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First published online 24 June 2008
doi: 10.1242/jcs.027698

Journal of Cell Science 121, 2339-2349 (2008)
Published by The Company of Biologists 2008
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Research Article

PKC{gamma} mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

Dineke S. Verbeek1,*, Joachim Goedhart2, Laurie Bruinsma1, Richard J. Sinke3 and Eric A. Reits1

1 Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
2 Section Molecular Cytology, Swammerdam Institute for Life Sciences, Centre for Advanced Microscopy, University of Amsterdam, The Netherlands
3 Department of Biomedical Genetics, University Medical Center Utrecht, University of Utrecht, The Netherlands

* Author for correspondence (e-mail: D.S.Verbeek{at}medgen.umcg.nl)

Accepted 22 April 2008


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disorder caused by mutations in the neuronal-specific protein kinase C gamma (PKC{gamma}) gene. Since most mutations causing SCA14 are located in the PKC{gamma} C1B regulatory subdomain, we investigated the impact of three C1B mutations on the intracellular kinetics, protein conformation and kinase activity of PKC{gamma} in living cells. SCA14 mutant PKC{gamma} proteins showed enhanced phorbol-ester-induced kinetics when compared with wild-type PKC{gamma}. The mutations led to a decrease in intramolecular FRET of PKC{gamma}, suggesting that they `open' PKC{gamma} protein conformation leading to unmasking of the phorbol ester binding site in the C1 domain. Surprisingly, SCA14 mutant PKC{gamma} showed reduced kinase activity as measured by phosphorylation of PKC reporter MyrPalm-CKAR, as well as downstream components of the MAPK signaling pathway. Together, these results show that SCA14 mutations located in the C1B subdomain `open' PKC{gamma} protein conformation leading to increased C1 domain accessibility, but inefficient activation of downstream signaling pathways.

Key words: Spinocerebellar ataxia, Protein kinase C gamma, GFP, FLIM, FRET


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The protein kinase C (PKC) family encompasses a group of serine/threonine kinases that have important roles in many cellular processes. This family consists of ten isoforms that are divided into three subclasses: conventional (PKC{alpha}, PKCβI, PKCβII and PKC{gamma}), novel (PKC{delta}, PKC{epsilon}, PKC{eta} and PKC{theta}) and atypical (PKC{zeta} and PKC{iota}). This classification is based on their sensitivity for the second messengers calcium and diacylglycerol. The conventional isoforms respond to both activators, whereas novel PKCs only react to diacylglycerol, and atypical PKCs are not activated by either calcium or diacylglycerol.

Recently, PKC{gamma} (also known as PRKCG) was identified as the disease-causing gene for the neurodegenerative disorder spinocerebellar ataxia type 14 (SCA14) (Chen et al., 2003Go). SCA14 is characterized by a slowly progressive cerebellar ataxia accompanied by slurred speech and abnormal eye movements. The ataxic syndrome is due to loss of Purkinje cells leading to cerebellar neurodegeneration. PKC{gamma} is the brain-specific member of the PKC family and is highly expressed in Purkinje cell soma and dendritic processes, and regulates expression of long-term depression at the parallel fiber of the Purkinje cell synapse, controlling synaptic plasticity (Daniel et al., 1998Go; Schrenk et al., 2002Go). Furthermore, PKC{gamma} activity seems to control the elimination of climbing fibers during cerebellar development because Purkinje cells from PKC{gamma} mutant mice persist with multiple climbing fibers into adulthood (Chen et al., 1995Go; Kano et al., 1995Go). These studies show the importance of PKC{gamma} activity in Purkinje cell development and neuronal connectivity; however, the molecular mechanisms by which PKC{gamma} controls these processes are still poorly understood.

Whereas many types of SCA are caused by coding polyglutamine repeat expansion mutations in the encoding proteins, missense mutations in PKC{gamma} were identified to cause SCA14 in families with different genetic backgrounds (Alonso et al., 2005Go; Chen et al., 2003Go; Dalski et al., 2006Go; Fahey et al., 2005Go; Klebe et al., 2005Go; Stevanin et al., 2004Go; van de Warrenburg et al., 2003Go; Vlak et al., 2006Go; Yabe et al., 2003Go). Up to now, 23 different mutations have been identified throughout the coding region of PKC{gamma}, although about 75% of all SCA14 mutations are located in the highly conserved C1B subdomain of the regulatory domain of PKC{gamma}. The impact of SCA14 missense mutations in this particular subdomain remains unknown, but we hypothesized that the mutated amino acids are possibly crucial for the structure of the C1B subdomain and could therefore induce conformational changes leading to altered regulation of PKC{gamma} activity.

