Archives of Insect Biochemistry and Physiology 61:65� (2006) Molecular Cloning and Expression of Protein Kinase C From Bombyx mori 1 1 Tomohide Uno, * Atsushi Nakao, 1 Nakada, and Osamu Itoh 1 2 Yoshihiro Fujiwara, 1 Chisato Katsurauma, Takuya Two partial cDNA clones (Protein kinase C alpha and Protein kinase C iota), each of which encoded a different member of PKCprotein family, were isolated using RT-PCR from mRNA of Bombyx mori. The full-length cDNAs were isolated using SMARTRACE. The cDNAs were expressed in HepG2 cells and the recombinant proteins were partially purified using an affinity chromatography. Protein kinase C alpha (BPKC alpha) showed a calcium-dependent kinase activity of histones. Whereas protein kinase C iota (BPKC iota) showed a calcium-independent activity. Bisindolyl maleimide I, a PKC inhibitor, inhibited these kinase activities. Furthermore, in vitro BPKC alpha interacted and phosphorylated two proteins expressed in the brain of Bombyx mori: Rab protein, which plays important roles in the vesicle transport in the brain, and bMBD2/3, which is a methyl DNA-binding protein and regulates transcription. These results suggest that these PKCs phosphorylate various substrate proteins and function in the brain of Bombyx mori. Arch. Insect Biochem. Physiol. 61:65�, 2006. � 2006 Wiley-Liss, Inc. KEYWORDS : Bombyx mori; brain; phosphorylation; protein kinase C INTRODUCTION PKCs share the same kinase domain and ATP binding site; they differ in their N-terminal domain that Protein kinase C (PKC) is a serine/threonine protein kinase that plays many roles in the regulation of growth and differentiation. Following acti- regulates the catalytic domains and carries the calcium and diacyl glycerol binding sites. In insects, PKC was partially purified from neu- vation, PKC is translocated to different cellular ral tissue of honey bee, Apis mellifera, and showed compartments where it phosphorylates substrate a calcium-dependent protein kinase activity (Alt- proteins (Nishizuka, 1986). PKCs are classified in felder et al., 1991). Furthermore, a specific substrate three groups according to their structural and en- for this PKC was purified to an apparent homoge- zymatic properties (Quest, 1996). The first group, neity conventional PKCs (cPKCs) composed of the (Muller, 1997). Various PKCs were identified us- bI, bII, g a, from neuronal tissue of the honey bee isoforms, require calcium, diacyl- ing a Drosophila genomic database (Morrison et al., glycerol, and negatively charged phospholipids for 2000). Genetic and immunostaining studies indi- their kinase activity; the second group of novel cate that insect PKC plays important roles in for- PKCs (nPKCs), aging behavior, learning, and memory (Humphries and d, e, q, and h, require diacylglycerol and negatively charged phospholipids; and the et al., 2003; Choi et al., 1991; Alshuaib et al., third group of atypical PKCs (aPKCs) 2004). Recent reports indicate that PKC is essen- i, z, l, and m only require negatively charged phospholipids. All 1 2 tial to cell polarity in epithelial cells, neuroblasts, Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku Hyogo, Japan Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan *Correspondence to: T. Uno, Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku Hyogo 657-8501, Japan. E-mail: email@example.com Received 5 January 2005; Accepted 2 August 2005 � 2006 Wiley-Liss, Inc. DOI: 10.1002/arch.20098 Published online in Wiley InterScience (www.interscience.wiley.com) 66 Uno et al. and oocytes (von Stein et al., 2005; Hutterer et al., Isolation of cDNA Clones 2004; Betschinger et al., 2005; Djiane et al., 2005), synapse development (Ruiz-Canada et.al., 2004), The fat