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¹ Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan
² Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan

	Abstract

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Protein kinase C (PKC) is translocated to various cellular regions in a subtype and stimulation-dependent manner. Thereafter, the activated PKC phosphorylates its substrate and causes subsequent cellular responses (PKC targeting). The 3-phosphoinositide-dependent protein kinase-1 (PDK1) has an essential role in the maturation of PKC by phosphorylating a threonine residue in the PKC activation loop. To elucidate the role of PDK1 in PKC targeting, we expressed mutant - or -PKC fused with GFP (- or -PKC-ALM (activation loop mutant)-GFP), whose threonine residue in the activation loop was replaced with alanine, and compared their P2Y receptor-mediated translocation with wild-type PKC-GFP in CHO cells. ATP (1 mM) induced the transient translocation of wild-type - or -PKC-GFP from cytoplasm to plasma membrane and following retranslocation from membrane to the cytoplasm. - or -PKC-ALM-GFP was also translocated to plasma membrane, which was, however, retained at the membrane for a longer period than wild type. Similar results were observed in kinase-negative PKC mutants, indicating that the phosphorylation by PDK1 affects the retranslocation step of PKC by regulating the kinase activity. The simultaneous monitoring of [Ca²⁺]_i and diacylglycerol (DG) levels with the translocation of PKC demonstrated that PKC-ALM induced the prolonged accumulation of DG, resulting in the prolonged retention of PKC-ALM at the plasma membrane. It is possible that PKC-ALM with decreased kinase activity could delay the conversion of DG at the plasma membrane. Our present study suggests that the activation loop phosphorylation plays an important role in receptor-mediated PKC targeting.

	Introduction

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Protein kinase C (PKC) is a family of serine/threonine kinases which plays important roles in signal transduction and the regulation of various cellular functions (Nishizuka 1995). PKC isoforms are classified into three groups based on the structures of their regulatory domains (Nishizuka 1988; Dempsey et al. 2000). Classical PKCs (cPKCs; , ßI, ßII, ), having two cysteine-rich domains (C1) and one C2 domain that bind diacylglycerol (DG) and Ca²⁺, respectively, are activated in a DG and Ca²⁺-dependent manner. Novel PKCs (nPKCs; , , , ), having two C1 domains and no C2 domain, are activated in a DG, but not in a Ca²⁺-dependent manner. Atypical PKCs (aPKCs; , /), having only one C1 domain, are insensitive to either DG or Ca²⁺. Previous live imaging studies using green fluorescent protein (GFP)-tagged PKC (PKC-GFP) demonstrated that PKCs are translocated to several cellular organelles in an isoform and stimulation-specific manner when activated by various stimulations, including receptor activation (Sakai et al. 1997; Feng et al. 1998; Ohmori et al. 1998; Shirai et al. 1998a, 1998b, 2000a; Wang et al. 1999; Kajimoto et al. 2001; Kashiwagi et al. 2002). Thereafter, PKCs recognize and phosphorylate their target substrates and cause the subsequent cellular responses (Ohmori et al. 1998, 2000). These findings suggest that the isoform and stimulation-specific translocation of PKC render the molecular basis underlying the multiplicity of PKC functions.

The maturation of PKC, the phosphorylation of multiple serine/threonine sites in PKC, is necessary for the full activation of PKC in response to various stimulations (Hofmann 1997; Parekh et al. 2000; Newton 2003). There are three phosphorylation sites in cPKC and nPKC, the ‘activation loop site,’ the ‘turn motif site’ and the ‘hydrophobic motif site.’ The most essential phosphorylation site for the PKC kinase activity is the threonine residue in the activation loop, which exists around the center of the protein kinase domain (Cazaubon et al. 1994; Orr & Newton 1994; Cenni et al. 2002; Liu et al. 2002). This threonine residue is phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Dutil et al. 1998; Le Good et al. 1998), which regulates the activities of other members of the AGC (cAMP-dependent, cGMP-dependent and PKC) superfamily of serine/threonine kinases, including protein kinase B (PKB), p70 ribosomal S6 protein kinase (S6K), serum and glucocorticoid-induced kinase (SGK) and so on, by phosphorylating threonine/serine residues in their activation loops (Toker & Newton 2000; Alessi 2001). In contrast, the turn motif and hydrophobic motif sites exist in the carboxyl-terminus of the kinase domain. The phosphate groups at these two carboxyl-terminal sites contribute to the stability of the active kinase state, subcellular localization and the interaction with PDK1 (Gysin & Imber 1996; Bornancin & Parker 1997; Edwards et al. 1999; Gao et al. 2001; Liu et al. 2002). The phosphorylation of these two sites depends on the kinase activity of PKC itself (autophosphorylation) (Dutil et al. 1994; Behn-Krappa & Newton 1999) after PDK1 initially phosphorylates the activation loop motif. Although the importance of activation loop phosphorylation in PKC kinase activity and function has been reported, the role of this phosphorylation in PKC translocation has remained unclear.

