Isoenzyme-specific Translocation of Protein Kinase C (PKC)βII and not PKCβI to a Juxtanuclear Subset of Recycling Endosomes

Elucidation of isoenzyme-specific functions of individual protein kinase C (PKC) isoenzymes has emerged as an important goal in the study of this family of kinases, but this task has been complicated by modest substrate specificity and high homology among the individual members of each PKC subfamily. The classical PKCβI and PKCβII isoenzymes provide a unique opportunity because they are the alternatively spliced products of the β gene and are 100% identical except for the last 50 of 52 amino acids. In this study, it is shown that green fluorescent protein-tagged PKCβII and not PKCβI translocates to a recently described juxtanuclear site of localization for PKCα and PKCβII isoenzymes that arises with sustained stimulation of PKC. Mechanistically, translocation of PKCβII to the juxtanuclear region required kinase activity. PKCβII, but not PKCβI, was found to activate phospholipase D within this time frame. Inhibitors of phospholipase D (1-butanol and a dominant negative construct) prevented the translocation of PKCβII to the juxtanuclear region but not to the plasma membrane, thus demonstrating a role for phospholipase D in the juxtanuclear translocation of PKCβII. Taken together, these results define specific biochemical and cellular actions of PKCβII when compared with PKCβI.

Members of the protein kinase C (PKC) 1 superfamily of lipiddependent serine-threonine kinases function as integral signaling intermediates in the transmission of numerous extracellular signals. PKC currently consists of 11 closely related isoenzymes that can be grouped into 3 subfamilies (classical, novel, and atypical) on the basis of lipid cofactor requirements and structural homology (1,2). A major form of cellular regulation of PKC involves the dynamic redistribution or "translocation" of PKC isoenzymes from cytosol to the plasma membrane. Membrane translocation of PKC is regulated through receptor-coupled lipid hydrolases that act on phosphoinositides and phosphatidylcholine to generate diacylglycerol (DAG) lipid second messengers. The recruitment and activation of PKC at membranes also link activation of the enzyme to the proximity of membrane protein substrates (3).
One of the major targets of PKC is phosphatidylcholinespecific phospholipase D (PC-PLD) (4). Activated PC-PLD hydrolyzes the phosphodiester bond of phosphatidylcholine to generate free choline and phosphatidic acid, which is converted to DAG through the actions of lipid phosphate phosphatases. DAG derived from phosphatidylcholine hydrolysis has been proposed to mediate the reciprocal regulation of PKC, but there are incomplete and conflicting data on this topic (5-7). One possible functional significance of PC-PLD-derived DAG arises from the sustained presence of DAG in the membrane for extended periods of time and in relatively high concentrations in comparison with DAG derived from phosphoinositide-specific PLC, and this phosphatidylcholine-derived DAG has been proposed to mediate long-term cellular processes that require sustained PKC activation (8).
The implication of specific PKC isoenzymes in distinct pathological processes has led to an intense effort to identify and elucidate the mechanisms that regulate isoenzyme-specific functions of PKC. To date, these efforts have been complicated by the large number of highly homologous PKC family members, expression of multiple PKC isoenzymes in each cell type, and the finding that there are only minor differences in substrate selectivity among different PKCs.
One of the best opportunities to study the mechanisms of isoenzyme specificity arises from the alternatively spliced gene products of the classical PKC (cPKC) ␤ gene. This alternative splicing creates two proteins, PKC␤I and PKC␤II, which display 100% identity over their first 621 amino acids but then diverge in the last 50 -52 carboxyl-terminal residues, which are encoded by two consecutive and alternatively spliced exons (9). Importantly, these divergences are 100% conserved across rat, rabbit, and human, suggesting that there are distinct functions encoded by both gene products. To that end, we previously identified residues in the carboxyl terminus of PKC␤II that are homologous to actin-binding proteins (10). We demonstrated that with long-term stimulation with phorbol esters, PKC␤II differentially interacts with the actin cytoskeleton, where it becomes activated and is then protected from phorbol 12-myristate 13-acetate (PMA)-induced down-regulation.
In another line of investigation, we have recently reported that sustained stimulation (45-60 min) of PKC with DAGmimicking phorbol and non-phorbol PKC agonists can induce the translocation of PKC␣ and PKC␤II to a subcomponent of the endosomal recycling compartment concentrated in a juxtanuclear location around the microtubule-organizing center/ centrosome (11). Kinetic studies with a fluorescence-conjugated transferrin ligand as a marker of membrane trafficking demonstrated that the cPKC-positive compartment functions to sequester membrane recycling components.