body was dissected from 50 silkworms and diuretic activity by diuretic hormone (Tobe et in sterile PBS (50 mM sodium phosphate, pH 7.5, al., 2005). and 150 mM NaCl), frozen rapidly in liquid nitro- In Bombyx mori, inhibitors specific to PKC in- gen, and stored at �癈 until use. Total RNA was hibited the secretion of prothoracicotropic hor- prepared by the acid guanidine phenol extraction mone, a neuropeptide related to metamorphosis, method using Sepasol (Nakalai Tesque, Kyoto, Ja- from the brain to the hemolymph (Shirai et al., pan). Day-2 fifth instar larvae were used for cDNA 1997). protein cloning. Poly (A) RNA was isolated from total RNA kinase phosphorylated BRab proteins, which regu- using a GENE Elute TM mRNA mini prep kit late neuro- (Sigma) according to the manufacturer抯 protocol. transmitters, or neuropeptides in the neural cell Powerscript reverse transcriptase (BD Biosciences, (Uno and Hiragaki, 2003; Uno et al., 2004). Thus, Palo Alto, CA) was used to synthesize cDNA. cDNA PKC is important to the neuropeptide or protein was amplified using degenerate PCR primers de- secretion of the insect brain. But there is no re- duced from the PKC amino acid sequences of other port to express insect protein kinase C in mam- species. The BPKC alpha cDNA was amplified us- malian cells and examine the characters of the ing primers cPKC-1 and cPKC-2. The BPKC iota expressed insect PKCs. cDNA was amplified using primers aPKC-1 and In the addition, vesicle partially transport of purified proteins, + In this study, two cDNAs (BPKC alpha and aPKC-2. PCR was performed in a 25-ml volume con- iota) of PKC were isolated from Bombyx mori. The taining 70 pmol of each of the primers, 0.25 U of cDNAs were expressed in mammalian cells. The KOD plus polymerase (Toyobo, Tokyo, Japan), 200 partially purified PKCs showed protein kinase ac- nM each of dNTP, 1 mM MgCl2, and 1� accessory tivities. buffer. The PCR reaction was carried out in a Gene Amp PCR system 9700 (Perkin Elmer, Shelton, CT) with an initial incubation for 2 min at 94癈, fol- MATERIALS AND METHODS lowed by 15 cycles of 15 s at 94癈, 15 s at 55癈 Materials (decreasing 1 degree per cycle), and 1 min at 68癈, Glutathione sepharose 4B, ECL, and [g- 32 P]ATP (>1,000 Ci/mmol) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Goat peroxidaseconjugated anti-mouse IgG and mouse anti-V5 monoclonal antibody were from Cell Signaling Technology (Beverly, MA). Ni-NTA superflow resin was from Qiagen, Hilden, Germany. Lipofectamine and pcDNA 3.1/V5-His-Topo were from Novagen, Madison, WI. Histone IIIs, EGTA, and Bisindolylmaleimide I were from Sigma (St. Louis, MO). In � and, finally, 20 cycles of 15 s at 94癈, 15 s at 40癈, and 1 min at 68癈. The amplified DNA fragments were subcloned into plasmid and sequenced using an ABI 310 or 3100 autosequencer (Perkin Elmer). The 5� and 3� regions of PKC cDNAs were obtained using the SMART RACE cDNA amplification kit (BD Biosciences) according to the manufacturer抯 instructions. The 5� and 3� region of BPKC alpha was amplified with primer cPKC-3 and cPKC-4, respectively. Nested PCR was performed with primers cPKC-5 Showa (5�) and cPKC-6 (3�). The 5� and 3� region of BPKC Shogetsu) of the silkworm, Bombyx iota was amplified with primer aPKC-3 and aPKC- mori. The larvae were reared on an artificial diet 4, respectively. Nested PCR was performed with (Silkmate 2M, Nosan Co., Yokohama, Japan) at primers aPKC-5 (5�) and aPKC-6 (3�). Finally, the 25癈 under 16L-8D lighting conditions and at 70% whole coding regions of the BPKC alpha and BPKC relative humidity. The other chemicals were of the iota were cloned using primer pairs cPKC-7 and purest grade commercially available. cPKC-8, and pairs aPKC-7 and