Previously, we demonstrated that - and -PKC-GFP were translocated from the cytoplasm to the plasma membrane and back to the cytoplasm (retranslocation) within 2 min after the stimulation of P2Y receptors with ATP in Chinese hamster ovary (CHO) cells (Sakai et al. 1997; Ohmori et al. 1998; Shirai et al. 2000a). In the present study, in order to investigate the role of PDK1 in the receptor-mediated translocation of PKC, we constructed the activation loop mutant (ALM) - and -PKC-GFP, in which the threonine residue phospho-rylated by PDK1 (:Thr⁵¹⁴ and :Thr⁵⁰⁵) was replaced with alanine, and expressed these mutants in CHO cells and compared the ATP-induced translocation of these mutants with that of wild-type PKC-GFP. We found that - and -PKC-ALM-GFP rapidly translocated to the plasma membrane, similar to wild-type PKC-GFP, but was retained at the plasma membrane for a longer period, unlike the wild-type PKC-GFP. We also demonstrated that the prolonged retention of PKC-ALM was caused by the decrease in kinase activity and the prolonged accumulation of DG at the plasma membrane.

	Results

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Expression and phosphorylation of wild-type and ALM PKC-GFP

We confirmed the expression and activation loop phosphorylation of wild-type and activation loop mutant (ALM) PKC-GFP (Fig. 1). The COS-7 cells, transfected with plasmids encoding each PKC-GFP protein, were analyzed by immunoblotting. As shown in Fig. 2A, wild-type - and -PKC-GFP were detected by anti-GFP antibody as specific bands with molecular masses of approximately 110 kDa. Similarly, - and -PKC-ALM-GFP were recognized as bands with slightly smaller molecular sizes than wild-type PKC-GFP. Antibodies specific for phosphorylated activation loop of - and -PKC recognized - and -PKC-GFP protein, respectively, but not - and -PKC-ALM-GFP (Fig. 2B), indicating that the activation loop of PKC-ALM-GFP is not phosphorylated.

View larger version (14K): [in this window] [in a new window]   Figure 1  Structures of wild-type and mutant PKC-GFP and PKC-DsRed2. The mutated amino acids are illustrated by circles. In the activation loop mutant (ALM) of PKC, a threonine residue in the activation loop of the kinase domain (:Thr514, : Thr505) was replaced with alanine. In the kinase negative (KN) mutant of PKC, a lysine residue (:Lys380, :Lys376), which is important for binding to ATP, was replaced with methionine.

View larger version (48K): [in this window] [in a new window]   Figure 2  Immunoblotting analysis of wild-type (WT) and ALM PKC-GFP transiently expressed in COS-7 cells. Left and right panels show the results of and -PKC-GFP, respectively. A.Immunoblotting analysis of GFP-fused PKCs with anti-GFP antibody. The antibody recognized wild-type - and -PKC-GFP with molecular sizes of approximately 110 kDa. The molecular sizes of - and -PKC-ALM-GFP were slightly smaller than those of the wild-type. B.Immunoblotting analysis with antibody specific for the phosphorylated activation loop. The anti-phospho-PKC (Thr514) antibody recognized wild-type -PKC-GFP with a molecular size equal to that shown in A, but not -PKC-ALM-GFP. Similarly, the anti-phospho-PKC (Thr505) antibody recognized wild-type but not ALM -PKC-GFP. WT: wild type, ALM: activation loop mutant.

We expressed wild-type and ALM PKC-GFP in CHO cells in order to observe purinergic receptor-mediated translocation. The intense fluorescence of -PKC-GFP was observed in the cytoplasm and faint fluorescence was seen in the nucleus (Fig. 3A). As reported previously (Shirai et al. 1998b), the application of ATP (1 mM) induced a rapid translocation of -PKC-GFP from the cytoplasm to the plasma membrane within 10 s after the stimulation, followed by a retranslocation from the membrane to the cytoplasm within 2 min (Fig. 3A and Video 1 (left) in supplemental videos). -PKC-ALM-GFP was also expressed in the cytoplasm and not in the nucleus before stimulation (Fig. 3B). ATP (1 mM) induced a rapid translocation of -PKC-ALM-GFP from the cytoplasm to the plasma membrane within 10 s after the stimulation, like -PKC-GFP. In contrast, after the translocation, -PKC-ALM-GFP was retained at the plasma membrane for more than 2 min. In several cells, the retranslocation from the membrane to the cytoplasm was not completed within 5 min, the observation period in this study (Fig. 3B and Video 1 (center) in supplemental videos).

View larger version (41K): [in this window] [in a new window]   Figure 3  ATP (1 mM)-induced translocation of wild-type, ALM - and -PKC-GFP in CHO cells. CHO cells expressing -PKC-GFP (A), -PKC-ALM-GFP (B), -PKC-GFP (C) or -PKC-ALM-GFP (D) were stimulated with ATP (1 mM). The sequential changes in the fluorescence of GFP-fusion protein were monitored. Images before (pre) and 0.5, 1, 2, 3 and 5 min after ATP stimulation are shown. Data shown are representative of at least five experiments. Bar = 10 µm.

