Spatial and temporal regulation of protein kinase D (PKD)
2000; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês
10.1093/emboj/19.12.2935
ISSN1460-2075
AutoresSharon A. Matthews, Teresa Iglesias, Enrique Rozengurt, Doreen A. Cantrell,
Tópico(s)Receptor Mechanisms and Signaling
ResumoArticle15 June 2000free access Spatial and temporal regulation of protein kinase D (PKD) Sharon A. Matthews Sharon A. Matthews Lymphocyte Activation Laboratory, Imperial Cancer Research Fund 44, Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Teresa Iglesias Teresa Iglesias Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Enrique Rozengurt Enrique Rozengurt Department of Medicine, UCLA School of Medicine and Molecular Biology Institute, 900 Veteran Avenue, Warren Hall, Los Angeles, CA, 90095-1786 USA Search for more papers by this author Doreen Cantrell Corresponding Author Doreen Cantrell Lymphocyte Activation Laboratory, Imperial Cancer Research Fund 44, Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Sharon A. Matthews Sharon A. Matthews Lymphocyte Activation Laboratory, Imperial Cancer Research Fund 44, Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Teresa Iglesias Teresa Iglesias Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Enrique Rozengurt Enrique Rozengurt Department of Medicine, UCLA School of Medicine and Molecular Biology Institute, 900 Veteran Avenue, Warren Hall, Los Angeles, CA, 90095-1786 USA Search for more papers by this author Doreen Cantrell Corresponding Author Doreen Cantrell Lymphocyte Activation Laboratory, Imperial Cancer Research Fund 44, Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Author Information Sharon A. Matthews1, Teresa Iglesias2, Enrique Rozengurt3 and Doreen Cantrell 1 1Lymphocyte Activation Laboratory, Imperial Cancer Research Fund 44, Lincoln's Inn Fields, London, WC2A 3PX UK 2Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK 3Department of Medicine, UCLA School of Medicine and Molecular Biology Institute, 900 Veteran Avenue, Warren Hall, Los Angeles, CA, 90095-1786 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2935-2945https://doi.org/10.1093/emboj/19.12.2935 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Protein kinase D (PKD; also known as PKCμ) is a serine/threonine kinase activated by diacylglycerol signalling pathways in a variety of cells. PKD has been described previously as Golgi-localized, but herein we show that it is present within the cytosol of quiescent B cells and mast cells and moves rapidly to the plasma membrane after antigen receptor triggering. The membrane redistribution of PKD requires the diacylglycerol-binding domain of the enzyme, but is independent of its catalytic activity and does not require the integrity of the pleckstrin homology domain. Antigen receptor signalling initiates in glycosphingolipid-enriched microdomains, but membrane-associated PKD does not co-localize with these specialized structures. Membrane targeting of PKD is transient, the enzyme returns to the cytosol within 10 min of antigen receptor engagement. Strikingly, the membrane-recycled PKD remains active in the cytosol for several hours. The present work thus characterizes a sustained antigen receptor-induced signal transduction pathway and establishes PKD as a serine kinase that temporally and spatially disseminates antigen receptor signals away from the plasma membrane into the cytosol. Introduction Antigen receptors are critical regulators of lymphocyte function. Antigen receptor signal transduction is mediated directly by tyrosine kinases that control a diverse network of signalling molecules, including enzymes involved in the control of inositol phospholipid metabolism such as phospholipase Cγ (PLCγ). The activation of PLCγ induces phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] hydrolysis, producing IP3 and diacylglycerol (DAG), which regulate intracellular calcium and activate the protein kinase C (PKC) family of serine/threonine kinases, respectively. There are multiple PKC isoforms, including classical PKCs (α, βI, βII and γ), which bind calcium, DAG and phospholipids; novel PKCs (δ, ϵ, η and θ), which are regulated by DAG and phospholipids; and the atypical PKCs (ξ and λ), which lack calcium- or DAG-binding domains (Newton, 1997; Mellor and Parker, 1998; Toker, 1998). The significance and potency of calcium and PKC signalling pathways for lymphocyte activation have been well documented (Berry and Nishizuka, 1990; Crabtree and Clipstone, 1994; Leitges et al., 1996; Crabtree, 1999). Recently, it has been demonstrated that antigen receptors stimulate the activity of protein kinase D (PKD; also known as PKCμ) (Sidorenko et al., 1996; Matthews et al., 