PKC remains inactive in a `closed' conformation via intramolecular interactions. This involves binding of the pseudo-inhibitory substrate that is localized at the N-terminus of the variable protein region (V1 domain) to the substrate-binding core in the catalytic domain (Pears and Parker, 1991Go). In addition, three phosphorylation events are required for PKC to become fully maturate, but still inactive. The first phosphorylation step is catalyzed by phosphoinositide-dependent protein kinase 1 (PDPK1), which is followed by two autophosphorylation events (Dutil et al., 1994Go; Dutil et al., 1998Go). Upon activation, PKC rapidly translocates from the cytoplasm to the plasma membrane and interacts with phospholipids, leading to the release of the pseudo-inhibitory substrate from the catalytic domain resulting in an `open' and active conformation (Newton, 2001Go). This translocation process is initiated by calcium binding to the C2 domain and the subsequently binding of diacylglycerol to the two C1 subdomains C1A and C1B (Newton, 2004Go; Oancea and Meyer, 1998Go; Rodriguez-Alfaro et al., 2004Go; Sakai et al., 1997Go). In the case of PKC{gamma}, C1A and C1B bind its ligand with comparable affinity in vitro (Quest et al., 1994Go; Quest and Bell, 1994Go). The V1-domain that contains the pseudo-inhibitory substrate controls the accessibility of the C1 domain for phorbol ester, which guarantees that PKC is sequentially activated by calcium and diacylglycerol signals (Oancea and Meyer, 1998Go). In addition, masking of the phorbol ester binding sites in the C1 domain is also regulated via intramolecular interactions between the C2 domain and the C-terminal V5 domain, because mutations in these domains make PKC{alpha} respond to DAG and increase the phorbol sensitivity of PKCβ (Stensman and Larsson, 2007Go).

Here we show that SCA14 mutations located in the C1B subdomain enhance PKC{gamma} translocation from cytoplasm to plasma membrane upon phorbol ester stimulation when compared with wild-type PKC{gamma}. These mutations apparently increase the accessibility of the C1 domain for phorbol ester by unmasking the phorbol ester binding sites of the C1 domain. Fluorescence lifetime imaging microscopy (FLIM) analysis using YFP-PKC{gamma}-CFP fusion proteins showed significantly lower intramolecular FRET signals when SCA14 mutations are present, demonstrating that the C1B subdomain mutations result in an `open' protein conformation. Surprisingly, the increased kinetics resulted in a decrease of PKC{gamma} kinase function upon activation. By using the PKC phosphorylation reporter MyrPalm-CKAR, we show that the mutations causing SCA14 reduce PKC{gamma} kinase activity in living cells. In addition, reduced extracellular signal-regulated kinase (ERK) nuclear translocation, and ERK phosphorylation was also observed in cells expressing mutant PKC{gamma}. Together, these findings show that reduced PKC{gamma} activity and aberrant mitogen-activated protein kinase (MAPK) signaling underlies SCA14.


Figure 1
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  		Fig. 1. Scheme and protein expression levels of wild-type and SCA14 mutant PKC{gamma}. (A) Schematic representation of the primary structure of PKC{gamma}, including the V1-domain containing the pseudoinhibitory substrate, the regulatory domain comprising the C1 and C2 domains and the catalytic domain comprising the C3 and C4 domains. SCA14 mutations used in this study are indicated in black. (B) Schematic representation of the constructs. Both full-length PKC{gamma} and the C1B subdomain are fused to GFP and RFP. (C) Western blot for PKC{gamma}, demonstrating equal expression of the different PKC{gamma} alleles (left). Similar expression levels of the C1B fusion proteins were also detected using anti-GFP (right). Actin was used as a loading control. (D) Confocal images of transiently transfected HeLa cells with either full-length PKC{gamma}-GFP or C1B-GFP fusion plasmid. SCA14 mutations do not alter the cellular localization and distribution of full-length PKC{gamma} and C1B proteins in HeLa cells. Scale bars: 10 µm.

