SEARCH

SEARCH BY CITATION

Keywords:

  • Aplysia ;
  • lipid binding;
  • novel protein kinase C;
  • phosphatidic acid;
  • phosphorylation;
  • translocation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine-glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine-alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation-dependent regulation of translocation.

Abbreviations used
Bis-1

bisindolymaleimide 1

Cal A

calyculin A

DAG

diacylglycerol

DOG

1,2-dioctanoyl-sn-glycerol

eGFP

enhanced green fluorescent protein

MBP

maltose binding protein

PA

phosphatidic acid

PC

phosphatidylcholine

PS

phosphatidylserine

Protein kinase Cs (PKCs) are a family of lipid-activated enzymes that play important roles in many cellular processes, including regulation of synaptic strength in the nervous system (Majewski and Iannazzo 1998; Sossin 2007). In Aplysia californica, behavioral sensitization, a form of learned fear, is mediated in part by an increase in the strength of the connections between sensory and motor neurons. This increase, called synaptic facilitation is mediated by the neurotransmitter serotonin (5HT) that can induce facilitation in isolated ganglia and in cocultures containing sensory and motor neurons (Byrne and Kandel 1996; Kandel 2001, 2012). In the Aplysia nervous system, there are two phorbol ester-regulated PKCs: PKC Apl I, homologous to the Ca2+-activated PKC family in vertebrates (α, β1, β2, and γ) also called conventional or classical PKCs (cPKCs), and PKC Apl II, which is homologous to the Ca2+-independent epsilon family of PKC in vertebrates (ε and η) called novel PKCs (nPKCs) (Kruger et al. 1991; Sossin et al. 1993; Sossin 2007) and both play a role in facilitation depending on the type of stimulation; PKC Apl II plays a role in the facilitation of depressed synapses (Manseau et al. 2001) and in facilitation after massed applications of 5HT (Sossin 1997; Jin et al. 2011), whereas PKC Apl I plays a role in facilitation when serotonin is applied together with sensory neuron firing (Zhao et al. 2006).

Both cPKCs and nPKCs have two C1 domains and one C2 domain, but the C2 domain of nPKCs is located N-terminal to the C1 domains and lacks the critical aspartic acid residues required for coordinating Ca2+ ions (Nalefski and Falke 1996). In cPKCs, the C2 domain mediates Ca2+-dependent binding to the membrane lipid phosphatidylserine (PS) (Newton 1995a,b). This binding is believed to be a primary step in kinase activation for classical PKCs (Oancea and Meyer 1998; Medkova and Cho 1999).

The function of the C2 domain of nPKCs is less clear (Farah and Sossin 2012). For PKC Apl II, removal of the C2 domain lowered the amount of lipid required to activate the enzyme, introducing the idea of C2 domain-mediated inhibition (Sossin et al. 1996; Pepio et al. 1998). Indeed, we later confirmed that the C2 domain of PKC Apl II interacts with its C1 domain to inhibit diacylglycerol (DAG) binding and that phosphatidic acid (PA) activates the kinase by binding to the C1b domain and removing C2 domain-mediated inhibition under physiological conditions (Farah et al. 2008). C2 domain-mediated inhibition has also been demonstrated in vertebrate novel PKCs (Stahelin et al. 2005; Melowic et al. 2007).

Another proposed role of the C2 domain of nPKCs is analogous to that in cPKCs and involves lipid binding. It has been previously reported that the C2 domain of PKCε can bind to PA and that PA binding to the C2 domain is required for translocation (Corbalan-Garcia et al. 2003; Jose Lopez-Andreo et al. 2003). For PKC Apl II, we have shown that while the C2 domain binds poorly to lipids, autophosphorylation of this domain greatly increases its binding affinity to phospholipids (Pepio and Sossin 2001), suggesting that C2 domain lipid binding may also play a role in translocation of PKC Apl II once the C2 domain is phosphorylated.

Autophosphorylation of PKCs may play a number of regulatory roles. There are two sites in the carboxy-terminal of PKCs, the turn site and the hydrophobic site, which can be autophosphorylated during maturation of the kinase (Keranen et al. 1995; Behn-Krappa and Newton 1999). However, it still remains unclear whether under physiological conditions these sites are autophosphorylated, or phosphorylated in trans by the TORC2 complex (Sarbassov et al. 2004). Other autophosphorylation sites have been suggested to regulate protein-protein interactions, such as 14-3-3 binding or HSP90 binding (Durgan et al. 2008; Gould et al. 2009). There also appears to be an important role for autophosphorylation in reversing translocation of PKC from the plasma membrane (Feng and Hannun 1998; Feng et al. 2000). This is seen most clearly by the increased localization of PKC on membranes after inhibition of PKCs by ATP-based inhibitors, or the increased membrane localization of kinase dead PKCs (Ohno et al. 1990; Stensman et al. 2004; Takahashi and Namiki 2007). This effect appears to be mediated by an increased affinity for DAG under these conditions (Takahashi and Namiki 2007). PKC Apl II is similar to other PKCs in this respect as its membrane localization is increased by ATP-based inhibitors, such as bisindolymaleimide 1 (Bis-1), although Bis-1 does not affect phosphorylation at the hydrophobic site under these conditions (Lim and Sossin 2006).

In the present study, we sought to elucidate the mechanisms by which post-translational modifications of the C2 domain regulate PKC Apl II translocation in vivo. We find that autophosphorylation of the C2 domain at Ser36 negatively regulates translocation after physiological stimulation in sensory neurons of Aplysia.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Plasmid construction and mutagenesis

The pNEX3 enhanced green fluorescent protein (eGFP)-PKC Apl II, monocistronic red fluorescent protein (mRFP)-PKC Apl II, eGFP-PKC Apl II R273H as well as the MBP-C2 fusion proteins have been described previously (Kaang 1996; Pepio and Sossin 1998, 2001; Manseau et al. 2001; Zhao et al. 2006; Farah et al. 2008). Mutations in eGFP-PKC Apl II S36E, eGFP-PKC Apl II S36A, eGFP-PKC Apl II S36E/R273H, eGFP-PKC Apl II S36E/D95A, eGFP-PKC Apl II S36E/I98N, MBP-PKC Apl II-C2 S36E/I98N, and MBP-PKC Apl II-C2 S36E/D95A were accomplished using overlap PCR, as has been described already (Pepio and Sossin 2001). All constructs were verified using sequencing.

Fusion protein synthesis and purification

Maltose-binding protein (MBP)-fusion proteins synthesis and purification has been previously described (Pepio and Sossin 2001; Farah et al. 2008).

Lipid preparation and lipid binding assay

Sucrose-loaded large unilamellar vesicles were prepared as described (Pepio and Sossin 2001). Lipid binding assay was carried out as previously described (Pepio and Sossin 2001).