In this study, we investigated the isoenzyme specificity of this novel translocation and found that whereas PKC␣ and PKC␤II translocated to the juxtanuclear compartment, PKC␤I did not. Further analysis showed that PKC␤II translocation to this compartment required kinase activity, implicating a substrate-dependent interaction. The specificity for PKC␤II versus PKC␤I and the requirement for kinase activity raised the possibility that PC-PLD may be involved in the process. Evidence is provided to implicate PLD in the selective translocation of PKC␤II to the juxtanuclear compartment. These results define a novel pathway of translocation of PKC␤II requiring differential activation of PLD. These data suggest that PKC␤II differentially activates PLD and that this is required for the mechanism of translocation to the subset of juxtanuclear recycling endosomes. The implications of these results are discussed. Cell Culture-HeLa cells were maintained in Dulbecco's modified Eagle's medium, and HEK 293 cells were maintained in Eagle's minimal essential medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin in a 5% CO 2 incubator at 37°C. Cells were passaged every 3-4 days to maintain cells in logarithmic growth.

Materials
Plasmid Construction-All recombinant DNA procedures were carried out following standard protocols. The wild type pBK-CMV-GFP-PKC-␤II and kinase-defective pBK-CMV-GFP-K371R-␤II cPKC constructs have been described previously (12). HA-tagged KR-hPLD1 was kindly supplied by Dr. Michael Frohman (State University of New York at Stony Brook, Stony Brook, NY). All DNA sequencing was performed at the Medical University of South Carolina DNA Sequencing Facility.
Indirect Immunofluorescence-HEK 293 cells were plated onto MatTek (Ashland, MA) confocal dishes at a density of 2.5-5.0 ϫ 10 Ϫ5 cells/dish and co-transfected 24 -48 h later with either HA-tagged KR-PLD1 or GFP-PKC ␤II/␤I or HA-tagged KR-PLD1 and GFP-PKC␤II or GFP-PKC␤I using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's recommendations. Twelve h after transfection, cells were treated with either ME 2 SO (0.01%) or 100 nM PMA for 1 h and fixed with 3.7% paraformaldehdye/10% methanol for 10 min at room temperature. After fixation, cells were permeabilized at Ϫ20°C with 100% methanol for 5 min. Cells were then washed with 1.5% FBS/phosphate-buffered saline (PBS) three times for 5 min each and blocked with 2.5% FBS/PBS for 1 h. Primary antibodies were incubated at a dilution of 1:50 or 1:100 in 1.5% FBS/PBS with 0.15% saponin for 2 h at room temperature or overnight at 4°C. After incubation with primary antibody, cells were washed three times for 5 min each with 1.5% FBS/PBS. TRITC-conjugated secondary antibody (Molecular Probes, Inc.) was incubated at a dilution of 1:50 or 1:100 in 1.5% FBS/PBS and 0.15% saponin for 1 h. Cells were washed a final three times for 5 min each with 1.5% FBS/PBS and visualized immediately with confocal microscopy.
Confocal Microscopy-Preparation of samples for immunofluorescence was as described above. Cells expressing GFP fusion proteins alone were viewed live in 10 mM HEPES-buffered media or viewed immediately after a 10-min fixation with 3.7% paraformaldehyde/10% methanol pre-warmed to 37°C. All confocal images were taken with an Olympus UltraView Spinning Disk Confocal IX-70 system with an Olympus 60X 1.4 NA lens and krypton/argon laser line. Each micro-scopic image is representative of 20 fields over a minimum of three experiments, and all images were taken at the equatorial plane of the cell. GFP fusion proteins were excited at 488 nm, and TRITC was excited at 543 nm. All fluorescent images were captured sequentially and combined within the UltraView software (PerkinElmer Life Sciences). Raw data images were cropped in Adobe Photoshop 5.0 for publication.