 

   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
PKC{gamma} protein expression and subcellular localization
In order to investigate the effects of SCA14 mutations on the intracellular distribution and kinetics of PKC{gamma} in living cells, we generated both green and red fluorescently tagged (GFP/RFP) fusion proteins of both wild-type and SCA14 mutant PKC{gamma}. In addition, we generated similarly tagged fusion proteins for the isolated C1B subdomains (Fig. 1B). The SCA14 mutations that were used in this study include G118D, V138E and C142S (van de Warrenburg et al., 2003Go; Vlak et al., 2006Go) (Fig. 1A), which are all located in the C1B subdomain. Both full-length PKC{gamma} and C1B were transiently expressed in HEK293T cells, followed by the analysis of cell lysates by western blotting, which showed that all constructs were efficiently expressed (Fig. 1C). Visualization of the intracellular distribution of the proteins in living cells showed that PKC{gamma} had a cytoplasmic distribution, whereas the much smaller C1B subdomain was distributed throughout the cytoplasm and nucleus (Fig. 1D). The three SCA14 mutations did not alter the cellular distribution of either full-length PKC{gamma} or the C1B subdomain. No massive aggregation was observed in cells expressing wild-type or mutant PKC{gamma}, as was shown by Seki and colleagues (Seki et al., 2005Go). Only a low percentage of cells showed accumulation of full-length PKC{gamma}-GFP protein in the perinuclear region of the cells, probably because of high levels of PKC{gamma} overexpression (data not shown).

SCA14 mutations located in the C1B subdomain enhance PKC{gamma} kinetics upon phorbol ester activation
Modelling and mutational studies have shown that multiple residues in the C1B subdomain of PKC{delta} are critical for Zn2+ ion coordination and phorbol ester binding, including the residues Val138 and Cys142 (Kazanietz et al., 1995Go; Quest et al., 1994Go; Xu et al., 1997Go). Val138Gly and Cys142Gly mutations completely abolish phorbol ester binding to the C1B subdomain. Given the homology between the different isoforms, we aimed at investigating whether the SCA14 mutations G118D, V138E and C142S would affect PKC{gamma} activation upon phorbol ester stimulation.

We started by measuring the kinetics of PMA-induced translocation of both wild-type C1B and mutant C1B subdomains in living cells. Initially, all experiments were performed using single plasmid transfections; however, to exclude cell-cell variation and to get a more accurate comparison between wild-type and mutant, co-transfection experiments were carried out with wild-type GFP and PKC{delta} mutant RFP plasmids. Similar results were obtained for both experiments (data not shown). HeLa cells were cotransfected with both wild-type C1B-GFP and mutant C1B-RFP to simultaneously image both translocation events using confocal microscopy. The translocation kinetics of G118D mutant C1B subdomain by PMA was similar to wild-type C1B (Fig. 2A,B). By contrast, no translocation of the V138E and C142S mutant C1B subdomains was observed upon PMA stimulation. Similar results were obtained when the GFP and RFP fluorophores were exchanged between the wild-type and mutant domains, excluding the possibility that this effect is fluorophore dependent (data not shown). Taken together, the results demonstrate that SCA14 mutations, which are all located in the C1B subdomain, apparently work via a different mechanisms, because some, but not all, of the disease-related mutations abolish PMA-induced translocation.


Figure 2
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  		Fig. 2. SCA14 mutations located in C1B affect C1B translocation kinetics upon phorbol ester stimulation. (A) Confocal images of HeLa cells cotransfected with C1B-GFP and SCA14 mutant C1B-RFP. PMA activation induces irreversible translocation of C1B-GFP and C1B G118D-RFP to the plasma membrane. By contrast, the V138E and C142S C1B-RFP did not respond to PMA activation. Images shown were recorded prior to (0) and 200 seconds after addition of 400 nM PMA. Scale bars: 10 µm. (B) Translocation analysis of cotransfected C1B-GFP and mutant G118D, V138E or C142S C1B-RFP showed that the V138E and C142S mutations impair phorbol ester (400 nM PMA)-induced membrane translocation. Each of the traces in B and C represent mean ± s.e.m. of 4-6 cells from at least three independent experiments.

