteins secreted into the medium (10) were also found to be phosphorylated (data not shown).
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FIG. 1. In vivo phosphorylation of NELL proteins expressed in COS-7 cells. COS-7 cells expressing either NELL1-FLAG or NELL2FLAG proteins were labeled with [ 32P]orthophosphate for 5– 6 h. The NELL-FLAG proteins in the cytoplasm were immunoprecipitated with an anti-FLAG antibody, subjected to SDS-PAGE, and then autoradiographed. The positions of NELL-FLAG proteins are indicated with arrows.
In vivo association of NELL proteins with PKC. NELL proteins were newly isolated as PKCbIinteracting proteins by the yeast two-hybrid screening (10). In the present studies, intracellular interaction of NELL proteins with PKCbI was analyzed by immunoprecipitation using COS-7 cells coexpressing the NELL-FLAG proteins and PKCbI-HA. In the anti-HA immunoprecipitates, the NELL2-FLAG protein was detected by Western blotting with an anti-FLAG antibody (Fig. 2, 3rd panel), confirming that the NELL2 protein associates in vivo with PKCbI. NELL2 protein in the anti-HA immunoprecipitates underwent phosphorylation in the presence of PKC activators (Ca 21, phosphatidylserine, and diolein) and [g- 32P]ATP (Fig. 2, 4th panel). Simultaneously, autophosphorylation of PKCbI was also observed. When a kinase negative mutant of PKCbI (K371M PKCbI-HA) was used instead of the wild-type PKCbI, the association with NELL2 protein was unaffected, but NELL2 protein was not phosphorylated under the same conditions, indicating that PKCbI can phosphorylate NELL2 protein but the kinase activity of PKCbI is not essential for the formation of the NELL2/PKCbI complex. Similarly, when the COS-7 cells coexpressing NELL1FLAG and PKCbI-HA were used for the immunoprecipitation assay, both the NELL1/PKCbI complex formation and the phosphorylation of NELL1 protein by PKCbI were observed (data not shown). Thus, it was assumed that the NELL proteins exhibit their functions by interacting with and being phosphorylated by PKC (presumably PKCbI). Mapping of regions involved in the NELL/PKC interaction. An essential region of NELL proteins for the interaction with PKCbI was surveyed by the yeast two-hybrid assay using the deletion mutants of NELL proteins as baits. As shown in Fig. 3, the EGF-like
domains of NELL1 and NELL2 proteins were identified to be essential for the interaction with the regulatory domain of PKCbI. Some groups of EGF-like domains have been demonstrated to participate in the Ca 21-dependent protein-protein interactions (12). NELL proteins possess three Ca 21-binding sites in their 2nd, 5th, and 6th EGF-like domains (10). Therefore, requirement of Ca 21 ion for the NELL/PKC interactions would be one of the foremost subjects to be investigated. Next, interactions of NELL proteins with other PKC isoforms were analyzed by the yeast two-hybrid assay (Fig. 4). When the regulatory domains of PKC isoforms (a, bI, g, d, «, and z) (1, 2) were used as baits, NELL2 protein was found to interact strongly with PKCbI, «, and z, weakly with PKCg and d, and not with PKCa. On the other hand, NELL1 protein interacted strongly with PKCd, weakly with PKCbI and z, and not with PKCa, g, and «. Furthermore, by using deletion mutants of PKCbI, «, and z, NELL2 protein was found to interact with the NH 2-terminal regions of PKCbI, «, and z, and NELL1 protein with those of PKCbI and z. Thus, the NELL proteins recognize the NH 2-terminal regulatory regions (in particular, the variable region,
FIG. 2. In vivo association of NELL2 protein and PKCbI. Either PKCbI-HA or its kinase negative mutant K371M PKCbI-HA was coexpressed with NELL2-FLAG protein in COS-7 cells. The lysates were immunoprecipitated (IP) and then blotted (Blot) with either an anti-FLAG or anti-HA antibody. 1st panel, detection of NELL2FLAG proteins in the anti-FLAG immunoprecipitates with an antiFLAG antibody. 2nd panel, detection of PKCbI-HA or K371M PKCbI-HA in the anti-HA immunoprecipitates with an anti-HA antibody. 3rd panel, detection of NELL2-FLAG proteins in the anti-HA immunoprecipitates with an anti-FLAG antibody. 4th panel, in vitro phosphorylation assay in the anti-HA immunoprecipitates. The positions of NELL2-FLAG protein and PKCbI-HA are indicated with arrows.
