68 Uno et al. Archives of Insect Biochemistry and Physiology 57:68–77 (2004) Phosphorylation of Rab Proteins From the Brain of Bombyx mori Tomohide Uno,* Atsushi Nakao, and Chisato Katsurauma Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins . These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases. Arch. Insect Biochem. Physiol. 57:68–77, 2004. © 2004 Wiley-Liss, Inc. KEYWORDS: small GTP-binding protein; Rab; Bombyx mori; brain; phosphorylation; protein kinase INTRODUCTION In eukaryotic cells, a variety of ras-related low Mr GTP-binding proteins (small GTP-binding protein) that regulate various cellular activities such as growth, differentiation, and intracellular transport have been described (Boguski and McCormick, 1993). Such small GTP-binding proteins exhibit 30–50% homologies with each other. Rab proteins, members of small GTP-binding protein, are associated with subcellular compartments in both the endocytic and exocytic pathway (Novick and Brennwald, 1993; Schimmoller et al., 1998; Pfeffer, 2001; Tuvim et al., 2001). These proteins are likely to be key regulatory components of pro- tein complexes catalyzing the fission and fusion of transport vesicles between distinct subcellular compartments. More than 50 members of the Rab family have been identified in mammals. It is thought that each Rab protein controls a specific and defined step in membrane trafficking. For instance Rab 1 is required for the transport of protein from the ER to the Golgi (Tisdale et al., 1992), Rab2 is associated with an intermediate compartment between the ER and Golgi (Tisdale and Balch, 1996), Rab3A is present on synaptic vesicles (Geppert et al., 1997), Rab5 controls membrane traffic from the plasma membrane to early endosomes (Christoforidis et al., 1999), Rab6 is distributed from medial Golgi to the TGN (Trans Golgi Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku Hyogo, Japan Abbreviations used: BRab = Bombyx mori Rab; CaM kinase = calcium/calmodulin dependent protein kinase; PKA = protein kinase A; PKC = protein kinase C. Contract grant sponsor: Ministry of Education, Science, Sports, and Culture of Japan; Contract grant number: 14760033. *Correspondence to: Tomohide Uno, Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku Hyogo 657-8501, Japan. E-mail: firstname.lastname@example.org Received 24 February 2004; Accepted 2 May 2004 © 2004 Wiley-Liss, Inc. DOI: 10.1002/arch.20014 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology Phosphoryation of Rab Proteins Network) (Martinez et al., 1994), Rab7 is associated with late endosomes (Mukhopadhyay et al., 1997), and Rab8 regulates the vesicular transport from the TGN to the plasma membrane (Huber et al., 1993). Many studies have demonstrated the importance of phosphorylation events in various aspects of cellular function. In brain, a number of kinases are stimulated upon calcium activation and are thought to play a key role in regulating the secretion process. Phosphorylations of Rab proteins in vivo or in vitro were reported. Rab1 and Rab4 proteins were phosphorylated by p34 cdc2 (Bailly et al., 1991). The activation of platelets by thrombin resulted in phosphorylation of Rab6 protein (Fitzgerald and Reed, 1999). These phosphorylations of Rab proteins were thought to regulate the transport of proteins via signal transduction. In insect, from a Drosophila melanogaster head, 9 Rabs were identified and cloned (Satoh et al., 1997). Further, 26 Rabs were identified from Drosophila genome library (Stenmark and Olkkonen, 2001). Genetic study indicates that Rab5, Rab7, and Rab11 are related to morphogen gradient formation and control Drosophila embryonic body plan during oogenesis (Dollar et al., 2002; Entchev and Gonzalez-Gaitan, 2002). Rab 6 is necessary for proper development of bristle shafts, part of the mechanosensory organs (Eggenschwiler et al., 1999). But little is known about the biochemical and functional features of Rab proteins of insects except in Drosophila. Furthermore, there is no report about phosphorylation of Rab proteins from insects. In this study, one cDNA clone of BRab8 protein from the brain of B. mori was newly expressed in Escherichia coli. BRab8 as well as two BRab proteins (BRab1 and BRab14), which were purified previously, were