 
Next, we examined whether the SCA14 mutations also affect PMA kinetics of full-length PKC{gamma}, because it has been shown that the C1A subdomain of PKC{gamma} can also bind PMA with comparable affinity as C1B (Ananthanarayanan et al., 2003Go). Therefore, we repeated the PMA-induced translocation experiment using full-length wild-type PKC{gamma}-GFP and mutant PKC{gamma}-RFP. Surprisingly, none of the SCA14 mutations impaired PMA-induced translocation, but resulted in enhanced translocation kinetics when compared with wild-type PKC{gamma} (Fig. 3A,B). This effect was also not dependent on the fused fluorophores, because exchange of the GFP and RFP fluorophores showed identical results (data not shown). Increased PMA-induced translocation has also been observed for a PKC{gamma} with a deleted V1 domain, which contains the pseudo-inhibitory substrate (Oancea and Meyer, 1998Go). Their results suggest that the accessibility of the C1 domain for phorbol ester is restricted by a `clamp' between the V1 domain and the catalytic domain. Removal of the V1 domain results in an `open' protein conformation, which leads to exposure of the phorbol binding sites in the C1 domain. To test whether SCA14 mutations in PKC{gamma} lead to similar translocation kinetics, we compared the kinetics of SCA14-mutant PKC{gamma}-GFP and deleted V1 mutant, deltaV1-PKC{gamma}-GFP upon PMA stimulation. Both mutants showed similar rates of translocation upon PMA stimulation, suggesting that SCA14 mutations may affect the regulation of the catalytic domain by the V1-clamp (Fig. 3C). In addition, we also tested whether the constitutively active pseudosubstrate mutant A24E-PKC{gamma} showed increased translocation kinetics upon PMA stimulation. The A24E mutation leads to constant exposure of the pseudosubstrate, leading to complete abolishment of the V1-clamp to the catalytic domain and induces therefore an `open' protein conformation (Pears et al., 1990Go). Indeed, the translocation kinetics of A24E-PKC{gamma} to the plasma membrane upon PMA stimulation was similar to deltaV1-PKC{gamma} and SCA14-mutant PKC{gamma} (data not shown).


Figure 3
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  		Fig. 3. SCA14 mutations located in C1B enhanced PKC{gamma} translocation kinetics upon phorbol ester stimulation. (A) Confocal images of HeLa cells cotransfected with wild-type PKC{gamma}-GFP and SCA14 mutant (G118D, V138E and C142S) PKC{gamma}-RFP. PMA activation induces irreversible translocation of PKC{gamma} to the plasma membrane. Images shown were recorded 140 and 440 seconds after addition of 400 nM PMA. Scale bars: 10 µm. (B) Translocation analysis showed that PMA (400 nM) stimulation resulted in an increase in translocation kinetics of all three SCA14 mutant PKC{gamma} proteins compared with wild-type PKC{gamma} to the plasma membrane. (C) Translocation analysis of wild-type PKC{gamma}-GFP, SCA14 mutant PKC{gamma}-GFP (G118D, V138E and C142S) and {Delta}VPKC{gamma}-GFP upon PMA (400 nM) stimulation. Each of the traces in B and C represent mean ± s.e.m. of 4-6 cells from at least three independent experiments.

 

These data suggest that the enhanced translocation kinetics of SCA14 mutant PKC{gamma} upon PMA stimulation might result from increased accessibility of the overall C1 domain for phorbol ester, as a result of conformation changes under the influence of disease-related mutations in the PKC{gamma} protein.