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FIG. 3. Delineated structures of NELL proteins and interaction with the regulatory domains of PKCbI analyzed by the yeast twohybrid assay. The yeast two-hybrid assay was done using various deletion mutants of NELL proteins fused with GAL4 AD (see Materials and Methods) and the full-length regulatory domain of PKCbI fused with GAL4 DBD as preys and baits, respectively. b-Gal activity of yeast transformants was assayed by the plate method as described in Materials and Methods (11, positive; 1, weakly positive; 2, negative). Based on the sequence comparison (10), the primary structures of NELL proteins are shown with residue numbers and divided into signal peptides (closed boxes), NH 2-terminal TSP-1-like modules (open boxes), vWF C domains (hatched boxes), and EGF-like domains (shaded boxes).
ulated spatiotemporarily during the embryogenesis, the NELL proteins (at least NELL2) have been suggested to participate in the growth and differentiation of neural cells (10). To induce such neural cell actions, NELL proteins containing EGF-like domains should interact with cellular proteins still unidentified. To examine whether NELL proteins function as ligands for the ordinary EGF receptor (ErbB) family, the proteins secreted into the culture medium of COS-7 cells were incubated with MDA-MB-453 cells that express all known EGF receptors (ErbB1–ErbB4) (19). However, no ErbB proteins were phosphorylated by NELL proteins added at a concentration of more than 30 ng/ml, despite that the tyrosine phosphorylation of ErbB1 was indeed induced by incubation with 1 ng/ml EGF (Higashiyama, S., unpublished results). Thus, further studies were needed to identify the cellular proteins interacting with NELL proteins, which must be different from the known ErbB proteins. In addition to the EGF-like domains, NELL proteins contain functional motifs related to TSP-1; a signal
V1) of PKC and interact with PKC in an isoformspecific manner. Intracellular localization of NELL proteins in COS-7 cells. We have reported that the rat NELL1 and NELL2 proteins expressed as homotrimeric glycoproteins in COS-7 cells are mainly found in the intracellular fractions and partially secreted into the culture medium (10). In addition, the two NELL proteins appeared to exist in different cytoplasmic sites, when analyzed by Western blotting of the NELL-expressing COS-7 cells (10). Therefore, intracellular localization of NELL proteins was further analyzed by an immunocytochemical fluorescence method using NELL-FLAG proteins detected indirectly with an anti-FLAG antibody. As shown in Fig. 5A, the NELL2 protein is present uniformly in the cytoplasm, whereas NELL1 is located at unique fibrillar structures (presumably the ER) within the cytoplasm (Fig. 5B). A recent immunocytochemical electron microscopic analysis of rat brain also indicated the localization of NELL proteins mainly around the ER of neuronal cells and partially in other intracellular compartments, existing in both nonglycosylated and glycosylated forms (Oyasu, M., Kuroda, S., Nakashita, M., Fujimiya, M., Kikkawa, U., and Saito, N., submitted). These results suggest that the NELL1 and NELL2 proteins are localized at distinct intracellular sites probably with different functions. DISCUSSION Because the NELL2 mRNA is expressed efficiently in the adult neural tissues exhibiting neuronal high plasticity (e.g., hippocampus) and its expression is reg-
FIG. 4. Delineated structures of the regulatory domains of PKC isoforms and interaction with NELL1 and NELL2 proteins analyzed by the yeast two-hybrid assay. The yeast two-hybrid assay was done using various deletion mutants of PKC regulatory domains fused with GAL4 DBD (see Materials and Methods) and the full-length NELL protein fused with GAL4 AD as baits and a prey, respectively. b-Gal activity of yeast transformants was assayed by the plate method as described in Materials and Methods (11, positive; 1, weakly positive; 2, negative). Based on the sequence comparison (1, 2), the primary structures of the regulatory domains of PKC isoforms are shown with residue numbers and divided into conserved regions (C1 and C2) (open boxes) and variable regions (V1–V3) (lines).