phosphorylated by mammalian and insect protein kinases. MATERIALS AND METHODS Materials Thrombin and [γ-32P] ATP (>1,000 Ci/mmol) were from Amersham Pharmacia Biotech (PiscatOctober 2004 69 away, NJ). CaM kinase II and PKA were from New England Bio Labs Inc (Beverly, MA). PKC was from Upstate Biotechnology (Lake Placid, NY). Ni-NTA superflow resin was from Qiagen (Chatsworth, CA). pET32a was from Novagen. Bisindolyl maleimide I was from Sigma (St. Louis, MO). The other chemicals were of the purest grade commercially available. Construction of Expression Plasmid and Expression in Escherichia coli The cDNA fragment containing the entire coding sequence of BRab8 was kindly provided by Dr. Mita of National Institute of Entomological and Sericultural Science. Sequence analysis was done using an ABI sequencer 377. Sequence homologies were done by BLAST search (Altschul et al., 1990). The cDNA fragments containing the entire coding sequence of BRab8 were amplified by PCR with primers containing BamHI or EcoRI. The amplified fragments were digested with BamHI and EcoRI. The digested fragment was inserted into the BamHI and EcoRI sites of an expression vector, pET32a. This cDNA was transformed into E. coli strain BL21. Transformed E coli cells (BL21) were incubated overnight in LB medium. The medium was diluted to 1:100 and incubated for 3 h. Expression of thioredxin-fusion protein was then induced by adding 1 mM isopropyl β-D-thiogalactopyranoside (IPTG), followed by an additional incubation for 24 h at 16°C. The cells were collected by centrifugation at 5,000g for 5 min, and then stored at –80°C. Purification of BRab Proteins All procedures were carried out at 4°C. The frozen cells (26.5 g) were suspended in Lysis buffer [50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole (pH 8.0)], disrupted by sonication, and then cleared by centrifugation at 12,000g for 40 min. The fusion protein was bound to Ni-NTA superflow resin and washed four times with 1 ml of the wash buffer [50 mM Na2HPO4, 300 mM NaCl, 20 mM imidazole (pH8.0)]. The beads were then washed 70 Uno et al. briefly in phosphate-buffered saline (PBS) [10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, and 1.8 mM KH2PO4 (pH7.3)]. The beads with bound protein were incubated with thrombin overnight at 22°C in PBS. Cleaved Rab8 proteins were recovered from the buffer. Not to overdigest with thrombin, DFP as protease inhibitor was added. The sample was concentrated on an Amicon microconcentrator and stored at –20°C. Proteins were determined using BSA (Fraction V, Sigma) as the standard by the method of Lowry et al. (1951). Expression and purification of BRab14 protein have been previously described (Uno and Hiragaki, 2003). Purification of BRab1 protein was done using the same method as BRab14 protein. SDS-PAGE: SDS-polyacrylamide gel analysis was performed according to the method of Laemmli (1970), using a 4.5% stacking gel and a 15% separating gel, at a constant current of 15 mA. The proteins in a gel were visualized with Coomassie Blue stain. Protein Kinase Assay The typical reaction mixture with a total volume of 20 µl contained 50 mM Tris-HCl (pH7.5), 6.25 mM MgCl2, 0.125 mM CaCl2, 2 mM dithiothreitol, mM EDTA , 0.1 or 0.5 µg protein, 0.2 unit protein kinase, and 10 µM [γ-32P] ATP. After incubation at 30°C for 15 min, the samples were treated with SDS-sample buffer. After electrophoresis, the gel was dried and analyzed by bioimaging analyzer (BAS 1000, Fujix). The position of BRab proteins on a gel was identified by Coomasie Brilliant Blue staining. PKC activity was assayed by adding phosphatidyl serine (10 mg/ml) and dioleine (1 mg/ ml) to the reaction mixture. CaM kinase activity was assayed by adding calmodulin (1.2 µM). Preparation of Brain Crude Homogenates From Bombyx mori Crude protein kinase fractions were obtained from the brain of B. mori. First, brains were rap- idly dissected out of 3-day-old larvae of 5th instar in Ringer‘s solution and homogenized in the homogenization buffer [25 mM Tris-HCl (pH7.5), 50 mM mercaptoethanol, 1 mM PMSF, and 0.25 M sucrose]. The homogenate (2 µg protein) was added to the reaction mixture for protein kinase assay. Purification of PKC From the Brain of B. mori The PKC active fraction from the brain was obtained, based