To analyze and compare the characteristic difference between wild-type and mutant PKC translocation statistically, we counted how many cells showed the retranslocation of PKC at a defined time point after the translocation started. We classified the state of PKC translocation as retained or retranslocated. In the retained cells, PKC was still present at the plasma membrane as seen in Fig. 3, 0.5 and 1 min after the drug application. In the retranslocated type cells, PKC had returned to the cytoplasm as shown in Fig. 3A, 2 and 3 min after the drug application. Table 1 shows the number of retranslocated cells/the total number of cells 0.5, 1, 2, 3 and 4 min after ATP-induced translocation of PKC-GFP started. Two and three minutes after the translocation started, all cells expressing wild-type PKC-GFP were retranslocated type, while most of the cells expressing PKC-ALM-GFP were the retained type. The number of cells with retranslocated PKC was significantly smaller in CHO cells expressing -PKC-ALM-GFP than in cells expressing wild-type -PKC-GFP. Similarly, the number of retranslocated type cells was significantly smaller in CHO cells expressing -PKC-ALM-GFP than wild-type -PKC-GFP. These results indicate that the lack of a phospho-rylated activation loop did not influence the ATP-stimulated translocation of PKC to the plasma membrane, but significantly retarded the retranslocation back to the cytoplasm. Similar results were obtained by the application of 1 mM UTP, a P2Y receptor-selective agonist, or a lower concentration (10 µM) of ATP (data not shown). These findings indicate that the translocation and retranslocation of PKC-GFP were mediated by the activation of P2Y purinoceptors, which endogenously exist in CHO cells, and were not nonspecific effects of ATP.

It has been reported that phosphorylation of the activation loop has a crucial role in the kinase activity of PKC (Cazaubon et al. 1994; Orr & Newton 1994). To elucidate the relationship between the kinase activity and retranslocation of PKC-GFP, we measured the kinase activities of immunoprecipitated PKC-GFP and PKC-ALM-GFP. The kinase activities of - and -PKC-ALM-GFP were markedly decreased, compared with those of wild-type - and -PKC-GFP (11.8% and 25.8% of wild-type PKC-GFP, respectively, Fig. 4A). To confirm that the lack of PKC activity retarded the retranslocation from the plasma membrane, we constructed kinase-negative - and -PKC-GFP (PKC-KN-GFP), in which the lysine residue in the ATP-binding site of PKC (:Lys³⁸⁰, :Lys³⁷⁶) was replaced with methionine (Fig. 1), and examined their ATP-induced translocation in transfected CHO cells. The translocation of - and -PKC-KN-GFP was very similar to that of PKC-ALM-GFP (Fig. 4B,C, respectively, and right panel of Videos 1 and 2, respectively, in supplemental videos) and retranslocation was significantly delayed, compared with wild-type - and -PKC-GFP (Table 1). These results suggest that the kinase activity of PKC plays an important role in the retranslocation step in receptor-mediated translocation of PKC.

View larger version (42K): [in this window] [in a new window]   Figure 4  Kinase activities of wild-type, ALM - and -PKC-GFP immunoprecipitated from COS-7 cells (A) and ATP (1 mM)-induced translocation of PKC-KN-GFP (: B, : C) in CHO cells. A. PKC-GFPs were transiently expressed in COS-7 cells by electroporation. The kinase activity of PKC-GFPs, immunoprecipitated with anti-GFP antibody from COS-7 cells, was assayed as described in Experimental procedures. Data are represented as the average of 5 independent experiments. Kinase activity of - and -PKC-ALM-GFP is indicated as a percentage of that of wild-type PKC-GFP. B, C. CHO cells expressing -PKC-KN-GFP (B) or -PKC-KN-GFP (C) were stimulated with ATP (1 mM). The sequential changes in the fluorescence of GFP-fusion protein were monitored. Images before (pre) and 0.5, 1, 2, 3 and 5 min after ATP stimulation are shown. Data shown are representative of at least 5 experiments. Bar = 10 µm.