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FIG. 5. Intracellular localization of NELL proteins in COS-7 cells. COS-7 cells expressing either NELL2-FLAG (A) or NELL1-FLAG (B) were stained by an immunocytochemical fluorescence method using an anti-FLAG antibody and observed under a confocal laser scanning microscopy.
peptide required for sorting to the ER, an NH 2terminal TSP-1-like module required for the heparinbinding activity, and five vWF C domains required for the homotrimeric oligomerization (10). In the present studies, the EGF-like domains have been demonstrated to interact with the NH 2-terminal V1 region of specific PKC isoforms. This is a novel protein-protein interaction identified for the first time between those domains contained in intracellular proteins and PKC. It is thereby suggested that the NELL proteins serve as intracellular signaling molecules by interacting with PKC through their EGF-like domains. For elucidation of physiological significance of the NELL/PKC interactions, cellular and intracellular colocalization of NELL proteins and PKC isoforms must be considered. PKCbII and « were previously shown to be present abundantly in the hippocampus and cerebral cortex by immunocytochemical analysis (20, 21). PKCbI is less contained in the hippocampus, while PKCz is widely distributed in the brain (22). Since the NELL2 protein is abundant in the hippocampal pyramidal cells showing neuronal high plasticity (10), PKCbII, «, and z are thus good candidates of PKC isoforms interacting with NELL2 (see Fig. 4). In contrast, the amount of NELL1 protein in the neural tissues examined is much lower than that of NELL2 (8, 10). Recently, we have found that the NELL1 mRNA is up-regulated in the cranial intramembranous bone and neural tissue, both of which are originated from neural crest cells during neurulation (9). Therefore, the NELL1 protein may interact with either PKCbI, bII, d, or z in such a restricted neural tissue (see Fig. 4). It is possible that both NELL proteins play important roles in the development and maintenance of neural tissues.
In the hippocampal neuronal cells, NELL2 protein was found mainly around the rough ER and partially in other intracellular compartments (Oyasu, M., Kuroda, S., Nakashita, M., Fujimiya, M., Kikkawa, U., and Saito, N., submitted). Although PKCg (23) and PKCh (24) have been reported to be localized around ER, no other PKC isoforms have been analyzed for the existence around ER, the NELL-interacting endogenous PKC isoforms remaining to be identified. In non-neural cancer cells, the NELL mRNAs were found to be expressed abundantly; the NELL1 mRNA in Burkitt’s lymphoma Raji cells and the NELL2 mRNA in colorectal adenocarcinoma SW480 cells (10). Also in the hemopoietic cells, the NELL mRNAs are expressed and developmentally regulated in the B lineage (25). These findings strongly suggest that the NELL proteins possess potential oncogenic activities in non-neural tissues. Because PKC isoforms play important roles in the cell growth, differentiation, oncogenesis, and apoptosis by phosphorylating specific proteins (1, 2), the intracellular NELL proteins are presumed to be phosphorylated by specific PKC isoforms and then exert their signaling functions, still unidentified though. ACKNOWLEDGMENTS We thank Professor S. Higashiyama for examining the ErbB phosphorylation assay using MDA-MB-453 cells. We are grateful to M. Oyasu, M. Nakashita, C. Tokunaga, M. Kawakami, K. Tatematsu, N. Nakagawa, and N. Kanayama for their technical assistance. We are also grateful to Professors T. Abe, S. Matsuhashi, K. Ting, N. Saito, U. Kikkawa, and Y. Nishizuka for their helpful advice.
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Involvement of Epidermal Growth Factor-like Domain of NELL Proteins in the Novel Protein–Protein Interaction with Protein Kinase C 1 Shun’ichi Kuroda 2 and Katsuyuki Tanizawa Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan
Received October 16, 1999
NELL proteins are the thrombospondin-1-like proteins that are strongly expressed in neural tissues, containing six epidermal growth factor (EGF)-like domains. By radiolabeling of the NELL proteinexpressing COS-7 cells with [ 32P]orthophosphate, here we demonstrate that NELL proteins are synthesized as phosphoproteins by interacting with protein kinases in the cells. By immunoprecipitation and in vitro phosphorylation assays, we have also found that NELL proteins expressed in COS-7 cells are associated with and phosphorylated by protein kinase C bI (PKCbI). Further analysis using various deletion mutants of NELL proteins by the yeast two-hybrid assay has revealed their EGF-like domains to be involved in the isoform-specific interaction with PKC. Conversely, the NH 2-terminal variable region of PKC isoforms has been found essential for the interaction with NELL proteins. Because NELL proteins are expressed mainly in the cytoplasm of neuronal cells, unlike most EGF-like domain-containing extracellular proteins, the novel protein–protein interaction identified here between the EGF-like domains of NELL proteins and PKC suggests that EGF-like domains of intracellular proteins can be a target of PKC that mediates various signaling pathways. © 1999 Academic Press