on the methods by Altfelder et al. (1991). The brain crude homogenate was centrifuged at 50,000g for 90 min. The supernatant was applied to a DEAE-Cellulofine A-500 column equilibrated with buffer A [25 mM Tris-HCl (pH 7.5), 50 mM mercaptoethanol, and 2 mM EDTA] at a flow rate of 1 ml/min. After washing with buffer A, proteins were eluted with a step-wise increasing NaCl. The eluates were phosphorylated in the reaction mixture containing Histone IIIS (100 pmol), 25 mM Tris-HCl (pH 7.5), 1 mg/ml diacylglycerol, 10 mg/ml phosphatidylserine, 6.25 mM MgCl2 and 0.125 mM CaCl2 and 10 µM [γ-32P] ATP. After incubation and electrophoretical analysis, active fractions were pooled; CaCl2 was added to a final concentration of 2.5 mM. The solutions were sequentially applied to a phosphatidyl serine-affinity column (Altfelder et al., 1991). The column was washed with buffer A containing 1 mMCaCl2 and the protein kinase activity was eluted with buffer A containing 10 mM EGTA. Immediately after elution bovine serum albumin was added to a final concentration of 1 mg/ml to the eluted sample to stabilize protein kinase activity. RESULTS AND DISCUSSION Isolation of cDNA clone for BRab8 Previously we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family. And these were expressed in E. coli and purified using an affinity chromatography (Uno et al., 1998; Uno and Hiragaki, 2003). One partial cDNA clone (BRab8), Archives of Insect Biochemistry and Physiology Phosphoryation of Rab Proteins which encodes a member of Rab protein family, was isolated using RT-PCR from a mRNA of the brain of B. mori (Uno and Hiragaki, 2003). This clone did not contain a coding region completely. So we searched using database (silk base), based on this partial DNA sequence. As a result, one clone, which encoded a polypeptide with 210 amino acids sharing 78% similarity with the Drosophila Rab8, was obtained (Fig. 1). The primary structure of every member of Rab protein contains 5 highly conserved sequence motifs (Fig. 1, Bourne et al., 1991). Four of these regions are required for GTP-binding and/or GTPase catalytic activity. The fifth is termed an effector site that contains the amino acids especially conserved in Rab-family proteins. As shown in Figure 1, 5 characteristic regions were almost conserved among three BRab proteins. In mammals, Rab8 is localized to the Fig. 1. Alignment of the BRab protein sequences. Amino acids conserved in most Rab proteins are indicated by asterisks. The Regions E and I–IV are boxed. Regions I– IV are conserved in all members of the small GTP-binding October 2004 71 trans-Golgi network, post-Golgi vesicles, and plasma membrane (Huber et al., 1993). In the brain of mammals, Rab proteins regulate many important processes such as transports of neurotransmitters, neuropeptides, and proteins in neuron. Rab8 is involved in the transport of proteins to the dendritic surface in neurons (Huber et al., 1995). Expression of cDNA for BRab8 in E. coli and Purification of the Expressed Protein We tried the expression of BRab8 in E. coli and the biochemical properties were examined. The cDNA for BRab8 was inserted into an expression vector pET32a and expressed in E. coli as a Histagged thioredxin fusion protein. The fusion protein recovered in the soluble fraction was bound with a Ni-NTA super flow resin. The fusion pro- protein superfamily and are essential for GTP-binding and GTPase activity of the proteins. Region E is the effector region specifically conserved within Rab family proteins. 72 Uno et al. Fig. 2. SDS-PAGE analysis of BRab8 protein. The arrows indicate molecular weight markers. Purified BRab8 protein (1 µg). tein bound to the resin was cut to remove Histagged thioredxin and soluble fraction was recovered. SDS-PAGE analysis showed that the purified protein was homogeneous (Fig. 2). The evaluated molecular mass on SDS-PAGE was 28 kDa. About 0.1 mg protein was recovered from 10-l culture of E. coli. Phosphorylation of BRab Proteins Using Mammalian Protein Kinase Rab proteins regulate many processes to transport neurotransmitters and neuropeptides in the brain (Tang, 2001). Recently in mammals, some protein kinases were found to phosphorylate Rab proteins and regulate protein transport (Chiariello et al., 1999; Fitzgerald