SCA14 mutant PKC{gamma} shows increased membrane targeting without affected C2 domain functioning
As PKC is sequentially activated by calcium (C2 domain) and phorbol ester (C1 domain), we wanted to determine the effect of a physiological stimulus on the translocation kinetics of PKC{gamma}. Therefore, we tested whether SCA14 mutations would affect histamine stimulation of PKC{gamma}, because activation of the histamine receptor induces the rapid release of endoplasmic-reticulum-stored calcium and production of diacylglycerol. HeLa cells were transiently cotransfected with wild-type (red) and SCA14-mutant (green) PKC{gamma} and imaged in real time using histamine stimulation and subsequent PMA and ionomycin treatment to obtain full PKC{gamma} translocation. Although no difference was detected in the translocation pattern between two wild-type PKC{gamma} protein fused to GFP and RFP (Fig. 4A), the amplitude of translocation of the SCA14 mutant PKC{gamma} was 1.5±0.03 (G118D; mean ± s.e.m.), 1.9±0.1 (V138E) and 1.4±0.05 (C142S) times significantly increased when compared with wild-type PKC{gamma} (G118D; V138E; C142S unpaired t-test, n=3-5; P=0.0002, P=0.0026 and P=0.0007, respectively) (Fig. 4B-E). The graphs are representatives of at least three independent experiments. These results indicate that the SCA14 mutations enhance the amount of PKC{gamma} protein at the plasma membrane upon stimulation of cells under physiological conditions.


Figure 4
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  		Fig. 4. SCA14 mutant PKC{gamma} shows increased membrane targeting but does not affect C2 domain functioning. (A) Translocation analysis of HeLa cells cotransfected with wild-type PKC{gamma}-RFP and wild-type PKC{gamma}-GFP. Both proteins induced rapid reversible translocation to the plasma membrane upon histamine stimulation. No difference was observed in translocation pattern between the two wild-type PKC{gamma} proteins fused to different fluorophores. Furthermore, wild-type PKC{gamma}-RFP was cotransfected with SCA14 mutant G118D (B), V138E (C) and C142S (D) PKC{gamma}-GFP. No difference was observed in membrane dissociation between wild-type and SCA14-mutant PKC{gamma}. Increased translocation amplitudes were observed for SCA14 mutant PKC{gamma} compared with wild-type PKC{gamma}. Graphs show the translocation curve of single cells, but represent three independent experiments. (E) Quantification of the amount of PKC{gamma} translocated to the plasma membrane upon histamine stimulation. Bars represent the ratio between the maximal amplitude of wild-type PKC{gamma} and the maximal amplitude of SCA14-mutant PKC{gamma}. Data are means ± s.e.m. of three experiments each on three cells (unpaired t-test, **P<0.01; ***P<0.001). (F) No differences were observed in C2 domain-induced translocation kinetics of full-length PKC{gamma}-GFP and mutant G118D, V138E or C142S PKC{gamma}-GFP in response to 5 µM A23187. The curves are mean ± s.e.m. of 3-5 cells and the data are representative of three experiments.

 
To resolve whether the enhanced membrane targeting of SCA14-mutant PKC{gamma} was solely caused by the increased accessibility of the C1 domain for phorbol ester or also by changes in Ca2+ binding to the C2 domain, we measured PKC{gamma} translocation to the plasma membrane upon Ca2+ ionophore stimulation. This ionophore activation will result in opening of Ca2+ pores in the plasma membrane leading to a rapid increase in intracellular Ca2+. No difference in translocation kinetics was observed between wild-type and SCA14 mutant PKC{gamma} (Fig. 4F), indicating that stimulation via the C2 domain was not affected.

Overall, these results show that SCA14 mutations located in the C1B subdomain principally affect the phorbol ester sensitivity of the C1 domain, which leads to increased membrane targeting of SCA14 mutant PKC{gamma}.

Unmasking of the C1 domain via SCA14 mutations is associated with altered PKC{gamma} conformation
Our kinetic studies suggest that the SCA14 mutations located in the C1B subdomain unmask the C1 domain, leading to exposure and increased accessibility of the phorbol ester binding sites. This could be the result of destabilization of the `closed' conformation, which will consequently provoke unmasking of the C1 domain. To examine the differences in PKC{gamma} protein conformation caused by C1B SCA14 mutations, we took advantage of fluorescence resonance energy transfer (FRET) analysis. It has been shown previously that intramolecular FRET can be used to probe protein conformation in single living cells by tagging the protein of interest with both a donor and an acceptor fluorophore (Giepmans et al., 2006Go; Miyawaki, 2003Go). The FRET signal is dependent on the distance and orientation of the two fluorophores and altered protein conformations of PKC{gamma} may lead to a changed distance between the N- and C-terminus and subsequent change in the FRET signal. PKC{gamma} FRET constructs were generated by tagging the N-terminus with EYFP and the C-terminus with ECFP.