The activation of the P2Y receptor in CHO cells generates DG and inositol-1, 4, 5-triphosphate (IP₃) through the activation of phospholipase C (PLC), which leads to the mobilization of Ca²⁺ from intracellular Ca²⁺ stores (Iredale & Hill 1993) and the activation of PKCs. It has been reported that both DG and Ca²⁺ are required for the retention of PKC at the plasma membrane during its translocation (Shirai et al. 2000a). In order to clarify whether the expression of PKC-ALM-GFP or PKC-KN-GFP affected the regulation of DG metabolism or [Ca²⁺]_i, we first examined the P2Y receptor-mediated change in [Ca²⁺]_i simultaneously with the observation of PKC-GFP translocation (Fig. 5A–C). We loaded calcium green-1, a fluorescent calcium indicator, into CHO cells expressing -PKC-GFP, -PKC-ALM-GFP or -PKC-KN-GFP. Figure 5(D–F) shows the time course of the GFP fluorescent ratio (plasma membrane fluorescent intensity/cytoplasm fluorescent intensity, green line) and in fluorescent intensity of calcium green-1 in the cytoplasm (blue line). In CHO cells expressing -PKC-GFP, [Ca²⁺]_i was immediately elevated by the application of ATP (1 mM) and gradually returned to the level before the stimulation within 2 min. The rise and fall of [Ca²⁺]_i were almost synchronous with the translocation and retranslocation of wild-type -PKC-GFP, respectively (Fig. 5A,D). The time course of [Ca²⁺]_i in -PKC-ALM-GFP- or -PKC-KN-GFP-expressing cells was similar to that observed in -PKC-GFP-expressing cells. However, the change in [Ca²⁺]_i was not synchronous with the translocation of ALM or KN -PKC-GFP. Two minutes after the stimulation, [Ca²⁺]_i had returned to the level before the stimulation, while PKC was still retained at the plasma membrane (Fig. 5B,C,E,F). These results suggest that the expression of ALM or KN PKC does not affect the regulatory system of [Ca²⁺]_i and that the prolonged retention of the mutant PKC-GFP is independent of the change in [Ca²⁺]_i.

View larger version (43K): [in this window] [in a new window]   Figure 5  Simultaneous imaging of [Ca2+]i using calcium green-1 (CG1) with ATP (1 mM)-induced translocation of wild-type, ALM or KN -PKC-GFP and in CHO cells. A–C. CHO cells expressing -PKC-GFP (A), -PKC-ALM-GFP (B) or -PKC-KN-GFP (C) were loaded with 5 µM calcium green-1, AM for 60 min at room temperature before the observation. Cells were stimulated with ATP (1 mM). The intensity of CG1 fluorescence (blue, lower panels) was monitored simultaneously with the changes in the fluorescence of PKC-GFP (green, upper panels). Images before (pre) and 0.5, 1, 2, 3 and 5 min after ATP stimulation are shown. Data shown are representative of at least three experiments. Bar = 10 µm. D–F. Plots represents the time course of the ratio of translocation (plasma membrane fluorescent intensity/cytoplasm fluorescent intensity, green line) and [Ca2+]i (fluorescent intensity of CG1 in the cytoplasm, blue line). The changes in the ratio and [Ca2+]i are plotted as a percentage of the maximum response. The plots of D, E and F represent the results obtained from the arrowed cell shown in A, B and C, respectively.

View larger version (49K): [in this window] [in a new window]   Figure 6  Structure of -C1-GFP (A) and ATP (1 mM)-induced translocation of -C1-GFP and -PKC-DsRed2 co-expressed in CHO cells (B–G). A. The structure of -C1-GFP (left) and translocation of -C1-GFP expressed in CHO cells, induced by ATP (1 mM) stimulation (right). -C1-GFP is composed of the C1 domain of -PKC (amino acids: 159–280) and GFP fused to the C-terminus of the C1 domain. B–D. CHO cells expressing -C1-GFP with -PKC-DsRed2 (B), -PKC-ALM- DsRed2 (C) or -PKC-KN-DsRed2 (D) were stimulated with ATP (1 mM). The changes in the fluorescence of PKC-DsRed2 (upper panels) and -C1-GFP (lower panels) were monitored. Images before (pre) and 0.5, 1, 2, 3 and 5 min after ATP stimulation are shown. Data are representative of at least five experiments. Bar = 10 µm. E–G. Plots represent the time course of the ratio of translocation (plasma membrane fluorescent intensity/cytoplasm fluorescent intensity) of PKC-DsRed2 (red line) and -C1-GFP (green line). The changes in the ratio are plotted as a percentage of the maximum response. The plots of E, F and G represent the results obtained from the arrowed cell shown in B, C and D, respectively.

View this table: [in this window] [in a new window]   Table 2  Period of translocation of -C1-GFP coexpressed with wild type (WT), ALM and KN mutant -PKC-DsRed2 induced by ATP (1 mM) in CHO cells

View larger version (37K): [in this window] [in a new window]   Figure 7  Structures of diacylglycerol kinase (DGK) and GFP-fused constitutively active mutant of DGK (GFP-DGK-CA, A) and ATP 1 mM-induced translocation of mutant -PKC-DsRed2 co-expressed with GFP (B, D) or GFP-DGK-CA (C, E) in CHO cells. A. Structures of diacylglycerol kinase (DGK) and a GFP-fused constitutively active mutant of DGK (GFP-DGK-CA). In order to make a constitutively active mutant of GFP-DGK, we connected the cDNAs encoding GFP and N-terminal-deleted DGK lacking an autoinhibitory motif (recoverin homology (RVH) motif and EF-hand motif, 1–268 amino acids). B–E. -PKC-ALM-DsRed2 was co-expressed with GFP (B) or GFP-DGK-CA (C) in CHO cells. Similarly, -PKC-KN-DsRed2 was co-expressed with GFP (D) or GFP-DGK-CA (E). ATP (1 mM) was applied to induce the P2Y receptor-mediated translocation. The sequential changes in the fluorescence of PKC-DsRed2 (upper panels) and GFP-DGK-CA (lower panels) were monitored. Images before (pre) and 0.5, 1, 2, 3 and 5 min after ATP stimulation are shown. Data shown are representative of at least five experiments. Bar = 10 µm.