1999). However, despite the clear functional link between PKD and antigen receptors, there has been no analysis of PKD localization in resting or activated lymphocytes. It has been reported that PKD functions to regulate Golgi organization and transport processes (Prestle et al., 1996; Jamora et al., 1999). It has also been described that PKD is localized partially with the Golgi compartment in a human hepatocellular carcinoma cell line and in human antral gastrin cells (Prestle et al., 1996; Moore et al., 1999). However, there has never been any integration of data about PKD activity and localization under physiological conditions of cell activation. In this context, the cellular activation of PKD is mediated by PKCs, which require lipid co-activators for catalytic activity, thereby restricting their activity to lipid-enriched subcellular locations, typically the plasma membrane. In contrast, PKD catalytic activity is regulated by protein phosphorylation (Iglesias et al., 1998b) and can be maintained in the absence of lipid co-activators (Zugaza et al., 1996, 1997; Matthews et al., 1997; Van Lint et al., 1998). PKD thus has the potential to be active away from membrane/lipophilic environments and could be active in the cytosol. One feature of PKD that might determine its subcellular localization is the presence of a conserved DAG-binding cysteine-rich domain (CRD) within its regulatory region that has previously been demonstrated to bind DAG and phorbol esters (Valverde et al., 1994; Van Lint et al., 1995; Dieterich et al., 1996). Classical and novel PKCs possess a CRD motif, which plays a critical role in recruiting these enzymes to the plasma membrane of various epithelial and fibroblast cell lines (Sakai et al., 1997; Feng et al., 1998; Oancea et al., 1998; Ohamori et al., 1998). CRD motifs additionally may target PKCs to other intracellular membranes, including the nuclear envelope (Oancea and Meyer, 1998; Oancea et al., 1998; Ohamori et al., 1998). However, the presence of a CRD domain is not necessarily sufficient for membrane targeting: recruitment of classical PKCs to the plasma membrane requires the binding of both DAG and calcium to regulatory CRD and C2 domains, respectively (Oancea and Meyer, 1998). Moreover, there are clearly other regulatory factors that can override the presence of a CRD domain to determine protein localization. Novel PKCs all have a CRD domain, but only θ is recruited to the plasma membrane during antigenic stimulation of T cells (Monks et al., 1997, 1998). In this context, the N-terminal regulatory region of PKD does not possess the calcium-binding C2 domain essential for effective membrane targeting of classical PKCs. PKD does, however, contain a putative pleckstrin homology (PH) domain (Valverde et al., 1994), which has the potential to play an important role in controlling the subcellular localization of PKD either through binding to lipid second messengers or via protein–protein interactions (Pitcher et al., 1995; Leevers et al., 1999; Lemmon, 1999). The objective of the present report was to use biochemical analysis and real-time confocal imaging of green fluorescent protein (GFP)-tagged PKD molecules in living cells to explore the localization of PKD in antigen receptor-activated cells. Importantly, this study integrates spatial and temporal aspects of PKD localization with PKD catalytic activity under physiological conditions of cell activation. Our results identify PKD as a signalling molecule that has two sites of action in lymphocytes: initially, PKD operates at the plasma membrane outside lipid microdomains during the initial activation response, but functions within the cytosol during sustained responses to antigen receptor ligation. Results Activation of PKD in B lymphocytes and in mast cells following antigen receptor ligation or phorbol ester activation A20 B-lymphoma cells were activated by cross-linking the B-cell antigen receptor (BCR) with F(ab)′2 fragments of anti-mouse IgG for various times (0–10 min). Cross-linking of the BCR complex was found to induce a rapid ∼7- to 8-fold increase in PKD catalytic activity (Figure 1A), a response that was maximal within 1 min of BCR triggering. Active PKD autophosphorylates on a C-terminal serine residue, S916 (Matthews et al., 1999), and antibodies that recognize S916-phosphorylated PKD can be used to monitor the activity of PKD. As shown in Figure 1A, the pS916 antiserum only weakly recognized PKD from unstimulated cells, but reacted strongly with active PKD isolated from BCR-activated A20 B lymphocytes. The antigen receptor present in mast cells is the high affinity receptor for IgE, the FcϵR1. The data presented in Figure 1B indicate that