aPKC-8, respectively. this study, we used a hybrid race (Kinshu or Shunrei � Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Protein Kinase C From Bombyx mori PCR was performed with an initial incubation for � � 2 min at 94 , followed by 10 cycles of 10 s at 94 C, � 15 s at 68 C (decreased at 1 degree per cycle), and � 3 min at 68 C, and, finally, 30 cycles of 10 s at � � � 94 C, 15 s at 58 C, and 3 min at 68 C. The primers used (Sigma) are (Y=T or C; R=A or G; M=A or C; D=A, G or T; N=A, C, G or T): 67 (pH7.6), 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1% NP40 and protease inhibitor cocktail], homogenized, and then cleared by centrifugation at 12,000g for 30 min. The fusion protein was bound to a Ni-NTA superflow resin equilibrated with buffer A. After washing, proteins were eluted with a step-wise increase in imidazole. The sample was � cPKC-1; MGNAAYGAYTTYATGGG, stored at � C. Proteins were determined using cPKC-2; TCYTTRCACATNCCRAARTC, BSA (Fraction V, Sigma) as the standard by the cPKC-3; TGAACGGAGGTGATCTGATG, method of Lowry et al. (1951). cPKC-4; CAACGTCATCGTCCTGAATG, cPKC-5; AATTCAAGGAACCCGTAGCC, Western Blotting cPKC-6; CACCGTTGTAGCCCTCATCT, cPKC-7; GCAGTCTTGCGAGTAGTTGCCTGTG, Proteins were separated on a 15% polyacryla- cPKC-8; CCTCTATTTCATTGTGCCATTCACG, mide gel and blotted using a standard procedure aPKC-1; GAYGARGAYATHGAYTGGGT, (Laemmli, 1970). As primary antibody, we used aPKC-2; TCYTTRCACATNCCRTARTC, 1:5,000 diluted anti-V5 antibody. As secondary an- aPKC-3; ACGTTGTCCAGCTTGAGGTC, tibody we used 1:5,000 diluted anti-mouse IgG aPKC-4; AGGACATCGACTGGGTTCAG, coupled to peroxidase. Protein bands were visual- aPKC-5; TGACGAACTCGATCACGAAG, ized using ECL Western blotting detection reagent. aPKC-6; CTTCCAAACACCGAGCAGAT, aPKC-7; TTCTTTTGAGGGCAACGGTTTATTA, GST-Pull Down Assay aPKC-8; TTCGCTCGCTCTCGCCATTTTGAAT. The cDNA fragments containing the entire codConstruction of Expression Plasmid and ing sequence of bMBD2/3 and BRab14 were am- Expression of cDNA for BPKCs in HepG2 Cells plified by PCR with primers containing BamHI or EcoRI as described previously (Uno et al., 2005). HepG2 cells were cultured in minimal essential The amplified fragments were digested with BamHI medium supplemented with 10% fetal bovine se- and EcoRI. The digested fragment was inserted into rum and antibiotics (100 U/ml penicillin and 100 the BamHI and EcoRI sites of an expression vector streptomycin) on plastic dishes (10-cm di- in Escherichia coli, pGEX6P2 (Amersham Pharama- ameter). The cDNA fragments containing the entire cia). The cDNA was transformed into E. coli strain coding sequence of BPKC alpha and BPK iota were BL21. Transformed E. coli cells (BL21) were pre- amplified by PCR and the amplified fragment was incubated at 37 C overnight in LB medium. The subcloned into an mammalian expression vector, medium was diluted to 1:100 and incubated at pcDNA3.1/V5-His-TOPO. This cDNA was trans- 37 C for 3 h. mg/ml � � formed into HepG2 cells using Opti MEM medium The culture was 3 liters. Expression of fusion (Gibco BRL, Gaithersburg, MD) and lipofectamine. protein was then induced by adding 1 mM isopro- Sixteen hours after transfection, the medium was pyl changed and cells were incubated for another 48 h. additional incubation for 24 h at 16 C . The cells The cells were collected by centrifugation at 5,000g were collected by centrifugation at 5,000g for 5 for 5 min, and then stored at � . min, and then stored at � C. The frozen cells � b-D-thiogalactopyranoside (IPTG), followed by � � were suspended in phosphate-buffered saline (PBS) Partial Purification of PKCs [140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, � All procedures were carried out at 4 C . The frozen cells were suspended in buffer A [10 mM Hepes Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. and 1.8 mM KH2PO4 (pH7.3)], disrupted for 1 min three times by sonication and then cleared by centrifugation at 12,000g for 30 min. 68 Uno et al. Fig. 1. Nucleotide and deduced amino acids sequences of BPKC alpha ( A) and BPKC iota ( B). The nucleotide and predicted amino acid sequences of the cDNA clone encoding the protein, BPKC alpha and BPKC iota, are shown. The arrows indicate the forward and reverse primers for RT-PCR. Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Protein Kinase C From Bombyx mori 69 The fusion protein was applied to a resin bed, 1 ml of glutathione sepharose equilibrated with PBS. The resin was washed with 20 ml of PBS four times and used for interacting with BPKCs translated in vitro. Glutathione beads bound to glutathione sulfotransferase (GST) were prepared using a similar method. Next, cDNAs for BPKC alpha and iota were amplified and subcloned into pcDNA3.1/V5-His-TOPO vectors as described above. V5-peptide-tagged BPKC proteins were expressed in vitro using the TNT-coupled reticulocyte system (Promega, Madison, WI; see Fig. 5, Input). In vitro translated BPKC (25 microliter) was diluted 40 times with PBS containing 0.2% NP40. The dilute was mixed with 25 microliter of fusion protein or GST on a glutathione S-sepharose resin at room temperature for 60 min. After washing four times with 1 ml of PBS containing 0.2% NP40, proteins were extracted from the resin with SDS-PAGE sample buffer, separated on a 15% Trisglycine SDS-PAGE gel, and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated with mouse anti-V5 antibody (1:5,000). Protein bands were visualized using ECL Western blotting detection reagent and goat peroxidase-conjugated anti mouse IgG as secondary antibody. Protein Kinase Assay The typical reaction mixture with a total volume of 20 TABLE 1. ml contained 50 mM Tris-HCl (pH 7.5), Percent Identities of Amino Acid Sequences Deduced From the Nucleotide Sequences of BPKCs Fragments to Those of the Corresponding Positions of PKCs* BPKC HPKC b b g d e z h q i m Figure 1 (continued) 69.6 BPKCi 48.5 l 68.0 50.4 ll 68.0 50.4 64.3 48.4 56.8 39.7 41.9 49.6 45.1 63.4 56.1 45.3 40.5 36.3 50.0 68.4 29.5 21.6 74.4 50.3 44.8 75.8 DPKC i a a a *Nucleotide sequences of PKCs were obtained from the GenBank database. HPKC from Homo sapiens, DPKC from Drosophila melanogaster. Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. 70 Uno et al. Fig. 2. Comparison of the deduced amino acid sequence of BPKC alpha ( A) and BPKC B) with those of BPKCs. iota ( DPKC and HPKC from Droso- phila melanogaster and Homo sapiens, respectively. The regions such as the catalytic domain (CD), cysteine-rich domain (CRD), and calcium-binding domain (C2) are boxed. 10 mg/ml phosphatidyl serine, 1 mg/ml dioleine, RESULTS 6.25 mM MgCl2, 0.125 mM CaCl2, 2 mM dithio- Isolation of cDNA Clones threitol, 0.1 mM EDTA , 0.1 or 0.5 mg protein, 0.2 mM [g-32P] ATP. After in- To obtain the partial sequences of PKCs of cubation at 30癈 for 15 min, the samples were Bombyx mori, we synthesized forward and reverse treated with SDS-sample buffer. After electrophore- oligonucleotide primers for the RT-PCR, based on sis, the gel was dried and analyzed by a bioimaging PKC alpha and PKC iota from the other species analyzer (BAS 1000, Fujix, Tokyo, Japan). The po- (Fig. 1). Next, we did RT-PCR using mRNA from sition of protein on a gel was identified by Coo- Bombyx mori. The amplified fragments were sub- masie Brilliant Blue staining. BRab14, BRab8, and cloned and sequenced. U protein kinase, and 10 bMBD2/3 proteins were purified as described previously (Uno et al., 