View this table: [in this window] [in a new window]   Table 3  Period of translocation of wild type (WT), ALM and KN mutant -PKC-DsRed2 coexpressed with GFP or GFP-DGK-CA induced by ATP (1 mM) in CHO cells

	Discussion

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Although the P2Y receptor-mediated translocations of - and -PKC-GFP were very similar, several subtle differences between them were also found in the present study. -PKC-GFP was translocated to the plasma membrane immediately after the stimulation, while the translocation of -PKC-GFP started 20–30 s after the stimulation (see Videos 1 and 2 in the supplemental videos). The retention period of -PKC-GFP at the plasma membrane was slightly longer than that of -PKC-GFP (Table 1). These differences may be derived from the different Ca²⁺ dependency between - and -PKC. Furthermore, we have recently reported that the overexpression of -PKC inhibited the Ca²⁺ oscillation mediated via metabotropic glutamate receptor type 5, while the overexpression of -PKC did not (Uchino et al. 2004), indicating that - and -PKC-GFP have different roles in the regulation of cellular functions in spite of their similarities in receptor-mediated translocation.

PDK1 conducts the activation loop phosphorylation of PKC, which is considered to be the first and crucial step in the maturation of PKC, followed by its autophosphorylation (Dutil et al. 1994; Behn-Krappa & Newton 1999). PDK1 is considered to be the only enzyme that phosphorylates the activation loop of PKC as the region was not phosphorylated in embryonic stem cells lacking PDK1 (PDK1–/– cells) (Balendran et al. 2000). Several studies have revealed that this phosphorylation is essential for the kinase activity of many subtypes of PKC (Cazaubon et al. 1994; Orr & Newton 1994; Cenni et al. 2002; Liu et al. 2002). In the present study, the kinase activity of -PKC-ALM-GFP was markedly decreased, compared with wild-type -PKC-GFP, while -PKC-ALM-GFP still had a kinase activity to some extent, which was only 25.8% of wild-type -PKC-GFP (Fig. 3A). Previous studies have demonstrated that the activity of -PKC is almost independent of its activation loop phosphorylation, supporting this difference in kinase activity between - and -PKC-ALM-GFP (Stempka et al. 1997, 1999). In addition to - and -PKC-ALM-GFP, - and -PKC-KN-GFP also displayed the prolonged retention at the plasma membrane. Feng and Hannun (1998) demonstrated that a kinase negative mutant of ßII-PKC was translocated from cytoplasm to membrane on stimulation with angiotensin II in HEK293 cells, followed by a prolonged retention of kinase-negative ßII-PKC at the plasma membrane for more than 10 min. The results in the present study were consistent with their result. Based on these findings, it is strongly suggested that a marked loss of kinase activity in PKC-ALM caused the prolonged retention of PKC at the plasma membrane during the retranslocation step, leading to the idea that the activation loop phosphorylation of PKC by PDK1 plays an important role in the receptor-mediated targeting of PKC by regulating PKC kinase activity.

We next investigated why the loss of PKC kinase activity results in the retention of PKC at the plasma membrane and a delayed retranslocation step. It is reported that both DG and Ca²⁺, products following the activation of G-protein- and PLC-coupled receptors, have crucial roles in the translocation of PKC, especially calcium and phospholipid-dependent classical PKC (Keranen & Newton 1997; Sakai et al. 1997; Shirai et al. 2000a). It is possible that the expression of PKC-ALM influences the regulatory system for [Ca²⁺]_i and DG level at the plasma membrane. To address these issues, we attempted to monitor the [Ca²⁺]_i or DG level, simultaneously with PKC translocation. As shown in Fig. 5, the expression of -PKC-ALM or -PKC-KN did not affect the receptor-mediated change in [Ca²⁺]_i, indicating that the prolonged retention of mutant PKCs was independent of [Ca²⁺]_i. This interpretation seems to be appropriate for understanding the mechanism of delayed retranslocation of -PKC-ALM as -PKC lacks C2 domain, a Ca²⁺-interacting domain of PKC, and is activated in a Ca²⁺-indipendent manner. This result is contradictory to the findings reported by Feng et al. (2000); Ca²⁺ has an important role in anchoring ßII-PKC to the plasma membrane as the deletion of C2 domain and calcium chelators shortened the retention period of kinase-negative ßII-PKC at the plasma membrane. To examine the involvement of C2 domain in the delayed retranslocation of -PKC-ALM, we constructed C2-deleted -PKC-ALM. This mutant also showed the prolonged retention at the plasma membrane and the retention period is not significantly different from that of -PKC-ALM-GFP (data not shown), suggesting that C2 domain is not involved in the delayed retranslocation.