Sf9 cell culture and confocal microscopy in Sf9 cells

Sf9 cells were cultured and transfected with plasmid DNA as previously described (Farah and Sossin 2011a). Cells expressing eGFP-PKC and mRFP-PKC constructs for PKC Apl II were examined using a Zeiss laser scanning microscope with an Axiovert 200 and a x63 oil immersion objective as previously described (Farah and Sossin 2011a). During imaging, 1,2-dioctanoyl-sn-glycerol (DOG) and/or DiC8-PA was added to the dish after 30 s, and a series of 12 confocal images was recorded for each experiment at time intervals of 30 s.

Aplysia cell culture preparation and microinjection of plasmid vectors

Sensory neuron cultures were prepared by following published procedures (Farah et al. 2008, 2009). Microinjection of plasmid DNA was performed as described (Farah and Sossin 2011a).

Drugs and reagents

Serotonin was purchased from (Sigma, St Louis, MO, USA) and used at a concentration of 10 μM. Bisindolymaleimide 1 and Calyculin A were purchased from (Calbiochem, San Diego, CA, USA) and used at concentrations of 1 μM and 100 nM, respectively.

Confocal microscopy of Aplysia neurons

For live cell imaging, neurons expressing eGFP-PKC constructs for PKC Apl II were imaged on a Zeiss laser scanning microscope with an Axiovert 200 and a ×40 oil immersion objective as previously described (Farah and Sossin 2011a). Confocal images were acquired before and after (Post) the addition of 5HT (10 μM) to the dish. When indicated, 5HT was washed away using artificial sea water media (10 mM HEPES, pH 7.5, 0.46 M NaCl, 10 mM KCl, 11.2 mM CaCl2, 55 mM MgCl2) and pictures were taken immediately post-wash (30 s post-wash). For Cal A experiments, cells were pre-treated with Cal A for 5 min and 5HT was then added in the presence of Cal A for 5 min. All experiments were performed at room temperature (20–23°C).

Image analysis and statistics

In Sf9 cells

The time series was analyzed using NIH Image J software as previously described (Zhao et al. 2006; Farah et al. 2008, 2009). An individual analysis of protein translocation for each cell was performed by tracing three rectangles at random locations at the plasma membrane and three rectangles at random locations in the cytosol. The translocation ratio was measured as the average intensity (membrane)/average intensity (cytosol) (Im/Ic) normalized to the degree of translocation before the addition of pharmacological agents (Post/Pre). As wild type (WT) PKC Apl II was co-expressed with a mutant PKC Apl II in Sf9 cells, the translocation ratio of the mutant was normalized to that of the WT protein for each individual cell and a paired Student's t-test was used on the non-normalized data. All data are presented as means ± SEM.

In neurons

Analysis of protein translocation was performed as described above for Sf9 cells except that strong red autofluorescence did not allow for us to co-express the WT PKC Apl II, and the mutant PKC Apl II in the same neurons so the translocation ratio for each protein was measured as the average intensity (membrane)/average intensity (cytosol) (Im/Ic) normalized to the degree of translocation before the addition of 5HT (Post-5HT/Pre-5HT). For the dissociation experiments, the degree of translocation following 5HT wash-off was normalized to the degree of translocation pre-wash i.e., after 5 min of 5HT treatment (Post-wash/Pre-wash). For quantification of the amount of PKC Apl II phosphorylation at the plasma membrane in neurons (Ser36 antibody), the entire membrane and cytoplasm of the sensory neuron were traced using NIH Image J, and the intensity was measured. For Cal A experiments, the translocation ratios in the control and the Cal A groups were normalized to the average translocation ratio in the control group from each experiment. The Student's t-test was used to compare translocation ratios in neurons except for Bis-1 experiments where the effect of the drug and the mutation was measured using a two-way analysis of variance (anova) test. All data are presented as means ± SEM.

Lipid-binding assay

One-way analysis of variance was used to compare binding of the different MBP-PKC Apl II-C2 fusion proteins to PS/PC vesicles. All data are presented as means ± standard errors of the means.

Immunocytochemistry

Sensory neurons expressing eGFP-PKC Apl II, eGFP-PKC Apl II S36A were treated with a single pulse of 10 μM 5HT for either 5 min or 45 min. For the Bis-1 experiments, cells expressing eGFP-PKC Apl II or eGFP-PKC Apl II S36E were treated either with Bis-1, 5-HT, or 5HT and Bis-1 for 45 min. Following treatment with the reagents, the cells were fixed for 30 min in 4% paraformaldehyde and 30% sucrose in 1× phosphate buffered saline (PBS). Cells were permeabilized in 0.1% Triton X-100 in 30% sucrose, 1× PBS for 10–15 min. The cells were then washed three times in 1× PBS, and then in NH4Cl for 15 min to quench free aldehydes. Prior to addition of the antibody, the cells were blocked for 30 min in 10% normal goat serum in 0.5% Triton X-100, 1× PBS. Samples were then incubated with the phospho Ser36 antibody previously described (Pepio and Sossin 2001) at a 1 : 200 dilution of a 1 mg/mL stock diluted in the blocking solution for 1 h, washed four times with 1× PBS, and then treated with Alexa Fluor 594 Goat anti-Rabbit IgG (Invitrogen, Burlington, Ontario, Canada) at a concentration of 1 : 500 diluted in the blocking solution. Cells were washed again in 1× PBS and visualized using a Zeiss laser scanning microscope as described above.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Phosphorylation at Ser36 is regulated downstream of 5HT in Aplysia sensory neurons

The C2 domain of PKC Apl II binds poorly to lipids, but phosphorylation of this domain at Ser36 located in loop 1 increases lipid binding and this increased lipid binding is also seen with a phosphomimetic mutation where Ser36 is converted to Glu (S36E) [Fig. 1; (Pepio and Sossin 2001)]. Ser36 was identified after in vitro autophosphorylation of PKC Apl II, and shown to be increased after phorbol ester stimulation in Sf9 cells (Pepio and Sossin 2001), but has not been shown to be phosphorylated during physiological activation of the kinase downstream of 5HT. We have previously shown that eGFP-tagged PKC Apl II translocates similar to endogenous PKC Apl II in response to 5HT in isolated sensory neurons (Zhao et al. 2006). Thus, to examine phosphorylation of PKC Apl II at Ser36 during physiological activation of the kinase, we expressed eGFP-PKC Apl II or eGFP-PKC Apl II S36A in sensory neurons and examined phosphorylation at Ser36 following 5 min or 45 min 5HT treatment followed by fixation and immunocytochemistry using a phospho Ser36 antibody previously described (Pepio and Sossin 2001). We have previously shown that PKC Apl II translocates to the plasma membrane in response to 5HT, not to the cytoskeleton, in Aplysia sensory neurons (Zhao et al. 2006; Farah et al. 2008; Nagakura et al. 2010; Farah and Sossin 2011a). Both eGFP-PKC Apl II and eGFP-PKC Apl II S36A translocated significantly to the membrane in response to 5HT (Fig. 2a and c and data not shown). For quantification of the amount of PKC Apl II phosphorylation at the plasma membrane, the entire membrane and cytoplasm of the sensory neuron were traced using NIH Image J, and the intensity was measured (arbitrary units). Immunoreactivity to the anti-phospho 36 antibody significantly increased after a 5 min treatment with 5HT compared with no treatment. There was an even greater increase after a 45 min treatment with 5HT. Despite a similar level of expression (data not shown), no increased immunoreactivity was seen when PKC Apl II S36A was expressed confirming the phosphospecificity of the antibody (Fig. 2a and c; quantified in Fig. 2b and d). The increase in phosphorylation was only seen at the plasma membrane where PKC is active, consistent with this being an autophosphorylation site (Pepio and Sossin 2001). The phospho 36 antibody was not sensitive enough to detect endogenous PKC Apl II in non-expressing neurons.

image

Figure 1. Comparison of the C2 domain sequence of Protein kinase C (PKC)ε from Rattus rattus and PKC Apl II from Aplysia californica. Loop 1 corresponds to the connection between β1 and β2 strands, and loop 3 corresponds to the connection between β5 and β6 strands. Arrows point to residues that were mutated in the C2 domain of PKC Apl II. Serine in position 36 was mutated to a glutamic acid, aspartic acid in position 95 was mutated to an alanine, and isoleucine in position 98 was mutated to an asparagine.