Abbreviations: PKC, protein kinase C; EGF, epidermal growth factor; TSP-1, thrombospondin-1; vWF, von Willebrand factor; ER, endoplasmic reticulum; NELL-FLAG, COOH-terminally FLAGtagged NELL protein; PKCbI-HA, NH 2-terminally HA-tagged PKCbI; GAL4 AD, GAL4 activating domain; GAL4 DBD, GAL4 DNA-binding domain. 1 This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (#09780576, #11780446, #11139241), research grants from KatoMemorial Bioscience Foundation, Association for the Progress of New Chemistry (ASPRONC), Takeda Science Foundation, and TERUMO Life Science Foundation (to S.K.). 2 To whom correspondence should be addressed at Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 5670047, Japan. Fax: 181-6-6879-8464. E-mail: [email protected]. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Protein kinase C (PKC) is a Ca 21/phospholipiddependent Ser/Thr protein kinase and is classified into at least 10 isoforms (1, 2), each of which bears a specific physiological function in various cellular activities, such as cell growth, differentiation, oncogenesis, and apoptosis, by interacting with their own target proteins (3– 6). Recently, we have identified new PKCinteracting proteins, NELL1 and NELL2, which are homologous to the NELL proteins that are expressed abundantly in neural tissues and contain six epidermal growth factor (EGF)-like domains (7–9), by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of PKCbI as a bait (10). The rat NELL proteins show about 55% identities with each other and possess, in addition to the EGF-like domains (11, 12), protein motifs similar to thrombospondin-1 (TSP-1); an NH 2-terminal TSP-1-like module (13) and five von Willebrand factor (vWF) C domains (14, 15) (see Fig. 3). In chick embryo, the NELL2 mRNAs are ubiquitously expressed in all tissues, but after hatching the expression is restricted only in neural tissues (7). We have also shown that the expression of NELL1 and NELL2 mRNAs in mouse embryo is rigorously regulated during embryogenesis (10). These findings have led to a suggestion that NELL proteins may play important roles in the development of neural tissues. The EGF-like domain is a polypeptide segment with 40 –50 amino acid residues including 6 conserved Cys residues and has so far been found in about 100 eukaryotic proteins related to cell proliferation, growth inhibition, and differentiation (11). These EGF-like domain-containing proteins mainly occur in the extracellular fractions, as membrane-associated or secreted proteins. However, some intracellular proteins are also known to contain EGF-like domains, e.g., NELL proteins occurring mainly in the cytoplasm (10), amphiregulin present in the nucleus (16), and prostaglandin endoperoxide H synthases existing in the endoplasmic reticulum (ER) and nuclear envelopes (17). Some groups of EGF-like domains have been demonstrated
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to participate in the Ca 21-dependent protein-protein interactions, although their interacting proteins have been unidentified (12). To identify proteins interacting with EGF-like domains, particularly with those contained in the intracellular proteins, we have dissected the NELL proteins newly isolated as PKC-interacting proteins by the yeast two-hybrid screening. We here report that the EGF-like domains of NELL proteins interact with PKC in an isoform-specific manner and suggest that those domains in the intracellular proteins may be a target of PKC that mediates various intracellular signaling pathways. MATERIALS AND METHODS Expression of epitope-tagged proteins in COS-7 cells. For expression of the COOH-terminally FLAG-tagged NELL proteins (NELL1FLAG, NELL2-FLAG), the plasmids pTB701-FLC-NELL1 and pTB701-FLC-NELL2 were used (10). For expression of the NH 2terminally HA-tagged form of PKCbI (PKCbI-HA) and kinasenegative PKCbI (K371M PKCbI-HA), plasmids pTB701-HA-PKCbI and pTB701-HA-K371M PKCbI (4) were used, respectively. These plasmids were transferred into COS-7 cells by electroporation using a Gene Pulser II (Bio-Rad Laboratories). COS-7 cells were cultured for 