Transphosphatidylation Assay-The cellular activity of PLD was assessed with a modified transphosphatidylation assay (13). Twelve h after transfection, HEK 293 cells were labeled overnight (12-16 h) in 35-mm dishes with 3.0 Ci/ml [ 3 H]palmitic acid in minimal essential medium supplemented with 10% FBS and 0.1% delipidated bovine serum albumin. Cells were then washed three times with PBS, and serum-free minimal essential medium/0.1% bovine serum albumin medium was added. Cells were allowed to recover in serum-free media at 37°C for 30 min before stimulation. Ten min before stimulation, 0.4% 1-butanol was added to the cells. Cells were stimulated with 100 nM PMA for the specified times and at the noted concentrations. All incubations were performed at 37°C. After stimulation, the culture media were removed, and the cells were washed rapidly three times with 1 ml of ice-cold PBS. Total cellular membranes were extracted via the method of Bligh and Dyer (14). Lipids were dried down and resuspended in 75 l of chloroform:methanol (2:1). Fifty l of the 75-l volume was loaded per lane. The TLC solvent system consisted of ethyl acetate:iso-octane:acetic acid (9:5:2). A phosphatidyl-butanol standard (Avanti Polar Lipids, Inc.) was included in parallel to confirm lipid species. The plate was sprayed with EN 3 HANCE Spray (PerkinElmer Life Sciences) to amplify the tritium signal and exposed for autoradiography for 24 h. The phosphatidyl-butanol band and the total remaining lipids were scraped and counted separately. The [ 3 H]phosphatidylbutanol band was compared with total labeled lipids to generate the percentage of increase over total labeled lipid. Each experiment was repeated three to five times.

Isoenzyme-specific Translocation of PKC␤II to a Subset of
Recycling Endosomes-To investigate the isoenzyme specificity of PKC␤ translocation to the subset of recycling endosomes centered around the microtubule-organizing center/centrosome described previously (Ref. 11), HeLa cells were transiently transfected with a GFP-tagged PKC␤I or PKC␤II construct and stimulated with PMA for 1 h. Confocal microscopic imaging revealed that in the absence of phorbol ester stimulation, both GFP-tagged cPKC isoenzymes displayed a diffuse cytoplasmic localization with exclusion from the nucleus (Fig. 1, A and B,   FIG. 1. Isoenzyme-specific translocation of GFP-PKC␤II to a subset of recycling endosomes. A, representative confocal images of HeLa cells transiently transfected with GFP-PKC␤II and treated 12 h after transfection with either 0.01% ME 2 SO or 100 nM PMA for 1 h. B, confocal images of HeLa cells transiently transfected with GFP-PKC␤I and treated with either 0.01% ME 2 SO or 100 nM PMA for 1 h. Each panel is representative of at least twenty ϫ60 fields, and each micrograph is a single image captured at the equatorial plane of the nucleus. panels 1). Addition of 100 nM PMA for 1 h induced translocation of GFP-PKC␤II to both the plasma membrane and the juxtanuclear location (Fig. 1A, panel 2). In contrast, stimulation of GFP-PKC␤I with PMA for 1 h resulted in the translocation of PKC␤I to the plasma membrane only, and no further translocation was evident (Fig. 1B, panel 2). These results suggest that amino acid residues encoded within the carboxyl terminus of PKC␤I and PKC␤II determine the specificity for translocation of PKC␤II to this subset of recycling endosomes.
To control for the possibility that the specificity of translocation for the two cPKC isoenzymes was related to different rates of translocation, a time course experiment was performed at 0, 1, 3, and 6 h. No juxtanuclear translocation of PKC␤1 was evident at any time point, suggesting that the differences between translocation were not related to rates of the process (data not shown).
Of note, in a previous study, Blobe et al. (10) stated that PKC␤II contained actin binding sequences within its carboxyl terminus not found in PKC␤I. Given the above-mentioned results, we examined the cellular actin pattern during translocation of PKC␤II to the juxtanuclear compartment. It was observed that upon stimulation with PMA for 1 h, there was a concentration of actin at the juxtanuclear location and that this overlapped with translocated GFP-PKC␤II (Fig. 2, A and B). These results reveal a significant effect of PMA on the reorganization of the actin cytoskeleton, and they demonstrate a co-localization of PKC␤II but not PKC␤I with the reorganized actin. Moreover, treatment of cells with cytochalasin D, which depolymerizes actin, caused partial unraveling of the pericentriolar actin and of the associated PKC␤II, which acquired a whorl-like appearance in the presence of cytochalasin D (Fig.  2C). These results confirm and extend the previous finding of in vitro and cellular association of PKC␤II (and not PKC␤I) with the actin cytoskeleton.