Download figure to PowerPoint

image

Figure 2. Protein kinase C (PKC) Apl II phosphorylation at Ser36 increases at the membrane following 5HT treatment. (a and c) Sensory cells expressing enhanced green fluorescent protein (eGFP)-PKC Apl II (a) or eGFP-PKC Apl II S36A (c) were treated either with a control solution, 5HT for 5 min or 5HT for 45 min. Cells were immediately fixed and immunostained with the phospho-specific Ser36 antibody and imaged for eGFP. (b and d) Quantification of PKC Apl II (b) or PKC Apl II S36A (d) phosphorylation at the plasma membrane and in the cytosol for the different conditions cited in (a) and (c). Phosphorylation of PKC Apl II at Ser36 significantly increases at the plasma membrane post 5 min 5HT and post 45 min 5HT (***< 0.001 and **< 0.01 by two-tailed unpaired Student's t-tests). Phosphorylation of PKC Apl II at Ser36 is also higher post 45 min 5HT than post 5 min 5HT (# 0.05 by two-tailed unpaired Student's t-tests). Error bars represent standard errors of the means; > 10 for each condition.

Download figure to PowerPoint

Knowing that Ser36 is phosphorylated in neurons, we next investigated the effect of the S36E mutation on 5HT-dependent translocation, as the phosphomimetic mutation replicates the increased binding to lipids seen in the phosphorylated protein (Pepio and Sossin 2001). We expressed eGFP-PKC Apl II S36E in sensory neurons and examined translocation in response to a single pulse of 5 min 5HT (10 μM). Contrary with our previous expectation that phosphorylation would increase translocation and the observation in Fig. 2 that PKC Apl II phosphorylated at S36 was enriched on the membrane, we found that the S36E mutation reduced 5HT-dependent translocation in sensory neurons (Fig. 3a; quantified in Fig. 3b). This result suggests that increased lipid binding of the phosphomimetic C2 domain previously observed in vitro does not correlate with translocation of the full-length phosphomimetic kinase in vivo. Furthermore, the enrichment of the phospho-specific antibody staining at the membrane does not appear to be caused by increased affinity of the phosphorylated protein for the membrane. The enrichment could be the consequence of fast removal of the phosphorylation in the cytoplasm. It may also be more difficult to visualize the phosphorylated protein in the cytoplasm as a result of both the diffuse distribution of PKC in the cytoplasm and increased background from pigment granules. It is also possible that the S36E mutation, while mimicking the effect of phosphorylation on lipid binding, differs from the endogenous protein in its affinity for the membrane.

image

Figure 3. S36E mutation inhibits, whereas S36A mutation increases 5HT-dependent translocation of Protein kinase C (PKC) Apl II in living sensory neurons. (a) Representative confocal fluorescence images of Aplysia sensory neurons expressing enhanced green fluorescent protein (eGFP)-PKC Apl II or eGFP-PKC Apl II S36E before (Pre 5HT) or 5 min following treatment with 5HT (Post 5HT). (b) The translocation ratio normalized to Pre 5HT is shown for the conditions cited in (a). Error bars represent standard errors of the means; > 9 for each condition. The translocation ratio of eGFP-PKC Apl II S36E was significantly lower than that of eGFP-PKC Apl II (***< 0.001 by two-tailed unpaired Student's t tests). (c) Representative confocal fluorescence images of Aplysia sensory neurons expressing eGFP-PKC Apl II or eGFP-PKC Apl II S36A before (Pre 5HT) or 5 min following treatment with 5HT (Post 5HT). (d) The translocation ratio normalized to Pre 5HT is shown for the conditions cited in (a). Error bars represent standard errors of the means; > 18 for each condition. The translocation ratio of eGFP-PKC Apl II S36A was significantly higher than that of eGFP-PKC Apl II (* 0.05 by two-tailed unpaired Student's t tests). (e) Confocal fluorescence images of Aplysia sensory neurons expressing eGFP-PKC Apl II or eGFP-PKC Apl II S36A before (Pre 5HT), 5 min following treatment with 5HT (Post 5HT), and immediately following the wash (Post-wash). (f) The translocation ratio post-wash was normalized to pre-wash (i.e., 5 min 5HT) and is shown for the conditions cited in (e). Error bars represent standard errors of the means; > 5 for each condition. There was significantly more eGFP-PKC Apl II S36A at the membrane following the wash compared to eGFP-PKC Apl II (**< 0.01 by two-tailed unpaired Student's t-tests). Data from wash experiments performed post 5 min 5HT or 45 min 5HT treatment was pooled together, as the results were the same for those two groups.

Download figure to PowerPoint

We next investigated the effect of blocking phosphorylation at Ser36 on PKC Apl II translocation in sensory neurons. We expressed eGFP-PKC Apl II S36A in isolated sensory neurons and examined translocation in response to a single pulse of 5HT (10 μM). The S36A mutation did not increase the basal association of PKC Apl II with the membrane (data not shown). However, the S36A mutation enhanced 5HT-dependent translocation of PKC Apl II in sensory neurons (Fig. 3c, quantified in Fig. 3d). This increased translocation might be explained by a decrease in the ability of eGFP-PKC Apl II S36A to be removed from the membrane after 5HT was washed off, as this phosphorylation is enriched in membrane-bound PKC. To test this idea, we washed off 5HT post 5 min or post 45 min treatment and the amount of kinase remaining at the membrane was quantified in neurons expressing eGFP-PKC Apl II or eGFP-PKC Apl II S36A. To control for the differential translocation of the two enzymes, the degree of translocation following 5HT wash-off was normalized to the degree of translocation pre-wash i.e., after 5 min of 5HT treatment (Post-wash/Pre-wash). As shown in Fig. 3e (quantified in Fig. 3f), there was significantly more eGFP-PKC Apl II S36A associated with the membrane post-wash compared to eGFP-PKC Apl II. Thus, phosphorylation at Ser36 regulates the level of PKC Apl II translocation during 5HT treatment, at least partly by promoting dissociation from the membrane. The increased translocation of eGFP-PKC Apl II S36A also argues against the possibility that the decreased translocation of eGFP-PKC Apl II S36E seen in Fig. 3a was as a result of the glutamic acid substitution being a poor mimic of the phosphorylated kinase. If this were the case, and the phosphorylated protein had a higher affinity for the membrane, the translocation of eGFP-PKC Apl II S36A would have been decreased; however, the opposite result was seen.