60 –72 h in a Dulbecco’s modified MEM (DMEM) medium supplemented with 10% (v/v) fetal bovine serum, and then used for expression assays. In vivo protein labeling with [ 32P]orthophosphate. About 5 3 10 7 COS-7 cells expressing either NELL1-FLAG or NELL2-FLAG were incubated for 5– 6 h in 5 ml of a phosphate-free DMEM medium (Life Technologies) supplemented with 10% (v/v) dialyzed FBS (Life Technologies) and 0.5 mCi/ml of [ 32P]orthophosphate (;9000 Ci/mmol, NEN). The NELL-FLAG proteins recovered in the cytoplasmic fractions were immunoprecipitated with 2 mg of an anti-FLAG monoclonal antibody (M2, Eastman Kodak) as described below, subjected to SDS-PAGE, and then autoradiographed. Immunoprecipitation. COS-7 cells from a 10-cm plate (about 5 3 10 7 cells) expressing either NELL1-FLAG or NELL2-FLAG were suspended in 500 ml of lysis buffer (4). Cleared lysates (500 ml) were incubated on ice for 1 h with 2 mg of an anti-FLAG or anti-HA (12CA5, Boehringer Mannheim) monoclonal antibody, and then mixed with 20 ml of protein G-Sepharose 4 fast flow beads (50% slurry, Pharmacia). After incubation at 4°C for 1 h with shaking, the beads were washed four times with lysis buffer, subjected to SDSPAGE, and then analyzed by Western blotting. In vitro phosphorylation assay. Cleared lysates (500 ml) prepared from the COS-7 cells (about 5 3 10 7 cells) coexpressing a NELLFLAG protein and PKCbI-HA were subjected to the immunoprecipitation using 2 mg of an anti-HA antibody and 20 ml of protein G-Sepharose 4 fast flow beads (50% slurry). The beads were mixed with 25 ml of the reaction mixture (20 mM Tris, pH 7.5, 10 mM MgCl 2, 1 mM CaCl 2, 20 mM ATP, 8 mg/ml phosphatidylserine, 0.8 mg/ml diolein) and 1 ml of [g- 32P]ATP (10 mCi/ml, .7000 Ci/mmol, Amersham). The reaction mixture was incubated at 30°C for 30 min, subjected to SDS-PAGE, and then autoradiographed. Yeast two-hybrid assay. Protein-protein interaction was analyzed by the yeast two-hybrid assay (18) using both of the NELL proteins fused with the GAL4 activating domain (GAL4 AD) and the regulatory domain of PKC fused with the GAL4 DNA-binding domain (GAL4 DBD) in the yeast strain CG-1945 (Clontech). Deletion mutants of PKC isoforms used for the yeast two-hybrid assay were as follows (4, 6): a-R, residues 1–336 of rat PKCa; b-R, residues 1–340 of rat PKCbI; b-V1, residues 1–19 of rat PKCbI; b-V1/C1, residues 1–159 of rat PKCbI; b-C1, residues 20 –159 of rat PKCbI; b-V2/V3,
residues 160 –340 of rat PKCbI; g-R, residues 1–349 of rat PKCg; d-R, residues 1–345 of rat PKCd; «-R, residues 1– 406 of rat PKC«; «-V1, residues 1–135 of rat PKC«; «-V1/C1, residues 1–297 of rat PKC«; «-C1/V3, residues 134 – 406 of rat PKC«; z-R, residues 1–250 of rat PKCz; z-V1, residues 1–113 of rat PKCz; z-V1/C1, residues 1–180 of rat PKCz; and z-C1/V3, residues 114 –250 of rat PKCz. Deletion mutants of NELL proteins used were as follows: NELL2, residues 1– 819 (full length) of rat NELL2; NELL2DN1, residues 443– 819 of rat NELL2; NELL2DN2, residues 663– 819 of rat NELL2; NELL1, residues 1– 810 (full length) of rat NELL1; NELL1DN1, residues 434 – 810 of rat NELL1; and NELL1DN2, residues 654 – 810 of rat NELL1. The activity of b-galactosidase (b-Gal) in yeast cells was detected by the plate assay method (18). Briefly, yeast transformants were transferred onto nylon membranes, permeabilized in liquid nitrogen, and placed on Whatman 3MM papers that had been soaked in Z buffer (4) containing 1 mg/ml 5-bromo-4-chloro-3-indolylb-D-galactoside (X-gal). After developing at 37°C for 10 h, the yeast cells forming either dark blue or blue colonies were classified into a positive (11) and a weakly positive group (1), respectively. The yeast cells forming white colonies were classified into a negative group (2). All measurements were repeated at least four times. Immunocytochemical observation of COS-7 cells expressing NELLFLAG proteins. COS-7 cells (about 5 3 10 5 cells) expressing either NELL1-FLAG or NELL2-FLAG were grown on cover glasses (1-cm diameter), washed with phosphate-buffered saline (PBS), and fixed with 4% (w/v) paraformaldehyde in PBS at room temperature for 30 min. The cells were washed again with PBS and permeabilized by incubating in PBS containing 0.25% (v/v) Triton X-100, 5% (v/v) normal goat serum, and 5% (w/v) skim milk at room temperature for 30 min. The anti-FLAG antibody was applied at a concentration of 0.4 mg/ml at 4°C and allowed to react overnight. A fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Amersham) was added at a concentration of 1 mg/ml at room temperature, and after 30 min the fluorescence was observed under a Zeiss LSM410 confocal laser scan microscope (Carl Zeiss).