Kinase Activity Is Required for Translocation of PKC␤II to the Juxtanuclear Compartment-To investigate the role of kinase activity in the translocation of PKC␤II to the juxtanuclear compartment, HeLa cells transfected with GFP-PKC␤II were stimulated with PMA, with or without a 30-min preincubation with 3 M Gö 6976, a specific inhibitor of the calcium-dependent PKC isoenzymes (15). As seen in Fig. 3A, panels 1 and 2, when the kinase activity of PKC␤II was inhibited with Gö 6976, PKC␤II translocated to the plasma membrane, but there was no further translocation to the juxtanuclear compartment. This requirement for kinase activity was further probed using a kinase-defective GFP-tagged PKC␤II mutant K371R, which is unable to bind ATP and phosphorylate PKC substrates. When stimulated with PMA for 1 h, GFP-K371R-␤II was observed to translocate to the plasma membrane, but no juxtanuclear accumulation was evident (Fig. 3B, panels 1 and 2). These data demonstrate that PKC␤II translocation to the plasma membrane occurs independent of kinase activity but that kinase activity is required for translocation to the juxtanuclear compartment.
PC-PLD Is Activated in a Time-and PKC-dependent Manner during the Sustained Stimulation of PKC with Phorbol Esters-PC-PLD1 is a major downstream target for calcium dependent, DAG/PMA-sensitive cPKC isoenzymes. Currently, there are some data demonstrating isoenzyme-specific differences in the cPKC regulation of PLD, including differences between PKC␤I and cPKC␤II (see "Discussion"). In addition, very recently, Hu and Exton (16) reported that the full-length rat PKC␣ isoenzyme binds and activates PC-PLD1 and that this was dependent on the presence of a phenylalanine residue (Phe 663 ) in the V5 carboxyl terminus of PKC␣. These data implicate a possible role for the V5 variable region of PKC in the PKC-PLD interaction. These observations, coupled with the requirement for kinase activity for PKC␤II translocation to the juxtanuclear compartment, raised the possibility that the mechanism of this translocation may involve the activation of PLD1.
To begin to explore this hypothesis, it was important first to establish whether PLD was activated in this system. Using a transphosphatidylation assay with 1-butanol to evaluate cellular PLD activity, HEK 293 cells were treated with 100 nM PMA. As seen in Fig. 4A, phosphatidyl-butanol accumulation, indicating PLD activity, was evident starting at 30 -40 min of PMA stimulation. Importantly, this activation was completely blocked by preincubation with 3 M of the selective cPKC inhibitor Gö 6976 (Fig. 4A). Interestingly, this time frame of PLD activation correlated closely with the translocation of PKC␤II to the juxtanuclear compartment.
To determine whether PKC␤I and PKC␤II regulated PLD differentially, HEK 293 cells were transiently transfected with each PKC isoenzyme, and the transphosphatidylation reaction was assayed. For each experiment, the efficiency of transfection was similar for both isoenzymes and ranged between 40% and 50% of total cell population. Interestingly, in cells that were transfected with PKC␤II, there was a higher basal level of PLD activity in the absence of phorbol stimulation when compared with PKC␤I. Upon stimulation with PMA, cells that expressed PKC␤II demonstrated a 50% increase in phosphatidyl-butanol accumulation (Fig. 4B). In contrast, there was no further stimulation of PLD in cells overexpressing PKC␤I. These data disclose significant isoenzyme specificity of activation of PLD by cPKC, and they also suggest the possibility that the isoenzyme specificity in translocation of PKC␤ isoenzymes to the juxtanuclear compartment may be related to the differential activation of PLD.
Translocation of PKC␤II to the Juxtanuclear Compartment Is Dependent upon PLD Activity-To investigate whether PLD activity is required for translocation of PKC to the juxtanuclear compartment, 1-butanol was utilized as an inhibitor of PLD activity. HEK 293 cells were preincubated with 0.4% 1-butanol for 10 min before a 1-h stimulation with PMA. Preincubation with 1-butanol had no effect on the translocation of PKC␤II to the plasma membrane, but there was a complete inhibition of the translocation of PKC␤II to the juxtanuclear location (Fig.  5A, panels 1 and 2). This inhibition was specific for the primary alcohol because the secondary alcohol 2-butanol did not block translocation (data not shown).