2005). As a result, the partial cDNA fragments coding PKCs were isolated from Bombyx mori. Next, Smart Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Protein Kinase C From Bombyx mori 71 Figure 2 (continued) RACE (Rapid Amplification of cDNA Ends) was insect PKCs (Fig. 2). Furthermore, BPKC iota had done to get the full-length cDNAs of BPKCs. Finally, a cysteine-rich domain (CRD) and BPKC alpha had two clones (BPKC alpha and BPKC iota) from this site and a calcium-binding site. Bombyx mori were identified and sequenced (Fig. 1). Their inserts are 2.52 and 2.98 k bp long, re- Expression of cDNA for BPKCs in Hep G2 spectively, including the poly A tail. BPKC alpha Cells and Partial Purification of the and BPKC iota bear an open reading frame corre- His-Tagged Protein sponding to aa 719 and 585, respectively. Compared to the other PKCs, BPKC alpha and BPKC The mammalian PKCs, which were expressed iota showed 74.4 and 75.8% similarity to Droso- in E. coli, do not show protein kinase activity, be- phila PKCs (Table 1). All of the protein kinase C抯 cause autophosphorylation of PKC is necessary to have a conserved motifs, which is a catalytic site change to the activated form (Flipuzzi et al.,1993; containing the ATP binding site(Quest, 1996). Dietrich et al., 1989). Probably, insect PKC expres- These sites of BPKCs were conserved among these sed in E. coli is thought not to show an activity, so Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. 72 Uno et al. we tried to express BPKCs in mammalian cells Inhibitions of activity by these reagents were ex- (HepG2 cells). At first, the cDNAs for BPKC alpha amined. As a result, activity of BPKC alpha was and BPKC iota were inserted into an expression inhibited by EGTA and BM (Fig. 4A, lanes 3 and vector and expressed in mammalian cultured cells 4), whereas BPKC iota was inhibited by BIM (Fig. as V5 and His-tagged fusion protein. The fusion 4B, lanes 3 and 4). As BPKC iota does not have a protein was extracted with buffer containing 1% calcium-binding site at the primary structure (Fig. NP40 from the cell and partially purified using a 2), the activity of the expressed BPKC iota was Ni-superflow column. Immunoblotting using an thought to be independent on calcium ion. antibody against the V5-tag peptide indicated that BPKC alpha and BPKC iota were detected (Fig. 3). Characteristics of BPKCs Phosphorylation of histones. To examine whether the expressed BPKCs in mammalian cells show protein kinase activity or not, the protein kinase activity was assayed. Histone III was used as a typical substrate. Both BPKC alpha and BPKC iota showed protein kinase activities of histone (Fig. 4; lanes 1 and 2). These results indicate that the two insect PKCs, which were expressed in mammalian cells, show protein kinase activities. Inhibition of Kinase Activity Using a Specific Inhibitor Kinase activity of mammalian PKC was inhibited by EGTA, calcium chelator, and bisindolylmaleimide I (BM), a PKC inhibitor (Beltman et al., 1996). Fig. 4. Phosphorylation of histones using BPKCs. BPKC Immunoblotting of par- [BPKC alpha (A) or BPKC iota (B)] was incubated in his- tially purified BPKCs. The cDNAs tones, 25 mM Tris HCl (pH 7.5), 1 mg/ml diacyl glyc- were expressed in HepaG2 cells as erol, 10 mg/ml phosphatidyl serine, 6.25 mM MgCl2, and V5- and His-tagged fusion proteins. 