RESULTS In vivo phosphorylation of NELL proteins expressed in COS-7 cells. When the plasmids carrying the COOH-terminally FLAG-tagged NELL genes were introduced into COS-7 cells, two types of FLAG-tagged proteins with molecular sizes of about 140-kDa and .400-kDa were found to be expressed in the cytoplasm by Western blotting (10). The .400-kDa protein was thought to be a homotrimeric form of the 140-kDa protein. In this study, COS-7 cells transiently expressing the NELL-FLAG proteins were labeled with [ 32P]orthophosphate for 5– 6 h. The cleared lysates obtained were mixed with an anti-FLAG antibody and the immunoprecipitates were subjected to SDS-PAGE, followed by autoradiography. As shown in Fig. 1, both of the 140- and .400-kDa proteins were found to be phosphorylated. The .400-kDa protein were converted to 140-kDa protein by prolonged denaturation before applying to SDS-PAGE (data not shown). This result indicates that the NELL proteins are phosphorylated in vivo by interacting with intracellular protein kinases. The NELL-FLAG proteins secreted into the medium (10) were also found to be phosphorylated (data not shown).
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FIG. 1. In vivo phosphorylation of NELL proteins expressed in COS-7 cells. COS-7 cells expressing either NELL1-FLAG or NELL2FLAG proteins were labeled with [ 32P]orthophosphate for 5– 6 h. The NELL-FLAG proteins in the cytoplasm were immunoprecipitated with an anti-FLAG antibody, subjected to SDS-PAGE, and then autoradiographed. The positions of NELL-FLAG proteins are indicated with arrows.
In vivo association of NELL proteins with PKC. NELL proteins were newly isolated as PKCbIinteracting proteins by the yeast two-hybrid screening (10). In the present studies, intracellular interaction of NELL proteins with PKCbI was analyzed by immunoprecipitation using COS-7 cells coexpressing the NELL-FLAG proteins and PKCbI-HA. In the anti-HA immunoprecipitates, the NELL2-FLAG protein was detected by Western blotting with an anti-FLAG antibody (Fig. 2, 3rd panel), confirming that the NELL2 protein associates in vivo with PKCbI. NELL2 protein in the anti-HA immunoprecipitates underwent phosphorylation in the presence of PKC activators (Ca 21, phosphatidylserine, and diolein) and [g- 32P]ATP (Fig. 2, 4th panel). Simultaneously, autophosphorylation of PKCbI was also observed. When a kinase negative mutant of PKCbI (K371M PKCbI-HA) was used instead of the wild-type PKCbI, the association with NELL2 protein was unaffected, but NELL2 protein was not phosphorylated under the same conditions, indicating that PKCbI can phosphorylate NELL2 protein but the kinase activity of PKCbI is not essential for the formation of the NELL2/PKCbI complex. Similarly, when the COS-7 cells coexpressing NELL1FLAG and PKCbI-HA were used for the immunoprecipitation assay, both the NELL1/PKCbI complex formation and the phosphorylation of NELL1 protein by PKCbI were observed (data not shown). Thus, it was assumed that the NELL proteins exhibit their functions by interacting with and being phosphorylated by PKC (presumably PKCbI). Mapping of regions involved in the NELL/PKC interaction. An essential region of NELL proteins for the interaction with PKCbI was surveyed by the yeast two-hybrid assay using the deletion mutants of NELL proteins as baits. As shown in Fig. 3, the EGF-like
domains of NELL1 and NELL2 proteins were identified to be essential for the interaction with the regulatory domain of PKCbI. Some groups of EGF-like domains have been demonstrated to participate in the Ca 21-dependent protein-protein interactions (12). NELL proteins possess three Ca 21-binding sites in their 2nd, 5th, and 6th EGF-like domains (10). Therefore, requirement of Ca 21 ion for the NELL/PKC interactions would be one of the foremost subjects to be investigated. Next, interactions of NELL proteins with other PKC isoforms were analyzed by the yeast two-hybrid assay (Fig. 4). When the regulatory domains of PKC isoforms (a, bI, g, d, «, and z) (1, 2) were used as baits, NELL2 protein was found to interact strongly with PKCbI, «, and z, weakly with PKCg and d, and not with PKCa. On the other hand, NELL1 protein interacted strongly with PKCd, weakly with PKCbI and z, and not with PKCa, g, and «. Furthermore, by using deletion mutants of PKCbI, «, and z, NELL2 protein was found to interact with the NH 2-terminal regions of PKCbI, «, and z, and NELL1 protein with those of PKCbI and z. Thus, the NELL proteins recognize the NH 2-terminal regulatory regions (in particular, the variable region,