To confirm this pharmacological inhibition, a catalytically deficient mutant PLD, KR-PLD1, was cotransfected along with GFP-PKC␤II into HEK 293 cells, and trafficking of PKC␤II to the juxtanuclear compartment was evaluated. When GFP-PKC␤II was stimulated with PMA for 1 h, the co-expression of KR-PLD1 did not inhibit the translocation of PKC␤II to the plasma membrane, but there was no translocation to the juxtanuclear compartment (Fig. 5B). The ability of the KR-PLD1 mutant to inhibit PLD was evaluated in a transphosphatidylation assay that was performed in parallel with the confocal studies. Transient transfection of KR-PLD1 reduced endogenous PLD activity by ϳ50% (Fig. 5C), thus confirming that KR-PLD1 inhibited cellular PLD activity. These data demonstrate that PLD activity is involved in the translocation of cPKC to a subset of recycling endosomes. DISCUSSION In the present study, we have identified a role for the carboxyl-terminal V5 variable region in the differential subcellular localization of PKC␤ isoenzymes to a recently defined novel intracellular target of translocation of PKC␤ that co-localizes with the microtubule-organizing center/centrosome (11). Investigation into the mechanism revealed that PKC␤II translocation to this juxtanuclear compartment, unlike translocation to the plasma membrane, required kinase activity of PKC. The results also revealed differential activation of PLD by PKC␤II and not PKC␤I. Finally, the results disclosed a critical role for PLD in the differential juxtanuclear translocation of PKC␤II. Thus, these data define an isoenzyme-specific function for cPKC in the regulation of PLD and, in turn, the reciprocal regulation of PKC by PLD through the modulation of PKC subcellular localization. Isoenzyme-specific differences in the subcellular localization and function of PKC␤I and PKC␤II have been reported previously. Using indirect immunofluorescence and monoclonal antibodies, several groups have observed an isoenzyme-specific association of PKC␤II with the actin cytoskeleton (17,18). In addition, Blobe et al. (10) identified an actin-binding sequence (ABS-1) located within the V5 region of PKC␤II that was absent from PKC␤I. This sequence was found to be necessary for the phorbol ester-induced translocation of PKC␤II to the actin cytoskeleton and for isoenzyme-specific functions associated with this translocation, including protection from down-regulation. Furthermore, Yamamoto et al. (19) identified specific and opposing functional roles for the PKC␤ isoenzymes in A10 vascular smooth muscle cells. They reported that upon overexpression of PKC␤I, there was a stimulation of cell proliferation, whereas PKC␤II overexpression was inhibitory to cell proliferation. This is supported by other data wherein the selective regulation of the expression of PKC␤ isoenzymes occurs through differential splicing in lymphoid cells (20,21). In addition to these studies, there are also a number of other studies that have proposed specific subcellular localization and/or functions for either one or the other isoenzyme, but in most cases there was no direct comparison between PKC␤I and PKC␤II, and it is not known whether the proposed functions are isoenzyme-specific (22). The results from this study define a very clear difference in the translocation of PKC␤I and PKC␤II such that only PKC␤II translocates to the juxtanuclear region.
PC-PLD is a major downstream target for PKC. Data from overexpression studies and in vitro experiments with purified or recombinant protein indicate that it is the calcium-dependent cPKC isoenzymes (␣ and ␤) that are the primary PKC regulators of PLD in the cell (23)(24)(25)(26). Additionally, there are some data to suggest possible isoenzyme-specific differences between PKC␤I and PKC␤II. For instance, a role for PKC␤1 has been suggested through the examination of PLD activity in cells that stably overexpressed PKC␤1 and displayed an enhanced formation of DAG in response to phorbol ester treatment (27,28). These results were further supported with studies that used purified PKC and a cell-free system to demonstrate a rank order of potency for PLD activation with "PKC beta 1 Ͼ alpha Ͼ gamma and beta 2" showing little or no activity (25). Interestingly, these studies are in apparent conflict with the current study. However, while researching the literature, it was discovered that there had been an inconsistency in the nomenclature that was applied initially to the PKC␤ isoenzymes. Whereas Ono et al. (29) proposed to name the 673-amino acid splice product, PKC␤II, and the 671-amino acid protein PKC␤I, Coussens et al. (30) referred to the 673amino acid splice product as "PKC beta I" and the shorter, 671-amino acid splice product as "PKC beta II." Currently, the accepted nomenclature is that the longer gene product is PKC␤II and the shorter one, PKC␤I (31).