0.125 mM CaCl2 and 50 The solubilized fraction was ap- min. The samples were treated with SDS-sample buffer. plied to a Ni-resin. The eluate was The gel was autoradiographed. Lane 1: -histone; Lane 2: elelctrophoresed and immunoblot- + histone; Lane 3: EGTA was added as calcium chelator; Fig. 3. mM[g- 32 P]ATP at 25癈 for 25 ted using anti-V5 antibody. Lane1, Lane 4: Bisinodolyl maleimide I (1 BPKC alpha; Lane2, BPKC iota. PKC inhibitor. Archives of Insect Biochemistry and Physiology mM) was added as a February 2006 doi: 10.1002/arch. Protein Kinase C From Bombyx mori 73 Interaction of Various Proteins BPKCs and proteins such as bMBP2/3 and BRab14 From the Brain were examined using the GST pull-down assay (Fig. 5). BPKC was translated as a V5-tagged protein in We found that various proteins from the brain vitro and incubated with GST-fusion protein of Bombyx mori were phosphorylated by protein ki- (Rab14 or bMBD2/3) bound on a glutathione nase C, which was partially purified from the brain sepharose. After washing, the bound BPKC was de- of Bombyx mori (Uno et al., 2004). tected using V5 antibody. BRab8 was not expressed Rab proteins regulate the transport of the neu- as a GST fusion protein in E. coli. Only BRab14 ropeptide and protein in the cell (Stenmark et al., was tried as fusion protein of rab proteins. As a 2001; Tang, 2001). Lately, it was reported that rab result, BPKC alpha and BPKC iota did not interact proteins were phosphorylated by various protein ki- with glutathione sulfotransferase (GST) as a con- nases such as PKC and the activation of protein ki- trol (Fig. 5; lane 2) but interacted with bMBD2/3 nase regulated the protein transport (Bailly et al., (Fig. 5; lane 3) and BRab14 (Fig. 5; lane 4). 1991; Chiariello et al., 1999; Fitzgerald and Reed, 1999). Mammalian PKC iota was reported to inter- Phosphorylation of Various act rab2 protein and regulate the protein transport Proteins by BPKC Alpha in the early secretory pathway (Tisdale, 2003), whereas insect MBD2/3 is a methyl DNA binding The extract from insect brain showed a calcium- proteins to bind methyl CpG containing DNA and dependent protein kinase activity of BRabs and regulate the transcription of the gene. DNA methy- bMBD2/3 (Uno et al., 2005). lation modified by DNA methyl transferase has been Calcium-dependent phosphorylation of pro- implicated in the regulation of a number of genetic teins in the brain of Bombyx mori was shown to be activities during embryo development and cell dif- important to neuropeptide secretion from the brain ferentiation (Li et al., 1992; Okano et al., 1999). into the hemolymph (Shirai et al., 1997). There- protein fore, the phosphorylation of BRab8, BRab14, and (MeCP2) plays important roles in brain develop- Mammalian methyl DNA-binding bMBP2/3 by BPKC alpha, which has a calcium- ment and is regulated by phosphorylation (Chen binding domain and shows a calcium-dependent et al., 2003). bMBD2/3 was phosphorylated by pro- activity, were examined in vitro using [g- tein kinase from the brain of Bombyx mori (Uno et (Fig. 6). As a result, BPKC alpha phosphorylated al., 2005). Thus, the interactions between the all of them. The expressed BPKCs were suggested Fig. 5. 32 P] ATP GST pull-down assay of BPKCs. cDNAs of BPKCs [BPKC alpha ( A ) or BPKC iota ( B )] were transcribed and translated as V5tagged proteins in vitro. Gluta- thone-sepharose binding GST (Lane 2 ), GST-bMBD2/3 (Lane 3), and GST-BRab14 proteins (Lane 4) were bound with the translated BPKC (Lane 1). BPKC proteins interacting these GST proteins were detected using V5 antibody. Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. 