To examine whether FRET can be used to distinguish between different conformations, we measured FRET efficiencies of the wild-type PKC{gamma}, which is in a `closed' conformation, and the A24E mutant which cannot bind the pseudosubstrate (N-terminus) to the substrate-binding grove and consequently remains in an `open' conformation. Although FRET was observed in these constructs, no differences in FRET efficiency were measured (data not shown). Since it is known that the ECFP and EYFP have the tendency to dimerize, the ECFP was replaced by a monomerized, brighter variant (Kremers et al., 2006Go), to abolish intramolecular dimerization. FLIM was used to measure FRET efficiencies (van Munster and Gadella, 2005Go) using SCFP3A as a control. Both phase and modulation lifetimes of SCFP3A were strongly reduced in the wild-type YFP-PKC{gamma}-SCFP3A protein, yielding a FRET efficiency of 29% (Fig. 5A-C). By contrast, the SCFP3A lifetime of the A24E-PKC{gamma} mutant was close to values of the unquenched SCFP3A with a relatively low FRET efficiency of 11% based on the phase lifetime (Fig. 5B,C) and 10% based on the modulation lifetime. These results fit with the model in which the N- and C-terminus are close together in the closed conformation of inactive wild-type PKC{gamma}, whereas the termini are further apart into an open conformation of the constitutively active A24E mutant. These results showed that intramolecular FRET can be used to distinguish between the closed and open PKC{gamma} conformation in unstimulated cells. When we monitored the changes in protein conformation upon activation of PKC by PMA, we consistently observed a FRET increase for all constructs (data not shown), whereas a decrease was expected, as this would correspond to the open active conformation. Similar observations have been reported for the PKC{delta} isoform (Braun et al., 2005Go). This can be explained by accumulation of PKC at the plasma membrane induced by PMA. Owing to the relative high surface concentration of PKC molecules at the plasma membrane, intermolecular aspecific FRET takes place. Therefore, this approach did not allow monitoring changes in conformation upon activation of PKC.


Figure 5
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  		Fig. 5. SCA14 mutations located in C1B open PKC{gamma} protein conformation. (A) Fluorescence lifetime imaging analysis of the indicated constructs, showing in each column, from left to right, the fluorescence intensity, false-color map of the fluorescence lifetime calculated from the phase shift (tau-phi) and histogram of the lifetime distribution with the same false-color scale as the lifetime map. Scale bar: 10 µm. (B) The table summarizes the average phase and modulation lifetimes (± s.d.). The number of cells measured is indicated by n. (C) FRET efficiency (mean ± s.e.m.; unpaired t-test, ***P<0.001) and quantification are calculated based on the phase lifetimes, using the SCFP3A lifetime as the control.

 
Subsequently, we incorporated the SCA14 mutations, G118D, V138E and C142S into the wild-type PKC{gamma} FRET construct. The SCA14 mutant PKC{gamma} gave rise to significantly lower FRET signals than wild-type PKC{gamma} (G118D; V138E; C142S, all P<0.0001, unpaired t-test, n=5) (Fig. 5A-C). The three SCA14 mutant PKC{gamma} displayed approximately similar FRET efficiencies ranging from 14% to 16% (Fig. 5C), based on the phase lifetime (and 11-12% based on the modulation lifetime). Interestingly, these FRET signals were similar to the value measured for A24E-PKC{gamma} (Fig. 5A,B). Although the FRET efficiency measured in the SCA14 mutants and A24E mutant is significantly reduced relative to wild-type PKC{gamma}, it cannot be concluded from these experiments that the conformations are identical. All proteins were properly expressed, excluding the possibility that the lower FRET signal was due to degradation of the SCA14-mutant PKC{gamma} proteins (data not shown).

In conclusion, these results demonstrate that SCA14 mutations located in the C1B subdomain change PKC{gamma} from a closed conformation into a different, most likely, open conformation in which the C- and N-termini are further apart.