and Reed, 1999). In insect, small GTP-binding proteins (20–30 kDa) are related to the secretion of PTTH (Prothoracicotropic hormone), one of neuropeptide, from the brain of B. mori to the hemolymph and some proteins (20–30 kDa) were phosphorylated by protein kinases in the process of PTTH secretion (Shirai et al., 1998). The position of small GTP-binding proteins on electrophoresis was similar to that of phosphorylated proteins (data not shown). Further BRab14 protein was phosphorylated by the crude protein kinase from the brain of rat in vitro (Uno et al., 2003). To clarify the protein kinase to phosphorylate Rab proteins, we phosphorylated Rab proteins using mammalian protein kinases (Fig. 3). PKC, PKA, and CaM kinase were used as protein kinases because these protein kinases were identified in the brain of insect and related to the behavior of insect (Humphires et al., 2003; Shanavas et al., 1998; Alfelder et al., 1991). As a result, PKC showed protein kinase activities of BRab8 and BRab14 proteins (Fig. 3, lanes 5 and 6). BRab8 and BRab14 proteins were faintly phosphorylated by PKA (Fig. 3, lanes 11 and 12). CaM kinase faintly phosphorylated BRab8 protein (Fig. 3, lane 8). BRab1 protein was not phosphorylated (Fig. 3; lanes 4, 7, and 10 ). Theses results indicate that BRab8 and BRab14 proteins were phosphorylated by protein kinases. Phosphorylation of BRab Proteins Using the Brain Extract From B. mori Next we examined whether the protein kinase in the brain of B. mori actually phosphorylates BRab proteins or not. The brains were dissected from the larvae and homogenized. The phosphorylation activity in the extract was examined. BRab8 and BRab14 proteins were phosphorylated (Fig. 4, lanes 5 and 8) and their activities were dependent on calcium ion because EGTA, calcium ion chelator, inhibited kinase activity (Fig. 4, lanes 6 and 9). BRab1 protein was not phosphorylated (Fig. 4, lane 2). These results indicate that BRab proteins were phosphorylated by calcium-dependent protein kinases in the brain of B. mori actually. In insects such as Drosophila and honey bee, PKC is known to play important roles in sensory sigArchives of Insect Biochemistry and Physiology Phosphoryation of Rab Proteins 73 Fig. 3. Phosphorylation of BRab proteins by mammalian protein kinases. The reaction mixture containing BRab proteins (BRab1, lanes 1, 4, 7, 10; BRab8, lanes 2, 5, 8, 11; BRab14, lanes 3, 6, 9, 12) and protein kinases (PKC, lanes 4–6; CaM kinase, lanes 7–9; PKA , lanes 10–12) were incubated with [γ-32P] ATP. The samples were treated with SDS-sample buffer. After electrophoresis, the gel was analyzed by bioimaging analyzer. (-): Control (-Protein kinase). Arrows 1 and 2 indicate the positions of bands (BRab 8 and BRab14, respectively) stained with CBB. naling, foraging behavior, and learning from a genetic and immunological approach (Humphries et al., 2003; Choi et al., 1991). As a new role of PKC in the brain of insect, PKC may regulate the function of Rab proteins by phosphorylation and control the secretion of neuropeptides, relating to metamorphosis and diapause, from the brain into the hemolymph. As mentioned above, calcium-regulated protein kinases in the brain of B. mori were suggested to phosphorylate BRab proteins. Actually in mammals, it is known that calcium is required for the vesicular transport (Pind et. al, 1994), but there is little data suggesting that Rab proteins are phosphorylated by calcium-regulated kinases. Furthermore, there is no data that insect Rab protein is phosphorylated by calcium-regulated protein kinase. PKC, one of calcium-regulated protein kinases, was identified in the brain of insect using partial purification (Altfelder et al., 1991). But no data have been published on isolation of PKC from the brain of B. mori. So based on the isolation of PKC from the brain of the honey bee, Apis mellifera, Fig. 4. Phosphorylation of BRab proteins using the brain homogenate. Crude protein kinase fraction was obtained from the brain of B. mori and incubated in the reaction mixture containing BRab proteins (BRab1, lanes 1–3; BRab8, lanes 4–6; BRab14, lanes 7–9) and [γ-32P] ATP. Calcium chelator, EGTA, was added to the reaction mixture at a final concentration of 10 mM (lanes 3, 6, and 9). Control (-homogenate; lane 1, lane 4, lane 7). Isolation of PKC From the brain of B. mori October 2004 74 Uno et al. 4). These results indicate that PKC was partially purified from the brain of B. mori. Phosphorylation of BRab Proteins Using PKC From the Brain of B. mori we partially purified PKC fraction using chromatographies from the brain of B. mori. First, the brain homogenate was applied to a DEAE-Cellulofine column chromatography and the protein kinase activities of the eluates were measured. The active fraction of PKC was applied to a phosphatidyl serine affinity chromatography. The isolated PKC showed a protein kinase activity for histone (Fig. 5, lane 2) and its activity was inhibited by a bisindolyl maleimide I, a PKC-specific inhibitor and calcium chelator, EGTA (Fig. 5, lanes 3 and Next, phosphorylations of BRab proteins were tried, using the isolated PKC fraction. As a result, BRab1 protein was not phosphorylated (Fig. 6, lane 3). BRab 14 and BRab8 proteins were phosphorylated by insect PKC (Fig. 6, lanes 5 and 7). These results indicate that insect PKC phosphorylates BRab8 and BRab14 proteins, similar to mammalian protein kinases. Rab8 mediates the polarized delivery of vesicles to cell surfaces. PKC activation by phorbol ester promotes a polarized transport of Rab8-specific vesicle (Hattula et al., 2002). Phosphorylation of BRab8 protein may change the interaction with the membrane of transport vesicles and finally change the distribution in the cell. Antisense oligonucleotide ananlysis demonstrated that Rab8 in hippocampal neurons relates to the membrane transport machinery that is involved in the neurite outgrowth (Huber et al., 1995). When the metamorphosis from the larvae via pupae to adult is done, the old axons in the brain are degradated, the neurite outgrowth is newly progressing, and finally the new neuronal network is Fig. 6. Phosphorylation of Rab proteins using PKC of Bombyx mori. PKC was incubated in the reaction mixture containing BRab proteins (BRab1, lane 3; BRab8, lane 5; BRab14, lane 7) and [γ-32P] ATP. Control; (-PKC, lanes 2– 6; -Rab, lane 1). Arrows 1 and 2 indicate the positions of bands (BRab 8 and BRab14, respectively) stained with CBB. Fig. 5. Protein kinase activity of PKC from the brain of B. mori. The homogenate of brains was centrifuged, the supernatant was applied to a DEAE-Cellulofine, and the active fraction was applied to an affinity column. The purified fraction was incubated in the reaction mixture containing Histone IIIS and [γ-32P] ATP (lane 2). Lane 1: (-PKC); lane 3: [+PKC specific inhibitor, bisindolyl maleimide I, (10 µM)]; lane 4: [+EGTA, (10 mM)]. Archives of Insect Biochemistry and Physiology Phosphoryation of Rab Proteins formed and the shape of the brain is changed. BRab8 may play important roles on neurite outgrowth in the process of metamorphosis. And modifications such as phosphorylation may change the functional characters of BRab8, regulate the membrane transport in the neurons of insects, and finally change the neuronal network during metamorphosis. Whereas in mammals the expression of Rab14 in the brain has been confirmed (Elferink et al., 1992), there have been no reports showing a physiological role of BRab 14 in the brain. In mammals, Rab14 protein was found to be included in phagosome by proteomic analysis (Garin et al., 2001). Calcium is necessary to a phagosome maturation in macrophage (Viera et al., 2002). So in mammals, phosphorylation of Rab14 protein by calcium-regulated kinases may regulate a phagosome function and a similar mechanism may exist in the brain. Lately, the crystal structure of Rab3, one of Rab proteins, was solved (Ostermeier and Brunger, 1999). The recognition sites of effector proteins, GDP/GTP binding sites, and the switching sites after GTP hydrolysis were presented. The effector proteins that interact for Rab8 proteins were specifically identified as coiled–coil protein or protein kinase to interact with microtubes and actins (Hattula et al., 2002; Hattula and Peranen, 2000; Chen et al., 2001; Ren et al., 1996). Phosphorylation of Rab8 protein may change the interaction for these effector proteins. So, studies are under way to determine the phosphorylated sites of Rab 8 protein. 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