FIG. 2. In vivo association of NELL2 protein and PKCbI. Either PKCbI-HA or its kinase negative mutant K371M PKCbI-HA was coexpressed with NELL2-FLAG protein in COS-7 cells. The lysates were immunoprecipitated (IP) and then blotted (Blot) with either an anti-FLAG or anti-HA antibody. 1st panel, detection of NELL2FLAG proteins in the anti-FLAG immunoprecipitates with an antiFLAG antibody. 2nd panel, detection of PKCbI-HA or K371M PKCbI-HA in the anti-HA immunoprecipitates with an anti-HA antibody. 3rd panel, detection of NELL2-FLAG proteins in the anti-HA immunoprecipitates with an anti-FLAG antibody. 4th panel, in vitro phosphorylation assay in the anti-HA immunoprecipitates. The positions of NELL2-FLAG protein and PKCbI-HA are indicated with arrows.
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FIG. 3. Delineated structures of NELL proteins and interaction with the regulatory domains of PKCbI analyzed by the yeast twohybrid assay. The yeast two-hybrid assay was done using various deletion mutants of NELL proteins fused with GAL4 AD (see Materials and Methods) and the full-length regulatory domain of PKCbI fused with GAL4 DBD as preys and baits, respectively. b-Gal activity of yeast transformants was assayed by the plate method as described in Materials and Methods (11, positive; 1, weakly positive; 2, negative). Based on the sequence comparison (10), the primary structures of NELL proteins are shown with residue numbers and divided into signal peptides (closed boxes), NH 2-terminal TSP-1-like modules (open boxes), vWF C domains (hatched boxes), and EGF-like domains (shaded boxes).
ulated spatiotemporarily during the embryogenesis, the NELL proteins (at least NELL2) have been suggested to participate in the growth and differentiation of neural cells (10). To induce such neural cell actions, NELL proteins containing EGF-like domains should interact with cellular proteins still unidentified. To examine whether NELL proteins function as ligands for the ordinary EGF receptor (ErbB) family, the proteins secreted into the culture medium of COS-7 cells were incubated with MDA-MB-453 cells that express all known EGF receptors (ErbB1–ErbB4) (19). However, no ErbB proteins were phosphorylated by NELL proteins added at a concentration of more than 30 ng/ml, despite that the tyrosine phosphorylation of ErbB1 was indeed induced by incubation with 1 ng/ml EGF (Higashiyama, S., unpublished results). Thus, further studies were needed to identify the cellular proteins interacting with NELL proteins, which must be different from the known ErbB proteins. In addition to the EGF-like domains, NELL proteins contain functional motifs related to TSP-1; a signal
V1) of PKC and interact with PKC in an isoformspecific manner. Intracellular localization of NELL proteins in COS-7 cells. We have reported that the rat NELL1 and NELL2 proteins expressed as homotrimeric glycoproteins in COS-7 cells are mainly found in the intracellular fractions and partially secreted into the culture medium (10). In addition, the two NELL proteins appeared to exist in different cytoplasmic sites, when analyzed by Western blotting of the NELL-expressing COS-7 cells (10). Therefore, intracellular localization of NELL proteins was further analyzed by an immunocytochemical fluorescence method using NELL-FLAG proteins detected indirectly with an anti-FLAG antibody. As shown in Fig. 5A, the NELL2 protein is present uniformly in the cytoplasm, whereas NELL1 is located at unique fibrillar structures (presumably the ER) within the cytoplasm (Fig. 5B). A recent immunocytochemical electron microscopic analysis of rat brain also indicated the localization of NELL proteins mainly around the ER of neuronal cells and partially in other intracellular compartments, existing in both nonglycosylated and glycosylated forms (Oyasu, M., Kuroda, S., Nakashita, M., Fujimiya, M., Kikkawa, U., and Saito, N., submitted). These results suggest that the NELL1 and NELL2 proteins are localized at distinct intracellular sites probably with different functions. DISCUSSION Because the NELL2 mRNA is expressed efficiently in the adult neural tissues exhibiting neuronal high plasticity (e.g., hippocampus) and its expression is reg-
FIG. 4. Delineated structures of the regulatory domains of PKC isoforms and interaction with NELL1 and NELL2 proteins analyzed by the yeast two-hybrid assay. The yeast two-hybrid assay was done using various deletion mutants of PKC regulatory domains fused with GAL4 DBD (see Materials and Methods) and the full-length NELL protein fused with GAL4 AD as baits and a prey, respectively. b-Gal activity of yeast transformants was assayed by the plate method as described in Materials and Methods (11, positive; 1, weakly positive; 2, negative). Based on the sequence comparison (1, 2), the primary structures of the regulatory domains of PKC isoforms are shown with residue numbers and divided into conserved regions (C1 and C2) (open boxes) and variable regions (V1–V3) (lines).