On the other hand, in cells expressing -PKC-ALM or -PKC-KN, -C1-GFP, an indicator of the DG level, was robustly translocated to the plasma membrane and retained there longer than in cells expressing wild-type -PKC (Fig. 6 and Table 2). This result suggests that the overexpression of PKC-ALM or PKC-KN, a mutant PKC without kinase activity, prevents the metabolism of DG and keeps the DG level high at the plasma membrane. These mutants may exert dominant negative effects on the functions of endogenous PKCs. This prolonged accumulation of DG would make PKC-ALM and PKC-KN retained at the plasma membrane for a longer period than wild-type PKC. This notion is further supported by the results that a constitutively active DGK (DGK-CA), which phosphorylates DG and converts it into PA, shortened the retention period of PKC-ALM and PKC-KN at the plasma membrane (Fig. 7 and Table 3). However, these periods of PKC-ALM and PKC-KN, co-expressed with GFP-DGK-CA, was still longer than that of wild-type PKC co-expressed with GFP (Table 3), indicating that other factors might be involved in this phenomenon. It has been demonstrated that autophosphorylation of two C-terminal serine/threonine residues (turn motif and hydrophobic motif) plays an essential role in the retranslocation step as the replacement of Ser⁶⁶⁰ and/or Thr⁶⁴¹ of ßII-PKC with alanine significantly inhibited the retranslocation of ßII-PKC from membrane to cytoplasm (Feng & Hannun 1998; Feng et al. 2000). In fact, we confirmed that C-terminal autophosphorylation sites of PKC-ALM-GFP and PKC-KN-GFP were not phosphorylated (data not shown). Therefore, no autophosphorylation as a result of decreased kinase activity might prolong the retention period of these mutant PKCs at the plasma membrane.

The results in the present study strongly indicate that the activated PKC would regulate the metabolic pathway of DG generated at the plasma membrane. DGK is known as one of the metabolic enzymes of DG. As described above, DGK phosphorylates DG and converts it into PA, another lipid mediator that cannot activate PKC (Topham & Prescott 1999; Kanoh et al. 2002). Therefore, DGK can modulate PKC activity by reducing the level of DG at the plasma membrane. Furthermore, we have previously demonstrated that the membrane retention period of -PKC-GFP was shortened by the co-expression of DGK and was prolonged by DGK inhibitor R59022 [GenBank] in case of its receptor-mediated translocation (Shirai et al. 2000b). From these findings, we speculate that the prolonged accumulation of DG, observed in the present study, is the result of the failure of DGK activation. This hypothesis is supported by our recent studies, demonstrating that -PKC directly interacted with DGK and phosphorylated two serine residues of DGK in a Ca²⁺-dependent manner. Furthermore, we have also revealed that the replacement of these two serine residues with glutamate that mimics the phosphorylation state of DGK, enhanced its kinase activity (Yanaguchi et al., submitted data). However, from the results of the present study, we cannot declare that PKC-ALM and PKC-KN prevent the activation of DGK in a dominantly negative fashion. It is possible that PKC-ALM or PKC-KN affects other factors, including DG lipase, which also regulates DG level at the plasma membrane. Further studies are necessary to fully elucidate how PKC regulates the DG metabolism.

In this study, we did not examine the effects of -PKC-ALM and -PKC-KN on the regulation of DG level because expressed -PKC-DsRed2 was unfortunately aggregated and did not respond to ATP stimulation. However, based on our previous report revealing that P2Y receptor-mediated translocation of -PKC was DG-dependent in CHO cells (Shirai et al. 2000a), we speculate that -PKC-ALM and -PKC-KN would also prevent the metabolism of DG, leading to the prolonged retention of these mutants at the plasma membrane.

We have elucidated that unphosphorylated and immature PKCs are retained at the plasma membrane for longer than phosphorylated and mature PKCs. However, the physiological significance of this phenomenon has remained unclear. As described before, the initial step in the maturation of PKC is thought to be a phosphorylation of the activation loop by PDK1. According to the theories proposed by Newton and others (Sonnenburg et al. 2001; Newton 2003), immature PKCs, not yet phosphorylated by PDK1, are supposed to undergo processing for maturation at the plasma membrane where PDK1 exists and is activated. Prolonged retention of immature PKCs at the plasma membrane may provide the opportunity for PDK1 to promptly recognize and phosphorylate target substrates, immature PKCs. In conclusion, we revealed that phosphorylation of the activation loop of PKC by PDK1 has an important role in the PKC-targeting process by regulating its kinase activity and DG level.

	Experimental procedures

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Materials

ATP was purchased from Research Biochemical International (Natick, MA). UTP was purchased from Wako Pure Chemical (Osaka, Japan). Anti-GFP rabbit polyclonal antibody and calcium green-1 AM were from Molecular Probes (Leiden, Netherlands). Anti-phospho-PKC (Thr⁵¹⁴) polyclonal antibody was from Biosource International (Camarillo, CA). Anti-phospho-PKC (Thr⁵⁰⁵) rabbit polyclonal antibody was from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA).