activation of the FcϵR1 induces a rapid ∼6-fold increase in PKD catalytic activity. Figure 1.BCR or FcϵR1 ligation induces PKD catalytic activity. (A) A20 B lymphocytes were left untreated (n/s) or were stimulated with 10 μg/ml F(ab)′2 fragments of anti-mouse IgG for various times (0–10 min). The cells were subsequently lysed before PKD was immunoprecipitated using the PA-1 antiserum and in vitro kinase assays performed. PKD activity was measured by autophosphorylation (IVK). Alternatively, total proteins from cell lysates were precipitated with cold acetone, separated by SDS–PAGE and subjected to western blot analysis using the pS916 antiserum and a pan C-terminal PKD antibody. Results are representative of three independent experiments. (B) RBL 2H3 cells were primed with 1 μg/ml IgE anti-DNP for 1 h at 37°C and then either antigenic cross-linking of the bound IgE was performed, using 500 ng/ml KLH-DNP, for various times (0–10 min) or the cells were left unstimulated (–). PKD was immunoprecipitated from cell lysates and the activity of PKD was measured by in vitro kinase assays (IVK). SDS–PAGE and western blot analysis of the cell lysates show equivalent amounts of PKD in all the samples. Results are representative of two independent experiments. (C) RBL 2H3 or A20 B cells were either left unstimulated (n/s) or were stimulated with 50 ng/ml PDBu for various times (0–10 min). PKD was immunoprecipitated from cell lysates and the activity of PKD was measured by in vitro kinase assays (IVK). Western blot analysis of the cell lysates shows equivalent amounts of PKD in all the samples. Results are representative of two independent experiments. Download figure Download PowerPoint The effects of stimulating B lymphocytes and mast cells with the phorbol ester phorbol 12,13 dibutyrate (PDBu), a pharmacological activator of PKC enzymes, on PKD activity were also measured. As shown in Figure 1C, PDBu treatment led to a rapid increase in the catalytic activity of PKD in both cell types. The kinetics and magnitude of this response (reaching a maximum 6- to 7-fold increase in PKD activity within ∼1 min) were essentially identical to those observed upon engagement of antigen receptors in these cells. Cellular localization of inactive PKD in resting mast cells and B lymphocytes To explore the spatial dynamics of PKD regulation in lymphocytes, a GFP-tagged version of wild-type PKD (GFP–PKD) was generated and expressed in either A20 B lymphocytes or the RBL 2H3 mast cell line. Addition of a GFP tag to the N-terminus of PKD did not affect basal PKD activity nor did it prevent antigen receptor-induced activation of PKD in B lymphocytes (Figure 2A) or in RBL 2H3 mast cells (data not shown). The localization of GFP-tagged PKD in quiescent and antigen receptor-activated cells was then analysed. The data in Figure 2B show mid-section confocal images taken from RBL 2H3 cells or from A20 B lymphocytes that had been transiently transfected with GFP–PKD. These images indicate that GFP–PKD was distributed evenly throughout the cytosol of these cells, with no apparent association with specific intracellular compartments, and was excluded from the nucleus. Importantly, immunofluorescence staining of fixed cells with a specific polyclonal PKD antibody confirmed that the endogenous PKD present in resting A20 B lymphocytes was distributed evenly throughout the cytosol of these cells (Figure 2C). It should be emphasized that the C-terminal epitope recognized by this antibody is unique to PKD and does not cross-react with the recently described serine kinase, PKCυ, which exhibits strong homology to PKD (Hayashi et al., 1999). The cytosolic distribution of PKD in B lymphocytes and mast cells demonstrated herein contrasts with the reported Golgi localization of PKD described in a hepatocellular carcinoma cell line and in human antral gastrin cells (Prestle et al., 1996; Moore et al., 1999). In further experiments, we noted that PKD localization in A20 B lymphocytes was clearly distinct from that of endogenous GM 130 (Figure 2C) or ectopically overexpressed GFP-tagged NAGT1 (Figure 2D), two well characterized Golgi-localized proteins (Nakamura et al., 1995; Shima et al., 1997). The data in Figure 2D show a comparison of the subcellular distribution of GFP–PKD or NAGT1–GFP in A20 B lymphocytes. Two-dimensional projections of sections in Z-series taken across the depth of the cells at 0.3 μm intervals clearly reveal the distinct pattern of PKD localization compared with this Golgi marker, demonstrating that PKD is not localized preferentially to the Golgi in B lymphocytes or in mast cells. Figure 2.Localization of PKD in resting A20 B lymphocytes and RBL 2H3 cells. (A) Top: schematic representation of the N-terminally GFP-tagged PKD construct. Bottom: A20 B lymphocytes were transiently transfected with 20 μg of wild-type GFP–PKD and left to recover overnight. Cells were left untreated (n/s) or were stimulated with 10 μg/ml F(ab)′2 fragments of anti-mouse IgG for various times (1–10 min). The cells were subsequently lysed before PKD (endogenous) and GFP–PKD (ectopically expressed) were immunoprecipitated using the PA-1 antiserum and in vitro kinase assays performed. PKD activity was measured by autophosphorylation (IVK). Western blot analysis of whole-cell lysates with a specific PKD antibody reveals PKD expression levels. Similar results were obtained in two independent experiments. (B) A20 B lymphocytes or RBL 2H3 mast cells were transiently transfected overnight with 10 μg of GFP–PKD cDNA. Shown are mid-section confocal images showing the expression of GFP–PKD in these cells. (C) Immunofluorescence staining of A20 B lymphocytes. Top: cells stained with a specific C-terminal PKD antibody (sc-935) to reveal the subcellular distribution of endogenous PKD. Middle: cells stained with secondary layer only. Bottom: immunofluorescence staining of the endogenous GM 130 present in A20 B lymphocytes using a GM 130 polyclonal antibody (Nakamura et al., 1995). Bar = 10 μm. (D) Comparison of the subcellular distribution of exogenously expressed GFP–PKD or NAGT1–GFP in A20 B lymphocytes. Images represent two-dimensional projections of sections in Z-series taken across the depth of the cells at 0.3 μm intervals. Download figure Download PowerPoint Antigen receptor ligation induces a transient relocalization of PKD to the plasma membrane To examine the effect of antigen receptor ligation on localization of PKD, RBL 2H3 cells expressing GFP–PKD were activated via the FcϵR1 and confocal images were taken of live cells in real time. The data (Figure 3A) demonstrate that activation of the FcϵR1 induces a rapid redistribution of GFP–PKD from the cytosol to the plasma membrane of mast cells. Membrane targeting of GFP–PKD was detectable 95%) and mast cells (>90%) observed. The results shown are representative of at least four independent experiments where each time 6–8 individual cells were imaged. Download figure Download PowerPoint Strikingly, GFP–PKD association with the plasma membrane of antigen receptor-stimulated cells was transient. Time-lapse confocal microscopy clearly demonstrated that GFP–PKD consistently dissociated from the plasma membrane of FcϵR1-activated mast cells (Figure 3A) or BCR-stimulated lymphocytes (Figure 3B) within ∼8–10 min of antigen receptor engagement. PKD localization in phorbol ester-stimulated B lymphocytes and mast cells The data presented in Figure 4A demonstrate that the phorbol ester PDBu induces translocation of GFP–PKD to the plasma membrane of RBL 2H3 cells. Membrane targeting of GFP–PKD was detectable within ∼6–8 min of PDBu treatment of RBL 2H3 cells and was maximal within ∼10–15 min (Figure 4A). Stimulation of A20 B lymphocytes with PDBu was also found to induce complete translocation of GFP–PKD from the cytosol to the plasma membrane of these cells within ∼10 min (Figure 4B). Image analysis of these cells for longer time periods found that the association of PKD with the plasma membrane was maintained for several hours in the continued presence of PDBu, with no evidence for the redistribution of PKD back into the cytosol (data not shown). The stable nature of PKD membrane translocation following phorbol ester treatment was in marked contrast to the transient plasma membrane residence of PKD observed in cells activated by antigen receptor engagement. Figure 4.Redistribution of GFP–PKD to the plasma membrane in A20 B lymphocytes and RBL 2H3 mast cells in response to phorbol ester treatment. RBL 2H3 cells (A) or A20 B lymphocytes (B), transiently expressing GFP–PKD, were imaged in real time before and after the addition of 50 ng/ml PDBu, using an inverted confocal microscope, at 37°C. Images taken at the indicated times are shown, with treatment carried out after the zero time point image had been acquired. Similar results were obtained in four independent experiments where at least 4–5 individual cells were analysed per experiment. Similar kinetics of translocation were observed in four independent experiments, where 4–5 individual cells were analysed each time. Download figure Download PowerPoint We also noted one other more subtle difference in PKD localization in PDBu-treated versus antigen receptor-activated RBL 2H3 cells. Here, PDBu stimulation caused the majority of PKD to redistribute to the plasma membrane. However, on close examination, a fraction of PKD was found