Inhibiting PKC increases 5HT-dependent translocation of PKC Apl II in sensory neurons

The above results suggest that autophosphorylation of PKC Apl II at Ser36 inhibits protein translocation, or increases removal of PKC from the membrane. To test this idea, we examined the effect of inhibiting PKC activation using Bis-1, an ATP-based inhibitor (Toullec et al. 1991), on PKC Apl II translocation. We expressed eGFP-PKC Apl II or eGFP-PKC Apl II S36E in sensory neurons and treated the cells either with a control solution (no drug), Bis-1, 5HT or 5HT and Bis-1 for 45 min. Cells were then immediately fixed and imaged for eGFP. These experiments could not be performed in live cells, as Bis-1 has strong autofluorescence that interfered with detection of the eGFP signal, and thus detection of the eGFP signal required fixation followed by washout of Bis-1. A two-way anova test (drug, mutation) was used to analyze the data. As shown in Fig. 4a (quantified in Fig. 4b), there was a significant effect of the drug (***< 0.001) and of the S36E mutation (***< 0.001) on PKC Apl II translocation. Most importantly, there was a highly significant effect of the interaction between the drug and the mutation (***< 0.001), showing that the drugs affected eGFP-PKC Apl II S36E significantly different than they affected eGFP-PKC Apl II. Notably, in the presence of 5HT, Bis-1 had a significant effect on PKC Apl II translocation (***< 0.001 by Bonferroni multiple comparisons test), but in the presence of 5HT, Bis-1 did not affect translocation of PKC Apl II S36E (p > 0.05 by Bonferroni multiple comparisons test). Although not significant in the anova (> 0.05 by Bonferroni multiple comparisons test), in some neurons, Bis-1 appeared to increase association of eGFP-PKC Apl II with the plasma membrane in the absence of 5HT, suggesting some autophosphorylation in resting cells and this was not seen with eGFP-PKC Apl II S36E (Fig. 4a and b). These results show that Bis-1 increases translocation of eGFP-PKC Apl II, but not that of eGFP-PKC Apl II S36E consistent with Bis-1 inhibiting autophosphorylation of PKC Apl II, but not eGFP-PKC Apl II S36E.

image

Figure 4. Bisindolymaleimide 1, a Protein kinase C (PKC) inhibitor, increases 5HT-dependent translocation of PKC Apl II in sensory neurons. (a) Sensory cells expressing enhanced green fluorescent protein (eGFP)-PKC Apl II or eGFP-PKC Apl II S36E were treated either with a control solution (no drug), Bis-1, 5HT or 5HT and Bis-1 for 45 min. Cells were immediately fixed and imaged for eGFP. (b) A two-way anova test was used to analyze the effect of the two variables, the drug and the mutation, on PKC Apl II translocation. There was a significant effect of both the drug and the mutation on translocation of PKC Apl II (***< 0.001 for both) and a significant effect of the interaction between the variables (***< 0.001). Bonferroni multiple comparisons test showed that in the presence of 5HT, Bis-1 had a significant effect on translocation of PKC Apl II (***< 0.001). However, in the presence of 5HT, Bis-1 did not have a significant effect on translocation of PKC Apl II S36E mutant (> 0.05). For PKC Apl II, translocation ratios in the 5HT group and the 5HT and Bis-1 group were significantly higher than the no drug group (*< 0.05 and ***< 0.001, respectively by Bonferroni multiple comparisons test). Translocation in the 5HT and Bis-1 group was also significantly higher than in the Bis-1 group for PKC Apl II (***< 0.001). For PKC Apl II S36E, translocation in the 5HT and Bis-1 group was significantly higher than the no drug group (**< 0.01), and translocation in the 5HT and Bis-1 group was also significantly higher than in the Bis-1 group (**< 0.01). Error bars represent standard errors of the means; > 7 for each condition. NS, not significant.

Download figure to PowerPoint

Inhibiting phosphatases inhibits 5HT-dependent translocation of PKC Apl II in living sensory neurons

We next examined whether inhibiting phosphate removal using calyculin A (Cal A), a potent phosphatase inhibitor, would affect the association of PKC Apl II with the plasma membrane. These experiments were performed in live cells, as Cal A is a clear drug, which does not interfere with detection of the eGFP signal. Sensory neurons expressing either eGFP-PKC Apl II or eGFP-PKC Apl II S36A were first treated with 5HT alone for 5 min. As reported above, 5HT lead to significant translocation of both PKC Apl II and PKC Apl II S36A to the plasma membrane (Fig. 5a; translocation ratios pre 5HT of 0.55 ± 0.03 and 0.63 ± 0.03, respectively; translocation ratios post 5HT of 1.66 ± 0.17 and 2.1 ± 0.32, respectively; ***< 0.001 for PKC Apl II and **< 0.01 for PKC Apl II S36A by one-tailed paired Student's t-tests). To test the effect of Cal A, sensory neurons expressing either eGFP-PKC Apl II or eGFP-PKC Apl II S36A were treated with Cal A for 5 min followed by 5HT and Cal A for another 5 min. The translocation ratios in the control and the Cal A groups were normalized to the average translocation ratio in the control group from each experiment. Treating the cells with Cal A alone for 5 min did not affect the basal association of PKC Apl II nor that of PKC Apl II S36A with the plasma membrane (data not shown). However, Cal A strongly inhibited 5HT-dependent translocation of PKC Apl II, but did not affect 5HT-dependent translocation of eGFP-PKC Apl II S36A mutant that could not be phosphorylated at Ser36 (Fig. 5a; quantified in Fig. 5b). These results suggest that phosphatases dephosphorylate PKC Apl II at Ser36 to promote the association of PKC Apl II with the membrane.

image

Figure 5. Calyculin A, a phosphatase inhibitor, inhibits 5HT-dependent translocation of Protein kinase C (PKC) Apl II in living sensory neurons. (a) Representative confocal fluorescence images of Aplysia sensory neurons expressing enhanced green fluorescent protein (eGFP)-PKC Apl II or eGFP-PKC Apl II S36A before (Pre 5HT) or 5 min following treatment with 5HT (Post 5HT) in the absence or presence of Cal A. In the Cal A groups, cells were pretreated with Cal A for 5 min followed by 5HT treatment in the presence of Cal A for another 5 min. (b) The translocation ratios in the control and the Cal A groups were normalized to the average translocation ratio in the control group from each experiment. Error bars represent standard errors of the means; > 6 for each condition. Cal A significantly inhibited translocation of PKC Apl II (***< 0.001 by two-tailed unpaired Student's t-tests).