Plasmid construction

Plasmids encoding wild-type rat g- and d-PKC-GFP were constructed as previously described (Sakai et al. 1997; Ohmori et al. 1998). Using these plasmids as templates, we constructed activation loop mutant (ALM) and kinase-negative (KN) PKC cDNAs by a PCR-based method (Weiner et al. 1994). For making PKC-ALM, Thr⁵¹⁴ in g-PKC and Thr⁵⁰⁵ in d-PKC, the sites phospho-rylated by PDK1 in the activation loop, were replaced with alanine residue. For making PKC-KN, Lys³⁸⁰ in g-PKC and Lys³⁷⁶ in d-PKC, ATP-binding sites in the kinase domains, were replaced with methionine residues. All mutant cDNAs were verified by sequencing. To construct mutant PKC-GFP fusion proteins, these mutant g- and d-PKC and GFP cDNAs were together subcloned into pTB701, an expression plasmid for mammalian cells (Ono et al. 1988). Wild type, ALM and KN g-PKC cDNAs were also subcloned into DsRed2-N1 (BD Biosciences Clontech, Palo Alto, CA), to construct PKC-DsRed2 fusion proteins. The structures of wild-type or mutant PKC-GFP and PKC-DsRed2 proteins are illustrated in Fig. 1.

Complementary DNA encoding the C1 domain of d-PKC (amino acids: 159–280) was amplified by PCR. The PCR products were subcloned into pTB701 with GFP cDNA to express a d-PKC C1 domain fused with GFP (designated as d-C1-GFP, Fig. 6A).

A plasmid containing pig diacylglycerol g (DGKg) cDNA was donated by Dr Kanoh (Sapporo Medical University School of Medicine, Japan). The cDNA of a constitutively active mutant of DGKg (DGKg-CA), in which the N-terminal auto-inhibitory domain (amino acids: 1–268) was truncated (Sakane et al. 1991; Jiang et al. 2000; Yamada et al. 2003), was constructed by a PCR-based method and subcloned into an expression plasmid, pEGFP-C3 (BD Biosciences Clontech), to construct a GFP-DGKg-CA fusion protein (Fig. 7A).

Cell culture

COS-7 cells were purchased from Riken Cell Bank (Tsukuba, Japan). The CHO-K1 cell strain was a gift from Dr Nishijima (National Institute of Health, Tokyo, Japan). COS-7 and CHO-K1 cells were cultured in Dulbecco's modified Eagle's Medium and Ham's F12 medium (Sigma Chemical, St Louis, MO), respectively, supplemented with 10% foetal bovine serum (FBS), 100 units/mL of penicillin and 100 mg/mL of streptomycin in a humidified atmosphere containing 5% CO₂ at 37 C.

Immunoblotting and kinase assay

Transient transfections of COS-7 cells were performed by electroporation. Plasmids (20–30 mg) encoding each type of PKC-GFP or PKC-DsRed2 were transfected into 6 ¥ 10⁶ cells using Gene Pulser (975 mF, 220 V; Bio-Rad Laboratories, Hercules, CA). Transfected cells were cultured in 9-cm-diameter culture dishes.

For immunoblotting, COS-7 cells, transfected with plasmids encoding each PKC-GFP or PKC-DsRed2, were cultured for 2 days and then harvested by 500 ¥ g centrifugation. The cell pellet was washed with 1 mL of homogenate buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA and 50 mM Tris/HCl, pH 7.4) and resuspended in 300 mL of lysis buffer (homogenate buffer containing 1% Triton X, 200 mg/mL of leupeputin, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF and 100 nM Calyculin A). After the sonication (UR-20P, TOMY SEIKO, Tokyo, Japan), protein levels were quantified using a Coomassie Protein Assay Reagent kit (Pierece, Rockford, IL). Twenty to 30 mg of protein of each sample was subjected to 7.5% SDS-PAGE, and the separated proteins were electrophoretically transferred on to polyvinylidine difluoride (PVDF) filters (Millipore, Bedford, MA). Nonspecific binding sites on PVDF filters were blocked by incubation with 5% skim milk in PBS-T (0.01 M phosphate-buffered saline containing 0.03% Triton X) for > 1 h at room temperature (RT). After being washed with PBS-T, the PVDF filters were incubated with anti-GFP polyclonal antibody (diluted 1: 2000), anti-phospho-PKCg (Thr⁵¹⁴) polyclonal antibody (1 : 1000) or anti-phospho-PKCd (Thr⁵⁰⁵) polyclonal antibody (1 : 1000) for > 1 h at RT. After further washing, the filters were incubated with HRP-conjugated anti-rabbit IgG antibody (1 : 10 000) for > 30 min at RT. After three more washes, the immunoreactive bands were visualized with a chemiluminescence detection kit (ECL‘ Western Blotting Detection Reagents, Amersham Biosciences, Buckinghamshire, England).