localized to membranes surrounding cytoplasmic granules (Figure 5A). In contrast, FcϵR1 stimulation of RBL 2H3 mast cells induced the total cellular pool of PKD to redistribute to the plasma membrane, with no detectable association with cytoplasmic granules (Figure 5B). Figure 5.Association of GFP–PKD with intracellular granules in phorbol ester- but not FcϵR1-stimulated RBL 2H3 mast cells. RBL 2H3 mast cells, transiently expressing GFP–PKD, were imaged in real time before and after treatment with PDBu (A) or following FcϵR1 ligation (B). PDBu induces association of GFP–PKD with both the plasma membrane and the membrane of cytosolic granules. In contrast, FcϵR1 ligation specifically induces association of GFP–PKD with the plasma membrane. In all experiments, phorbol ester treatment gave a slow translocation to the plasma membrane while FcϵR1 stimulation resulted in a rapid plasma membrane translocation. The number of experiments was six and four, respectively, where 4–5 individual cells were analysed in each experiment. Download figure Download PowerPoint Sustained activation of PKD by antigen receptor engagement PKD is thus localized transiently to the plasma membrane of B lymphocytes and mast cells following antigen receptor ligation, but subsequently returns to the cytosol in a dynamic cycle that is complete within 8–10 min following receptor stimulation. To examine whether the dissociation of PKD from the plasma membrane was accompanied by a reduction in its kinase activity, the kinetics of PKD activation were compared with those of PKD membrane targeting. The data presented in Figure 1 indicated that substantial PKD activity could be detected after 10 min of antigen receptor engagement in both B lymphocytes and mast cells, at times when PKD dissociation from the plasma membrane was complete (see Figure 3). To investigate the long-term kinetics of PKD activation following antigen receptor ligation, A20 B lymphocytes were stimulated with F(ab)′2 for a period of up to 2 h. PKD activity was then measured at the indicated time points by in vitro kinase assays and by western blot analysis of total cell lysates using the pS916 antibody (which selectively recognizes active PKD). As Figure 6A indicates, BCR-induced activation of PKD in A20 B lymphocytes is an extremely sustained response: maximal activity was seen within 1 min of antigen receptor ligation and was maintained at high levels throughout the 10 min period that PKD is found associated with the plasma membrane. Importantly, both PKD catalytic activity and S916 phosphorylation remained high for at least 2 h post-receptor triggering, at times when PKD had fully dissociated from the plasma membrane (Figure 3 and data not shown). Figure 6.Sustained PKD activity following BCR ligation. (A) A20 B lymphocytes were plated in complete medium at a density of 1.5 × 107 cells/ml in a 24-well plate and left to recover at 37°C, 5% CO2 for 1 h before stimulation. Subsequently, the cells were left untreated (n/s) or were stimulated with 10 μg/ml F(ab)′2 fragments of anti-mouse IgG for various times (2–120 min). The cells were lysed and PKD immunoprecipitated using the PA-1 antiserum. In vitro kinase assays (IVK) were performed using PKD autophosphorylation as the measure of catalytic activity. Alternatively, proteins in the lysates were precipitated with cold acetone, separated by SDS–PAGE and subjected to western blot analysis using the pS916 antiserum and a pan C-terminal PKD antibody. Results are representative of three independent experiments. (B) A20 B lymphocytes were left untreated (n/s) or were stimulated with 10 μg/ml F(ab)′2 fragments of anti-mouse IgG for various times (0–30 min) or with 50 ng/ml PDBu for 15 min. Particulate (P; membrane) and soluble (S; cytosolic) fractions were prepared from the cells, as described in Materials and methods, and proteins in the resulting fractions were precipitated with cold acetone, separated by SDS–PAGE and subjected to western blot analysis using the pS916 antiserum to detect active PKD. Results are representative of two independent experiments. Download figure Download PowerPoint The confocal images presented in Figure 3 indicated that active PKD does not localize to specific intracellular structures following its dissociation from the plasma membrane in antigen receptor-stimulated cells. We examined this further by biochemical fractionation experiments. As shown in Figure 6B, active PKD was found in the particulate (membrane) fraction of these cells within 2 min of BCR stimulation, but within ∼15 min the PKD activity within this compartment had diminished. In contrast, active PKD could be detected within the soluble (cytosolic) compartment of B