Download figure to PowerPoint

Lipid binding to the C2 domain in vitro does not correlate with protein translocation in vivo

How does phosphorylation of the C2 domain inhibit PKC translocation? We previously showed that the C2 domain phosphorylation at Ser36 regulates binding to lipids. However, the above results seem difficult to attribute to increased lipid binding to the C2 domain of PKC Apl II, which would be predicted to increase, not decrease translocation to the plasma membrane. To investigate this idea, we turned to Sf9 cells, as it is easier to manipulate lipids in this system. Indeed, very high concentrations of exogenous lipids are required to translocate PKC Apl II in neurons, compared with Sf9 cells (Dunn et al. 2012). Another advantage of Sf9 cells is that we can easily co-express eGFP and mRFP-tagged kinases in the same cell to directly assess effects of mutations, while strong red autofluorescence minimized our abilities to do this experiment in Aplysia neurons (Farah et al. 2008). We have previously shown that neither the fluorescent protein added, nor competition between the PKCs in the same cell, affect the ability to compare the translocation of these enzymes (Farah et al. 2008). To confirm the inhibitory role of phosphorylation of the C2 domain, we co-expressed mRFP-PKC Apl II and eGFP-PKC Apl II S36E in Sf9 cells and examined translocation in response to DOG (0.5 μg/mL), a cell-permeable analog of diacylglycerol (Farah et al. 2008). As shown in Fig. 6a (quantified in Fig. 6b), PKC Apl II S36E translocation was significantly inhibited compared with that of PKC Apl II, although the effect of the mutation appeared smaller than in neurons. This effect was also concentration-dependent and was not observed at higher DOG concentrations (2.5 and 5 μg/mL; data not shown). Furthermore, the S36E-mediated inhibition could be removed at this concentration of DOG (0.5 μg/mL) by adding subthreshold concentrations of the cell-permeable analog of PA, DiC8-PA whose effect is to decrease the DOG concentration required for translocation (Fang et al. 2001; Farah et al. 2008). These results suggest that one role of phosphorylation at Ser36 is to lower the kinase's affinity for diacylglycerol.

image

Figure 6. S36E mutation inhibits 1,2-dioctanoyl-sn-glycerol (DOG)-mediated translocation in Sf9 cells. (a) Confocal fluorescence images of Sf9 cells co-expressing mRFP-PKC Apl II and eGFP-PKC Apl II S36E at different points of the time-lapse experiment (Pre DOG being 0 s and Post DOG being 60 s). DOG (0.5 μg/mL) was added to the dish after 30 s of recording. (b) The translocation ratios at 60 s of mRFP-PKC Apl II and eGFP-PKC Apl II S36E are shown in the presence of DOG (0.5 μg/mL). The translocation ratio of Protein kinase C (PKC) Apl II S36E was normalized to that of PKC Apl II for each individual cell (**< 0.01 by two-tailed paired Student's t-tests). Error bars represent standard errors of the means; = 19.

Download figure to PowerPoint

We next examined mutations in the C2 domain that are predicted to affect lipid binding. Lipid binding is thought to require loop 3 in the C2 domain as mutations in this loop modulate translocation in a vertebrate orthologue of PKC Apl II, PKCε (Ochoa et al. 2001; Jose Lopez-Andreo et al. 2003; Schechtman et al. 2004). Phosphorylation of Ser36 may act to liberate this domain and increase lipid binding (Pepio and Sossin 2001). To test this idea, we examined the effect of mutations in this region. Jose Lopez-Andreo and colleagues have shown that mutating Ile89 to an asparagine in loop 3 of PKCε (Fig. 1) decreases translocation in response to DOG and to DiC8-PA, in RBL-2H3 cells (Jose Lopez-Andreo et al. 2003). They reasoned that Ile89 plays an important role in membrane targeting of the kinase by enhancing the affinity of the C1 domain to DAG or even participating directly in the interaction. We mutated the equivalent residue (Ile98) in PKC Apl II and tested the effect of this mutation on binding of MBP-PKC Apl II C2-S36E to PS/PC lipid vesicles. I98N mutation did not affect binding of MBP-PKC Apl II S36E to lipids (data not shown). We further examined whether I98N mutation could rescue Ser36-mediated inhibition of translocation by co-expressing mRFP-PKC Apl II and eGFP-PKC Apl II S36E/I98N in Sf9 cells and analyzing translocation in response to different concentrations of DOG. The I98N mutation did not affect translocation of either eGFP-PKC Apl II or eGFP-PKC Apl II S36E and did not affect the inhibition of translocation mediated by the S36E mutation (data not shown). Next, we mutated Asp95 in PKC Apl II to an alanine. Schechtman et al. (2004) have shown that mutating the equivalent residue (Asp86 in Fig. 1) in PKCε to Ala resulted in increased translocation rate upon activation in CHO-Hir cells. Somewhat surprisingly, we found that this mutation abolished the increased lipid binding observed in MBP-PKC Apl II C2-S36E protein. As previously shown (Pepio and Sossin 2001), S36E mutation increases binding of an MBP-C2 domain fusion protein to PS/PC lipid vesicles while the D95A mutation removed this increased binding of the S36E mutant to lipids (Fig. 7a quantified in Fig. 7b). To investigate the effect of this mutation on translocation of PKC Apl II in Sf9 cells, we first co-expressed mRFP-PKC Apl II and eGFP-PKC Apl II D95A in this system and analyzed translocation in response to different concentrations of DOG. The D95A mutation alone did not affect translocation of PKC Apl II (data not shown). We also examined the effect of D95A on translocation of PKC Apl II S36E by co-expressing mRFP-PKC Apl II and eGFP-PKC Apl II S36E/D95A in Sf9 cells and examining translocation in response to DOG (0.5 μg/mL). As shown in Fig. 7c (quantified in Fig. 7d), a similar decrease in translocation of PKC Apl II S36E/D95A compared to PKC Apl II was seen, despite the lack of lipid binding of the MBP-PKC Apl II C2-S36E/D95A mutant. Thus, increased lipid binding does not correlate with the inhibition seen with the phosphomimetic mutation in cells.

image

Figure 7. D95A mutation inhibits binding of the MBP-PKC Apl II-C2 S36E to PS/PC lipid vesicles, but does not affect kinase translocation in Sf9 cells. (a) Sucrose-loaded lipid vesicle assay for C2 domain membrane binding. Maltose-binding protein (MBP) fusion proteins were expressed in bacterial DH5α cells, purified, pre-centrifuged to remove aggregates, and then incubated with sucrose-loaded vesicles as described under ‘Materials and Methods’. MBP-PKC Apl II-C2 (WT), MBP- PKC Apl II-C2 S36E or MBP- PKC Apl II-C2 S36E/D95A were incubated with sucrose-loaded vesicles consisting of 60% phosphatidylserine and 40% phosphatidylcholine (PS/PC) for 10 min at 15°C. The sucrose vesicles were subsequently sedimented in a Beckman TLA-100 ultracentrifuge at 100 000g for 30 min, and the pellet (P) and supernatant (S) fractions were separated and analyzed by PAGE. (b) Graphic illustration of quantitated results from (a). Data are displayed as the percentage of total protein found in the bound (P) fraction for each construct cited in (a). One-way analysis of variance was used to compare binding of MBP-PKC Apl II-C2 fusion proteins to PS/PC lipid vesicles. Whereas very little of the wild type fusion protein domain is sedimented, a considerable amount of the S36E protein does sediment (* 0.05). The D95A mutation reduced sedimentation of the S36E protein significantly (# 0.05). Error bars represent standard errors of the means; > 4. (c) Confocal fluorescence images of Sf9 cells co-expressing mRFP-PKC Apl II and eGFP-PKC Apl II S36E/D95A at different points of the time-lapse experiment (Pre 1,2-dioctanoyl-sn-glycerol (DOG) being 0 s and Post DOG being 60 s). DOG (0.5 μg/mL) was added to the dish after 30 s of recording. (d) The translocation ratios at 60 s of mRFP-PKC Apl II and eGFP-PKC Apl II S36E/D95A are shown in the presence of DOG (0.5 μg/mL). The translocation ratio of Protein kinase C (PKC) Apl II S36E/D95A was normalized to that of PKC Apl II for each individual cell (* 0.05 by two-tailed paired Student's t-tests). Error bars represent standard errors of the means; = 9.