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FIG. 5. Intracellular localization of NELL proteins in COS-7 cells. COS-7 cells expressing either NELL2-FLAG (A) or NELL1-FLAG (B) were stained by an immunocytochemical fluorescence method using an anti-FLAG antibody and observed under a confocal laser scanning microscopy.
peptide required for sorting to the ER, an NH 2terminal TSP-1-like module required for the heparinbinding activity, and five vWF C domains required for the homotrimeric oligomerization (10). In the present studies, the EGF-like domains have been demonstrated to interact with the NH 2-terminal V1 region of specific PKC isoforms. This is a novel protein-protein interaction identified for the first time between those domains contained in intracellular proteins and PKC. It is thereby suggested that the NELL proteins serve as intracellular signaling molecules by interacting with PKC through their EGF-like domains. For elucidation of physiological significance of the NELL/PKC interactions, cellular and intracellular colocalization of NELL proteins and PKC isoforms must be considered. PKCbII and « were previously shown to be present abundantly in the hippocampus and cerebral cortex by immunocytochemical analysis (20, 21). PKCbI is less contained in the hippocampus, while PKCz is widely distributed in the brain (22). Since the NELL2 protein is abundant in the hippocampal pyramidal cells showing neuronal high plasticity (10), PKCbII, «, and z are thus good candidates of PKC isoforms interacting with NELL2 (see Fig. 4). In contrast, the amount of NELL1 protein in the neural tissues examined is much lower than that of NELL2 (8, 10). Recently, we have found that the NELL1 mRNA is up-regulated in the cranial intramembranous bone and neural tissue, both of which are originated from neural crest cells during neurulation (9). Therefore, the NELL1 protein may interact with either PKCbI, bII, d, or z in such a restricted neural tissue (see Fig. 4). It is possible that both NELL proteins play important roles in the development and maintenance of neural tissues.
In the hippocampal neuronal cells, NELL2 protein was found mainly around the rough ER and partially in other intracellular compartments (Oyasu, M., Kuroda, S., Nakashita, M., Fujimiya, M., Kikkawa, U., and Saito, N., submitted). Although PKCg (23) and PKCh (24) have been reported to be localized around ER, no other PKC isoforms have been analyzed for the existence around ER, the NELL-interacting endogenous PKC isoforms remaining to be identified. In non-neural cancer cells, the NELL mRNAs were found to be expressed abundantly; the NELL1 mRNA in Burkitt’s lymphoma Raji cells and the NELL2 mRNA in colorectal adenocarcinoma SW480 cells (10). Also in the hemopoietic cells, the NELL mRNAs are expressed and developmentally regulated in the B lineage (25). These findings strongly suggest that the NELL proteins possess potential oncogenic activities in non-neural tissues. Because PKC isoforms play important roles in the cell growth, differentiation, oncogenesis, and apoptosis by phosphorylating specific proteins (1, 2), the intracellular NELL proteins are presumed to be phosphorylated by specific PKC isoforms and then exert their signaling functions, still unidentified though. ACKNOWLEDGMENTS We thank Professor S. Higashiyama for examining the ErbB phosphorylation assay using MDA-MB-453 cells. We are grateful to M. Oyasu, M. Nakashita, C. Tokunaga, M. Kawakami, K. Tatematsu, N. Nakagawa, and N. Kanayama for their technical assistance. We are also grateful to Professors T. Abe, S. Matsuhashi, K. Ting, N. Saito, U. Kikkawa, and Y. Nishizuka for their helpful advice.
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