Importantly, a very recent report by Hu and Exton (16) identified residues in the carboxyl terminus of PKC␣ as critical to the activation of PLD by PKC. It is noteworthy that the carboxyl domain of PKC␣ shows higher homology to the carboxyl domain of PKC␤II than PKC␤I. These results underscore the importance of the variable region in the PKC-PLD relationship. Coupled with the results from this study implicating PLD in the juxtanuclear translocation of PKC␤II, the differential activation of PLD by PKC␤II and PKC␤I provides a mechanistic explanation for the selective translocation of PKC␤II to the juxtanuclear region.
In addition, these results raise the possibility that sustained activation (30 -60 min) of DAG/PMA-responsive PKC may disclose novel isoenzyme-specific functions arising from differential subcellular localization. This could potentially lead to disparate isoenzyme effects secondary to differential down- FIG. 5. Translocation of GFP-PKC␤II requires PLD activity. A, representative confocal images of cells transiently transfected with GFP-PKC␤II (panels 1 and 2) and treated with PMA for 1 h with (panel 2) and without (panel 1) a 10-min preincubation with 1-butanol. Each panel is representative of at least twenty ϫ60 fields, and each micrograph is a single image captured at the equatorial plane of the nucleus. B, immunofluorescence of cells transiently co-transfected with GFP-PKC␤II and HA-tagged KR-PLD1. Cells were treated with either 0.01% ME 2 SO or 100 nM PMA for 1 h. KR-PLD1 was detected with anti-HA and a secondary antibody conjugated to rhodamine. C, the effects of KR-PLD1 on endogenous PLD activity. Cells were transiently transfected with either pGCN-vector or KR-PLD1, and 12 h after transfection, cells were subjected to steady-state labeling with 3 Ci of [ 3 H]palmitate overnight. Cells were washed and then treated with 100 nM PMA for 1 h. Phosphatidylbutanol formation was quantitated by scraping and scintillation counting. These results are representative of three separate experiments. regulation and the differential access to compartmentalized substrates. It is interesting that we found the compartment to overlap with actin because this is predicted from the study of Blobe et al. (10), who demonstrated biochemically that the differential interaction of PKC␤II with actin prevented PMAinduced down-regulation.
In our previous study, we showed that the juxtanuclear translocation of PKC␤II and PKC␣ regulates the kinetics of the recycling endosomes such that a component of those endosomes becomes sequestered in this juxtanuclear region. The findings of isoenzyme specificity of translocation and interaction with PLD and the role of PLD in the juxtanuclear translocation of PKC␤II therefore suggest specific roles for the PKC␣/PKC␤II-PLD pathway in regulation of membrane recycling.
One interesting question raised by the current findings is the mechanism by which PLD participates in/regulates juxtanuclear translocation of PKC. The results with the K/R mutant of PLD, which appears to function as a dominant negative, establish the role of PLD in this process. The observation that overexpression of the KR-PLD1 reduces cellular PLD activity by 50% but completely blocks PKC translocation to the juxtanuclear compartment suggests that there is a requirement for a specific subcellular pool of PLD activity in the translocation event. That is, KR-PLD1, which in our system localizes in a pattern similar to wild-type PLD1 ( Fig. 5; data not shown), may act to specifically inhibit the PLD1 isoform in that specific compartment but may not inhibit PLD2. Moreover, the results with 1-butanol demonstrate a specific requirement for PLD activity and, presumably, the production of its product, phosphatidic acid. It would be of interest to determine the mechanism by which phosphatidic acid regulates this translocation. (Whereas the current results do not distinguish whether it is phosphatidic acid or a subsequent metabolism to DAG that is required for this translocation, it is presumed that PMA would circumvent the requirement for DAG, thus pointing to a role for phosphatidic acid itself.) In conclusion, these studies identify specific differences in the function of PKC␤I and PKC␤II relating to activation of PLD and juxtanuclear translocation. Moreover, understanding this juxtanuclear translocation promises novel insight into a novel paradigm of PKC function whereby sustained activation results in selective translocation to a novel compartment with selective functions.