Download figure to PowerPoint

Another possibility is that phosphorylation at Ser36 is regulating C1-C2 domain interactions to increase C2 domain-mediated inhibition of binding of DAG to the C1 domain (Farah et al. 2008). We have recently shown that this appears to be one mechanism used by a peptide derived from the C2 domain to inhibit PKC translocation (Farah and Sossin 2011b). Mutating Arg273 to a Histidine in the C1b domain of PKC Apl II both blocks binding to PA and removes C2 domain-mediated inhibition (Farah et al. 2008). This mutation also removes the inhibitory effect of the peptide derived from the C2 domain (Farah and Sossin 2011b). As the R273H mutation removes C2-domain-mediated inhibition, we examined if the effect of the phosphomimetic mutation could be rescued by the R273H by co-expressing mRFP-PKC Apl II and eGFP-PKC Apl II S36E/R273H in Sf9 cells and examining translocation in response to DOG (0.5 μg/mL). As shown in Fig. 8a (quantified in Fig. 8b), the R273H mutation did not remove the S36E-mediated inhibition suggesting that the S36E mutation does not work by strengthening C2-domain-mediated inhibition.

image

Figure 8. The R273H mutation does not remove the S36E-mediated inhibition. (a) Confocal fluorescence images of Sf9 cells co-expressing mRFP-PKC Apl II and enhanced green fluorescent protein-PKC Apl II S36E/R273H at different points of the time-lapse experiment (Pre 1,2-dioctanoyl-sn-glycerol (DOG) being 0 s and Post DOG being 60 s). DOG (0.5 μg/mL) was added to the dish after 30 s of recording. (b) The translocation ratios at 60 s of mRFP-PKC Apl II and eGFP-PKC Apl II S36E/R273H are shown in the presence of DOG (0.5 μg/mL). The translocation ratio of Protein kinase C (PKC) Apl II S36E/R273H was normalized to that of PKC Apl II for each individual cell (* 0.05 by two-tailed paired Student's t-tests). Error bars represent standard errors of the means; = 12.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In this study, we showed that autophosphorylation of the C2 domain inhibits translocation in the novel PKC Apl II. A mutation that blocked autophosphorylation (S36A) enhanced translocation, whereas a phosphomimetic mutation (S36E) reduced translocation to the plasma membrane following application of 5HT. Inhibiting PKC activation increased 5HT-dependent translocation, whereas inhibiting phosphatases inhibited 5HT-dependent translocation and both of these effects were inhibited by mutating Ser36. Our results suggest an important role for C2 domain phosphorylation in regulating protein dissociation from the plasma membrane, which may be important for kinase inactivation during synaptic plasticity.

How does phosphorylation of Ser36 affect translocation?

There are a number of possible mechanisms for the regulation of translocation by phosphorylation of the C2 domain; however, some of them can be eliminated by present or past experiments. First, increased lipid binding of the phosphomimetic C2 domain does not correlate with translocation of the full-length phosphomimetic kinase in vivo suggesting that lipid binding to the C2 domain does not underlie the effect of C2 domain phosphorylation on PKC translocation in the complex environment of cells. In agreement with this, we have previously shown that the lipid dependence for activation of the purified kinase is not affected by mutations at Ser36 (Pepio and Sossin 2001). Second, inhibition of translocation by the phosphomimetic can be rescued by increased DOG in Sf9 cells, suggesting that the effect is mediated by changing the affinity of the kinase for DOG. As this is similar to the effect of C2-domain-mediated inhibition, an attractive model would be that phosphorylation increases inhibition mediated by the C2 domain. However, removing this inhibition with the R273H mutation did not rescue the effect of the phosphomimetic inhibition. Phosphorylation may regulate the affinity for DOG through a distinct interaction between the C2 and C1b domain, or by changing affinity for DOG through inducing a conformational change in the enzyme. It is also possible that C2 domain phosphorylation may regulate translocation through protein-protein interactions. C2 domains are known to regulate translocation through binding to receptor for activated C kinases (Mochly-Rosen 1995; Mochly-Rosen and Gordon 1998; Kheifets and Mochly-Rosen 2007). However, the role for receptor for activated C kinase binding in translocation of PKC Apl II remains unclear, as removing the C2 domain increases translocation of the kinase. There may be unidentified proteins that bind to PKC through the C2 domain and act to remove PKC from the membrane. These could include proteins that bind to the phosphorylated domain and assist in redistribution of the protein to the cytoplasm, or effects of phosphorylation on other unknown co-factors. For example, tyrosine phosphorylation downstream of 5HT activation is important for translocation of PKC Apl II in sensory neurons, but the mechanism by which this affects translocation is not known (Nagakura et al. 2010). It is possible that Ser36 phosphorylation may negatively interact with this pathway.

Autophosphorylation as a regulator of PKC reverse translocation

Inhibiting PKC activation using Bis-1 increased translocation in sensory neurons suggesting that autophosphorylation regulates protein translocation. This effect was blocked by the S36E mutation suggesting that autophosphorylation of this residue is important for the increased translocation of the enzyme. These results are consistent with our previous reports showing that Bis-1 is also effective at translocating endogenous PKC Apl II to the membrane (Lim and Sossin 2006). Our present results do not rule out the possibility of other autophosphorylation sites regulating PKC Apl II. Indeed, we have previously demonstrated that PKC Apl II could autophosphorylate at Ser2 located in the C2 domain (Pepio and Sossin 2001). Furthermore, autophosphorylation of the conventional PKC Apl I at Thr613 located in the catalytic domain was suggested to reduce the ability of PKC Apl I to translocate to membranes (Nakhost et al. 1999). This site is conserved in PKC Apl II and may also regulate kinase translocation. Translocation by ATP-based inhibitors is common for classical PKCs (Takahashi and Namiki 2007), although not well characterized previously in novel PKCs. For classical PKCs, it was shown that this effect reflected an increased response to endogenous DAG (Takahashi and Namiki 2007) and this is consistent with the decreased translocation in response to DOG observed in the S36E mutant.