SCA14 mutant PKC{gamma} shows reduced kinase activation in living cells
Since SCA14 mutant PKC{gamma} display enhanced PMA kinetics and exhibit a different protein conformation, we hypothesized that C1B SCA14 mutations alter the phosphorylation capacity and thus activity of PKC{gamma}. In order to measure PKC{gamma} activity in living cells, we used the PKC phosphorylation reporter, MyrPalm-CKAR, which is a membrane-bound intramolecular FRET reporter (Violin et al., 2003Go). Immediate phosphorylation of the PKC substrate sequence upon PKC activation causes a conformational change that reduces the amount of FRET between the CFP and YFP fluorophores. Unfortunately, the PKC-substrate peptide sequence is not selectively accessible for PKC{gamma} but also permits phosphorylation of PKC{alpha} and PKCβ. Therefore, we first tested whether activation of expressed wild-type PKC{gamma} would lead to lower FRET ratios than activation of endogenous PKC ({alpha} and β) levels in HeLa cells. HeLa cells were transiently transfected with MyrPalm-CKAR or cotransfected with wild-type PKC{gamma}-RFP. First, we stimulated the cells with histamine, which only induced partial phosphorylation of MyrPalm-CKAR, but did not show much difference in FRET ratio and magnitude of activation between cells overexpressing wild-type and endogenous PKC in response to histamine (Fig. 6A). Since cells react rather heterogeneously to stimulation by histamine, we decided to subsequently add PMA to obtain the maximal amplitude of the reporter corresponding to maximal phosphorylation. Cells overexpressing PKC{gamma} showed significant lower FRET ratios (24.2±0.4; mean ± s.e.m.) (P<0.0001, unpaired t-test, n=5) compared with cells that did not express PKC{gamma} (15.3±0.2), demonstrating that we could detect PKC{gamma}-specific CKAR phosphorylation (Fig. 6A). We then investigated whether changes in PKC{gamma} protein conformation due to unmasking of the C1 domain are reflected in a different MyrPalm-CKAR FRET ratio. Surprisingly, significantly lower FRET efficiencies (G118D; V138E; C142S: 20.6±0.4; 18.4±0.5; 18.0±0.6; mean ± s.e.m.; P=0.0002; P=0.0001; P<0.0001, unpaired t-test, n=3-5) were observed in cells expressing SCA14 mutant PKC{gamma} than cells containing wild-type PKC{gamma} upon PMA stimulation, indicating less MyrPalm-CKAR phosphorylation (Fig. 6B). These results suggest that the SCA14 mutant PKC{gamma} has less capability to phosphorylate cellular substrates and thus subsequently exhibit reduced kinase activity in living cells. This finding was confirmed in cell lysate of cells overexpressing wild-type or SCA14 mutant PKC{gamma} using an anti-phosphoserine-PKC-substrate antibody. We observed an overall reduction in kinase activity in cell lysates of SCA14 mutant PKC{gamma} when compared with wild-type PKC{gamma}, because a number of unidentified PKC targets showed a reduction in phosphorylation upon PMA stimulation (data not shown). To examine whether reduced SCA14 mutant PKC activity upon activation is the result of increased basal membrane activity due to the open conformation, we used the PKC inhibitor Gö6976 to block basal PKC activity. Upon addition of Gö6976 to unstimulated cells expressing either wild-type PKC{gamma} or SCA14 mutant PKC{gamma}, we observed similar increased YFP/CFP FRET ratios in both cases (data not shown). This result suggests that the MyrPalm-CKAR reporter is constitutively phosphorylated to a similar extent by either wild-type PKC{gamma} or SCA14 mutant PKC{gamma}.