74 Uno et al. to phosphorylate various substrates from the brain and regulate protein transport or transcription of gene in the brain by calcium-dependent signal transduction. DISCUSSION Insect PKCs were isolated and analyzed from Drosophila and other insects (Altfelder et al., 1991; Morrison et al., 2000). A genetic approach indicates that insect PKCs relate learning and behavior (Rui-Canada et al., 2005; Humphries et al., 2003; Choi et al., 1991). Recently, it was reported that insect PKC is related to the cell polarity of eye, asymmetric cell division, and signal transduction Fig. 6. for diuretic peptides (Betschinger et al., 2005; alpha was incubated in proteins (Lane 1: substrate; Lane Djiane et al., 2005; von Stein et al., 2005; Tobe et al., 2005). PKC regulates many important signal Phosphorylation of BRabs and bMBD2/3. BPKC 2: BRab8; Lane 3: BRab14; Lane 4: bMBD2/3), 25 mM Tris HCl (pH 7.5), 1 mg/ml diacyl glycerol, 10 mg/ml phosphatidyl serine, 6.25 mM MgCl 2, and 0.125 mM transactions in the insect brain. There is little data on phosphorylate substrates of the insect brain in vitro. We examined whether CaCl 2 and 50 mM [g- 32 P]ATP at 25 癈 for 25 min. The samples were treated with SDS-sample buffer. The gel was autoradiographed. insect PKCs phosphorylate substrates in vitro or not. As shown in Figure 1, two cDNAs for PKC of insect (BPKC alpha and BPKC iota) were isolated using RT-PCR and RACE. PKC alpha phosphory- the cultured cells (Hattula et al., 2002). Insect PKC lates in a calcium-dependent manner containing may regulate the neuropeptide secretion to cause signal transduction (Quest, 1996) and atypical phenomena such as metamorphosis and diapause PKC containing PKC iota regulates cell polarity by phosphorylating rab8 proteins. Rab proteins and synaptic plasticity (Betschinger et al., 2005; have conserved GTP binding, GTPase, and effec- Djiane et al., 2005). These BPKCs were expressed tor-binding domains (Stenmark et al., 2001; Oster- in mammalian cells and partially purified. BPKC meier and Brunger, 1999). The determination of alpha showed a calcium-dependent kinase activ- phosphorylated residues of rab8 is in progress to ity examine the functional change of rab8 by phos- for histone, whereas BPKC iota showed a calcium-independent activity (Fig. 4). Further- phorylation. more, GST pull-down experiments (Fig. 5) indi- Insect MBD protein binds methylated DNA dur- cate that in vitro translated BPKCs interacted with ing embryogenesis and represses the transcription two insect proteins, which are BRab, a small GTP- from the methylated gene (Ballestar et al., 2001). binding protein, and bMBD2/3, a methyl DNA- Mammalian Methyl DNA binding protein regulates binding the transcription of neurohormone such as Brain protein. BPKC alpha phosphorylated BRab and bMBD2/3 proteins (Fig. 6). Rab proteins regulate the neuropeptide or neu- Derived Neuropeptide Factor (BDNF) in the brain (Chen et al., 2003). rotransmitter transport in the brain (Tang, 2001; bMBD2/3 has conserved motifs such as methyl Stenmark et al., 2001). Specifically, Rab8 regulates DNA binding and transcriptional repression do- membrane traffic in developing neurons (Huber mains interacting with various nuclear proteins. et al., 1995). The distribution of mammalian Rab8 Similar to rab proteins, the determination of phos- in the cell is changed by adding PKC activator in phorylated amino acid residues will clarify the re- Archives of Insect Biochemistry and Physiology February 2006 doi: 10.1002/arch. Protein Kinase C From Bombyx mori 75 lationship between phosphorylation and the tran- Rab5a, Rab5b and Rab5c are differentially phosphorylated scriptional regulation by bMBD2/3 protein. in vitro. FEBS Lett 453:20�. BPKC alpha phosphorylated rab proteins and bMBD2/3 proteins in vitro. In vivo calcium-dependent phosphorylation of these proteins in the brain must be ascertained. Immunoprecipitation of rab and bMBP2/3 using 32 Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, Jaenisch R, Greenberg ME. 2003. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science 302:885�9. 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