Autophosphorylation of the C2 domain might be a common feedback mechanism for PKCs to regulate their translocation to membranes and their activation. Indeed, Ser36 is conserved in the novel PKCη and autophosphorylation at this site may act as a means of self-regulation for the kinase (Littler et al. 2006). Furthermore, PKCα can also phosphorylate itself in the C2 domain at Thr250 and this autophosphorylation has been used to monitor its activation in vivo (Ng et al. 1999). This site is conserved in PKCβ and PKCγ and thus is likely to regulate their activation as well. It will be interesting to see if this site regulates reverse translocation. Previous reports suggested that autophosphorylation of the turn or hydrophobic site may also be required for removal of PKC from the membrane (Feng and Hannun 1998; Feng et al. 2000; Stensman et al. 2004). However, as kinases without phosphorylation of these sites are less active, these results could be explained by the lack of autophosphorylation of other sites in these kinases.

Comparison to past results

We published earlier that phosphorylation at Ser36 increased PKC translocation (Pepio and Sossin 2001). It is important to note the difference between our present study and the previous one. The previous study examined phorbol dibutyrate (PDBu) mediated translocation following baculovirus mediated expression of PKCs in Sf9 cells and found a modest decrease in translocation of PKC Apl II S36A compared with the wild type protein with no difference in the translocation of PKC Apl II S36E. PDBu mediated translocation differs from physiological translocation in a number of ways: (i) PDBu translocates PKC Apl II mainly to the cytoskeleton, not to the plasma membrane (Nakhost et al. 2002); (ii) PDBu has a much higher affinity for PKC Apl II than DOG or DAG and thus changes in the affinity for DAG would not be affected; and (iii) PDBu interacts with both the C1a and C1b domains of PKC Apl II (unpublished data), whereas DOG only binds to the C1b domain (Farah et al. 2008). Moreover, baculoviral mediated protein expression leads to higher expression level of PKC Apl II than the transfection technique used in the present study. This could lead to higher levels of basal autophosphorylation and presumably titration of any possible PKC binding proteins. Thus, we believe that these previous in vitro results do not reflect the physiological translocation of the enzyme in their complex environment in vivo.