lymphocytes up to 30 min after BCR stimulation, indicating that active PKD does not associate strongly with intracellular membranes following its dissociation from the plasma membrane. Importantly, PKD isolated from PDBu-treated B lymphocytes was active in the particulate fraction of these cells at these times (Figure 6B and data not shown), in agreement with the confocal data presented above demonstrating that PKD stably associates with the plasma membrane in response to phorbol ester treatment. Accordingly, these confocal images and biochemical data suggest that there are two phases of PKD regulation following antigen receptor stimulation in B lymphocytes and mast cells: an initial activation/recruitment to the plasma membrane followed by a sustained period of PKD activity within the cytosol. Membrane targeting of GFP–PKD is mediated by the DAG-binding CRD The structural basis for the membrane recruitment of a classical PKC isoform, PKCγ, has been explored in detail recently and requires the co-ordinate binding of calcium to the C2 domain followed by DAG binding to the CRD domain (Oancea and Meyer, 1998). The N-terminal regulatory region of PKD does not contain a C2 domain, but does contain a DAG-binding CRD domain and a putative PH domain (Valverde et al., 1994). To determine the relative contributions of the PH domain and the CRD to antigen receptor-induced PKD translocation, GFP-tagged versions of different PKD CRD or PH domain mutants were made. These were expressed transiently in A20 B lymphocytes, and a comparison of the distribution of these molecules in quiescent and BCR-stimulated cells was made by real-time confocal imaging of live cells. The GFP–PKD ΔCRD mutant contains a deletion of the entire CRD domain, whilst the GFP–PKD P287G mutant contains a proline to glycine substitution within the CRD. In both cases, the integrity of the CRD domain of PKD is destroyed and the binding of phorbol esters/DAG to PKD is prevented (Iglesias et al., 1998a). As shown in Figure 7, GFP–PKD ΔCRD and GFP–PKD P287G were distributed evenly throughout the cytosol of quiescent A20 cells and excluded from the nucleus, indistinguishably from wild-type GFP–PKD. Following BCR ligation, wild-type GFP–PKD transiently translocated from the cytosol to the plasma membrane, as observed previously. In contrast, real-time confocal imaging studies of cells showed that BCR ligation was repeatedly unable to induce the plasma membrane redistribution of either GFP–PKD ΔCRD or GFP–PKD P287G (Figure 7). Hence the BCR-induced translocation of PKD to the plasma membrane was dependent on the integrity of the DAG/phorbol ester-binding CRD domain of PKD. Figure 7.Plasma membrane targeting of GFP–PKD in response to BCR triggering is mediated by the DAG-binding CRD. A20 B lymphocytes were transiently transfected with different GFP–PKD mutations: a deletion of the CRD (ΔCRD); a point mutation within CRD (P287G); a deletion of the PH domain (ΔPH); and a kinase-deficient PKD mutant (D733A). Confocal images of representative living cells are shown at different time points before and after BCR ligation at the times indicated. Transient plasma membrane association of the kinase-dead GFP–PKD D733A and the GFP–PKD ΔPH mutants was observed following BCR ligation, comparable with wild-type GFP–PKD. The kinetics of the subcellular redistribution of the ΔPH and the D733A GFP–PKD mutants were similar to those of wild-type GFP–PKD, with maximal membrane association seen at ∼1–2 min before returning to the cytosol within ∼7–10 min in >80% of the cells analysed. The cytosolic localization of GFP–PKD ΔCRD and GFP–PKD P287G was not altered upon BCR ligation in >95% or >75% of the BCR-stimulated cells imaged, respectively. Translocation of GFP–PKD mutants was visualized in at least six independent experiments, where 4–6 individual cells were analysed before and during BCR stimulation in each experiment. Download figure Download PowerPoint To determine whether the PH domain of PKD was involved in modulating its subcellular localization, A20 B lymphocytes were transiently transfected with a PKD mutant containing a deletion of the entire PH domain (GFP–PKD ΔPH). The confocal images in Figure 7 show a homogeneous distribution of GFP–PKD ΔPH throughout the cytosol and the nucleus of resting B lymphocytes, unlike wild-type or CRD mutants of PKD whose localization was restricted to the cytosolic compartment. In response to BCR stimulation, an accumulation of GFP–PKD ΔPH at the plasma membrane was observed with an accompanying depletion of the cytosolic pool of GFP–PKD ΔPH (Figure 7). The plasma membrane targeting of GFP–PKD ΔPH was transient
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