For kinase assays, COS-7 cells were transfected with plasmids encoding each PKC-GFP and cultured for 3 days. After the harvest and lysis of cells as described above, immunoprecipitated protein samples were prepared as follows. The lysed protein samples were centrifuged at 19 000 ¥ g for 30 min at 4 C, and the supernatant was rotated with anti-GFP polyclonal antibody at 4 C for 2 h, then with protein A Sepharose (Amersham Biosciences) at 4 C overnight. Samples were centrifuged at 2000 ¥ g for 5 min at 4 C, and pellets were washed three times with PBS (–). Finally, a 1/10 volume of immunoprecipitated pellet was used for the kinase assay. The assay of PKC-GFP was conducted using a PepTag assay for non-radioactive detection of PKC kit (Promega, Madison, WI). According to the manufacturer's directions, 1.35 mM CaCl₂ and 200 mg/mL phosphatidylserine were used as PKC activators. The amounts of immunoprecipitated PKC-GFP were quantified by immunoblotting a 1/10 volume of immunoprecipitated pellet with anti-GFP antibody. The relative intensities of immunoreactive bands were calculated using NIH Image software. The kinase activity of PKC-GFP was normalized to the amount of immunoprecipitated PKC-GFP.

Observation of translocation

Plasmids (2.5 mg) were transfected into CHO cells (1 ¥ 10⁵ cells) by lipofection using Fugene‘ 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's directions. Transfected CHO cells were spread on to poly D-lysine-coated glass bottom culture dishes (MatTek Corp., Ashland, MA) and cultured for 2 days. Before the observation, the culture medium was replaced with 0.9 mL of HEPES buffer (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES and 10 mM glucose, pH 7.3).

The fluorescence of GFP and DsRed2 was monitored with a confocal laser scanning fluorescent microscope (LSM510META, Carl Zeiss, Esslingen, Germany) at 488-nm argon laser excitation using a 505–530 nm band pass barrier filter and 542-nm HeNe laser excitation using a 560 nm long pass barrier filter, respectively. Translocations of GFP or DsRed2-fused proteins were triggered by a direct application of 0.1 mL of ATP or UTP solution at a 10 times higher concentration into HEPES buffer to obtain the appropriate final concentration. Images were recorded every 5 s for 5 min before and after the stimulation. All experiments were performed at RT.

As previously described, PKC is translocated from cytoplasm to the plasma membrane and retranslocated back to the cytoplasm upon purinergic receptor stimulation in CHO cells (Sakai et al. 1997; Ohmori et al. 1998; Shirai et al. 2000a). To evaluate and compare the characteristic difference between wild-type and mutant PKC translocation, we classified the state of PKC translocation as ‘retained’ or ‘retranslocated,’ as described in results. Results using this analytical method are shown in Tables 1–3. The statistical significance at a defined time point after the translocation started was calculated with Fisher's exact test.

For monitoring the intracellular Ca²⁺ level ([Ca²⁺]_i), cells were loaded with 5 mM calcium green-1 AM for 60 min at RT before the observation. A stable calcium green-1 AM solution was prepared by adding a 1/10 volume of 10% cremophore EL (Sigma, St. Louis, MO) to a stock solution of calcium green-1 AM (1 mM in dimethyl sulfoxide). The emissions of GFP and calcium green-1 were both excited with an argon laser at 488 nm and were separated by emission fingerprinting (Carl Zeiss, LSM510META). The fluorescent intensities of GFP, DsRed2 and calcium green-1 in 1–7 mm² regions of interest (ROIs) were measured on images using the Carl Zeiss LSM510 software. The averaged fluorescent intensity of GFP- or DsRed2-fused protein at the cytoplasm or plasma membrane was calculated from three to five different ROIs in a single cell. The fluorescent ratio was defined as the plasma membrane fluorescent intensity/cytoplasm fluorescent intensity. The fluorescent intensity of calcium green-1 was calculated from three to five different ROIs in the cytoplasm. These values were plotted to generate a time course of fluorescent protein fused-PKC translocation and [Ca²⁺]_i (Figs 5–7).

	Supplementary material

Top Abstract Introduction Results Discussion Experimental procedures Supplementary material References

 
Video 1: Translocation of wild-type g-PKC-GFP (left), g-PKC-ALM-GFP (center) and g-PKC-KN-GFP (right) induced by 1 mM ATP in CHO cells. Video 2: Translocation of wild-type d-PKC-GFP (left), d-PKC-ALM-GFP (center) and d-PKC-KN-GFP (right) induced by 1 mM ATP in CHO cells. Sequential 64 images obtained every 5 s using a confocal laser scanning fluorescent microscope were compacted to the 10-s movies and saved as Quick Time movie files.

	Acknowledgements

 
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from Takeda Science Foundation and the Japanese Smoking Research Association. This work was carried out with equipment of the Research Center for Molecular Medicine, Hiroshima University School of Medicine.

	Footnotes

 
Communicated by: Kozo Kaibuchi

*Correspondence: E-mail: nsakai{at}hiroshima-u.ac.jp

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