Physiological significance of C2 domain phosphorylation

The role of phosphorylation in the C2 domain appears to be most important for removal of the kinase after the end of stimulation. What would be the significance of faster removal of PKC Apl II from the membrane? PKC Apl II activation is known to regulate synaptic plasticity and consequent memory formation in Aplysia californica (Manseau et al. 2001; Farah et al. 2009). In particular, the amount of time PKC spends on the membrane appears to be critical for the type of plasticity generated. We have previously shown that PKC Apl II translocation desensitizes differently during spaced and massed training paradigms (Farah et al. 2009). Spaced training is known to be superior at generating long-term memories than massed training (Sutton et al. 2002). During spaced training (or 5 × 5 min applications of 5HT spaced by 15 min washes in cultures), PKC translocation is down-regulated through PKA signaling, whereas massed training (one continuous application of 90 min 5HT), leads to persistent translocation of PKC (Farah et al. 2009). To distinguish spaced training from massed training, PKC Apl II has to quickly inactivate after 5HT is washed out. Indeed, when 5HT is applied for 10 min instead of 5 min, facilitation switches from one dependent on PKA, to one dependent on PKC, presumably due to the longer time of PKC activation (Ghirardi et al. 1992; Jin et al. 2011). In the present study, we show that phosphorylation at Ser36 increases at the plasma membrane during application of 5HT consistent with an autophosphorylation of PKC Apl II at this site regulating its own dissociation from the plasma membrane and consequent inactivation. Regulation of membrane residence time of PKC via this mechanism may be important for its role in learning and memory formation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by Canadian Institutes of Health Research (CIHR) Grant MOP 12046. WSS is a James McGill Professor and an FRSQ Chercheur National.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • Behn-Krappa A. and Newton A. C. (1999) The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation. Curr. Biol. 9, 728737.
  • Byrne J. H. and Kandel E. R. (1996) Presynaptic facilitation revisited: state and time dependence. J. Neurosci. 16, 425435.
  • Corbalan-Garcia S., Sanchez-Carrillo S., Garcia-Garcia J. and Gomez-Fernandez J. C. (2003) Characterization of the membrane binding mode of the C2 domain of PKC epsilon. Biochemistry 42, 1166111668.
  • Dunn T. W., Farah C. A. and Sossin W. S. (2012) Inhibitory responses in Aplysia pleural sensory neurons act to block excitability, transmitter release, and PKC Apl II activation. J. Neurophysiol. 107, 292305.
  • Durgan J., Cameron A. J., Saurin A. T., Hanrahan S., Totty N., Messing R. O. and Parker P. J. (2008) The identification and characterization of novel PKCepsilon phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily. Biochem. J. 411, 319331.
  • Fang Y., Vilella-Bach M., Bachmann R., Flanigan A. and Chen J. (2001) Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 294, 19421945.
  • Farah C. A. and Sossin W. S. (2011a) Live-imaging of PKC translocation in Sf9 cells and in aplysia sensory neurons. J. Vis. Exp. 50, e2516, doi: 10.3791/2516.
  • Farah C. A. and Sossin W. S. (2011b) A new mechanism of action of a C2 domain-derived novel PKC inhibitor peptide. Neurosci. Lett. 504, 306310.
  • Farah C. A. and Sossin W. S. (2012) The role of C2 domains in PKC signaling. Adv. Exp. Med. Biol. 740, 1012. in press.
  • Farah C. A., Nagakura I., Weatherill D., Fan X. and Sossin W. S. (2008) Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons. Mol. Cell. Biol. 28, 47194733.
  • Farah C. A., Weatherill D., Dunn T. W. and Sossin W. S. (2009) PKC differentially translocates during spaced and massed training in Aplysia. J. Neurosci. 29, 1028110286.
  • Feng X. and Hannun Y. A. (1998) An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane. J. Biol. Chem. 273, 2687026874.
  • Feng X., Becker K. P., Stribling S. D., Peters K. G. and Hannun Y. A. (2000) Regulation of receptor-mediated protein kinase C membrane trafficking by autophosphorylation. J. Biol. Chem. 275, 1702417034.
  • Ghirardi M., Braha O., Hochner B., Montarolo P. G., Kandel E. R. and Dale N. (1992) Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in Aplysia sensory neurons. Neuron 9, 479489.
  • Gould C. M., Kannan N., Taylor S. S. and Newton A. C. (2009) The chaperones Hsp90 and Cdc37 mediate the maturation and stabilization of protein kinase C through a conserved PXXP motif in the C-terminal tail. J. Biol. Chem. 284, 49214935.
  • Jin I., Kandel E. R. and Hawkins R. D. (2011) Whereas short-term facilitation is presynaptic, intermediate-term facilitation involves both presynaptic and postsynaptic protein kinases and protein synthesis. Learn. Mem. 18, 96102.
  • Jose Lopez-Andreo M., Gomez-Fernandez J. C. and Corbalan-Garcia S. (2003) The simultaneous production of phosphatidic acid and diacylglycerol is essential for the translocation of protein kinase Cepsilon to the plasma membrane in RBL-2H3 cells. Mol. Biol. Cell 14, 48854895.
  • Kaang B. K. (1996) Parameters influencing ectopic gene expression in Aplysia neurons. Neurosci. Lett. 221, 2932.
  • Kandel E. R. (2001) The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 10301038.
  • Kandel E. R. (2012) The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB. Mol. brain 5, 14.
  • Keranen L. M., Dutil E. M. and Newton A. C. (1995) Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr. Biol. 5, 13941403.
  • Kheifets V. and Mochly-Rosen D. (2007) Insight into intra- and inter-molecular interactions of PKC: design of specific modulators of kinase function. Pharmacol. Res. 55, 467476.
  • Kruger K. E., Sossin W. S., Sacktor T. C., Bergold P. J., Beushausen S. and Schwartz J. H. (1991) Cloning and characterization of Ca(2+)-dependent and Ca(2+)-independent PKCs expressed in Aplysia sensory cells. J. Neurosci. 11, 23032313.
  • Lim T. and Sossin W. S. (2006) Phosphorylation at the hydrophobic site of protein kinase C Apl II is increased during intermediate term facilitation. Neuroscience 141, 277285.
  • Littler D. R., Walker J. R., She Y. M., Finerty P. J. Jr., Newman E. M. and Dhe-Paganon S. (2006) Structure of human protein kinase C eta (PKCeta) C2 domain and identification of phosphorylation sites. Biochem. Biophys. Res. Commun. 349, 11821189.
  • Majewski H. and Iannazzo L. (1998) Protein kinase C: a physiological mediator of enhanced transmitter output. Prog. Neurobiol. 55, 463475.
  • Manseau F., Fan X., Hueftlein T., Sossin W. S. and Castellucci V. F. (2001) Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia. J. Neurosci. 21, 12471256.
  • Medkova M. and Cho W. (1999) Interplay of C1 and C2 domains of protein kinase C-alpha in its membrane binding and activation. J. Biol. Chem. 274, 1985219861.
  • Melowic H. R., Stahelin R. V., Blatner N. R., Tian W., Hayashi K., Altman A. and Cho W. (2007) Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Ctheta. J. Biol. Chem. 282, 2146721476.
  • Mochly-Rosen D. (1995) Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 268, 247251.
  • Mochly-Rosen D. and Gordon A. S. (1998) Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J. 12, 3542.
  • Nagakura I., Dunn T. W., Farah C. A., Heppner A., Li F. F. and Sossin W. S. (2010) Regulation of protein kinase C Apl II by serotonin receptors in Aplysia. J. Neurochem. 115, 9941006.
  • Nakhost A., Dyer J. R., Pepio A. M., Fan X. and Sossin W. S. (1999) Protein kinase C phosphorylated at a conserved threonine is retained in the cytoplasm. J. Biol. Chem. 274, 2894428949.
  • Nakhost A., Kabir N., Forscher P. and Sossin W. S. (2002) Protein kinase C isoforms are translocated to microtubules in neurons. J. Biol. Chem. 277, 4063340639.
  • Nalefski E. A. and Falke J. J. (1996) The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci. 5, 23752390.
  • Newton A. C. (1995a) Protein kinase C. Seeing two domains. Curr. Biol. 5, 973976.
  • Newton A. C. (1995b) Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270, 2849528498.
  • Ng T., Squire A., Hansra G. et al. (1999) Imaging protein kinase Calpha activation in cells. Science 283, 20852089.
  • Oancea E. and Meyer T. (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95, 307318.
  • Ochoa W. F., Garcia-Garcia J., Fita I., Corbalan-Garcia S., Verdaguer N. and Gomez-Fernandez J. C. (2001) Structure of the C2 domain from novel protein kinase Cepsilon. A membrane binding model for Ca(2+)-independent C2 domains. J. Mol. Biol. 311, 837849.
  • Ohno S., Konno Y., Akita Y., Yano A. and Suzuki K. (1990) A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation. J. Biol. Chem. 265, 62966300.
  • Pepio A. M. and Sossin W. S. (1998) The C2 domain of the Ca(2+)-independent protein kinase C Apl II inhibits phorbol ester binding to the C1 domain in a phosphatidic acid-sensitive manner. Biochemistry 37, 12561263.
  • Pepio A. M. and Sossin W. S. (2001) Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain. J. Biol. Chem. 276, 38463855.
  • Pepio A. M., Fan X. and Sossin W. S. (1998) The role of C2 domains in Ca2+-activated and Ca2+-independent protein kinase Cs in aplysia. J. Biol. Chem. 273, 1904019048. [published erratum appears in J Biol Chem 1998 Aug 28;273(35):22856].
  • Sarbassov D. D., Ali S. M., Kim D. H., Guertin D. A., Latek R. R., Erdjument-Bromage H., Tempst P. and Sabatini D. M. (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14, 12961302.
  • Schechtman D., Craske M. L., Kheifets V., Meyer T., Schechtman J. and Mochly-Rosen D. (2004) A critical intramolecular interaction for protein kinase C epsilon translocation. J. Biol. Chem. 279, 1583115840.
  • Sossin W. S. (1997) An autonomous kinase generated during long-term facilitation in Aplysia is related to the Ca(2+)-independent protein kinase C Apl II. Learn. Mem. 3, 389401.
  • Sossin W. S. (2007) Isoform specificity of protein kinase Cs in synaptic plasticity. Learn. Mem. 14, 236246.
  • Sossin W. S., Diaz A. R. and Schwartz J. H. (1993) Characterization of two isoforms of protein kinase C in the nervous system of Aplysia californica. J. Biol. Chem. 268, 57635768.
  • Sossin W. S., Fan X. and Saberi F. (1996) Expression and characterization of Aplysia protein kinase C: a negative regulatory role for the E region. J. Neurosci. 16, 1018.
  • Stahelin R. V., Digman M. A., Medkova M., Ananthanarayanan B., Melowic H. R., Rafter J. D. and Cho W. (2005) Diacylglycerol-induced membrane targeting and activation of protein kinase Cepsilon: mechanistic differences between protein kinases Cdelta and Cepsilon. J. Biol. Chem. 280, 1978419793.
  • Stensman H., Raghunath A. and Larsson C. (2004) Autophosphorylation suppresses whereas kinase inhibition augments the translocation of protein kinase Calpha in response to diacylglycerol. J. Biol. Chem. 279, 4057640583.
  • Sutton M. A., Ide J., Masters S. E. and Carew T. J. (2002) Interaction between amount and pattern of training in the induction of intermediate- and long-term memory for sensitization in aplysia. Learn. Mem. 9, 2940.
  • Takahashi H. and Namiki H. (2007) Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors. Biochem. J. 405, 331340.
  • Toullec D., Pianetti P., Coste H. et al. (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 1577115781.
  • Zhao Y., Leal K., Abi-Farah C., Martin K. C., Sossin W. S. and Klein M. (2006) Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation. J. Neurosci. 26, 88478856.