Figure 6
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  		Fig. 6. SCA14 mutations in C1B reduce PKC{gamma} kinase activity in living cells. (A) HeLa cells were transiently transfected with either MyrPalm-CKAR alone or cotransfected with wild-type PKC{gamma}-RFP or the different SCA14 mutant PKC{gamma}-RFP. Intramolecular FRET was measured by determining the ratio of the YFP/CFP fluorophore intensities of the MyrPalm-CKAR molecule. Upon phosphorylation, less FRET from CFP to YFP will be measured because conformational changes increase the distance between the N- and C-terminus. MyrPalm-CKAR shows reduced phosphorylation, as was shown by less loss of FRET in the presence of SCA14 mutant PKC{gamma} (G118D, V138E and C142S) when compared with wild-type PKC{gamma} upon stimulation with 500nM PMA. The data traces were corrected by the background images acquired prior to adding ligand. The corrected traces were normalized to 1 by dividing the traces by the average baseline FRET ratio. (B) Bars represent the FRET efficiencies of the MyrPalm-CKAR reporter in the absence and presence of wild-type PKC{gamma} and SCA14 mutant PKC{gamma} (unpaired t-test, ***P<0.001). (C) Western blot analysis shows reduced MARCKS phosphorylation in whole cell lysates of SCA14-mutant PKC{gamma} upon PMA stimulation (400 nM) compared with cells containing wild-type PKC{gamma} using anti-phospho-MARCKS antibody. Top panel shows equal PKC{gamma} protein levels in all cell lysates. (D) Quantification of band intensity. Bar graph represents the phosphorylation of MARCKS normalized to the amount of MARCKS and PKC{gamma}, and related to the value obtained for cells expressing wild-type PKC{gamma}. Data in B and D represent mean ± s.e.m.

 
Since these experiments only report on the phosphorylation of an artificial substrate or unknown PKC targets, we also wanted to examine whether relevant cellular target proteins were also affected by reduced phosphorylation. Therefore, we studied the phosphorylation status of the known intracellular PKC target myristoylated alanine-rich C-kinase substrate (MARCKS). MARCKS is a multifunctional protein implicated in cell motility, membrane trafficking and mitogenesis, because its activity regulates growth cone adhesion and pathfinding (Gatlin et al., 2006Go; Myat et al., 1997Go). We determined the phosphorylation level of endogenous MARCKS in lysates of cells transfected with either wild-type or SCA14 mutant PKC{gamma} upon PMA activation, using an anti-phospho-MARCKS antibody. Immunoblotting showed no basal phosphorylation of MARCKS, but when PMA was added, reduced MARCKS phosphorylation in cells expressing SCA14 mutant PKC{gamma} was observed compared with that in cells containing wild-type PKC{gamma} (Fig. 6C,D).

These experiments demonstrate that SCA14 mutations located in the C1B subdomain affect PKC{gamma} kinase activity, resulting in reduced phosphorylation of downstream targets.

SCA14 mutant PKC{gamma} affects downstream MAPK signaling
The MAPK pathways that activate ERK play an important role in cell mobility, cell migration, dendrite spine plasticity and long-term potentiation in learning and memory functions (De Zeeuw et al., 1998Go; Pullikuth and Catling, 2007Go; Robinson et al., 1998Go). Misregulation of cell migration has already been associated with neurodegeneration (Hafezparast et al., 2003Go; Vallee et al., 2001Go) and as result, we hypothesized that aberrant MAPK and ERK signaling may be affected in SCA14. The movement of ERK2 into the nucleus has been suggested to be important for the long-term consequences of ERK activation (Horgan and Stork, 2003Go). Therefore, we determined ERK2 cytoplasmic-nuclear shuttling using confocal microscopy. GFP-ERK2 was transiently transfected in HeLa cells either with wild-type PKC{gamma}-RFP or SCA14 mutant PKC{gamma}-RFP and stimulated them with PMA. PKC activation induced a transient GFP-ERK2 nuclear translocation within 10 minutes of addition of PMA (Fig. 7A,B). Translocation analysis showed a significant reduction in nuclear accumulation (G118D; V138E; C142S: P=0.0024; P=0.0009; P=0.0011, unpaired t-test, n=3) of GFP-ERK2 in the nucleus of cells coexpressing SCA14 mutant PKC{gamma} (G118D; V138E; C142S: 2.0±0.04; 1.7±0.03; 1.7±0.06; mean ± s.e.m.) compared with cells coexpressing wild-type PKC{gamma} (3.0±0.1) or cells only expressing GFP-ERK2 (2.9±0.1) (Fig. 7B,C). To exclude the idea that reduced nuclear ERK translocation was caused by basally higher ERK levels in the nucleus of cells coexpressing SCA14 mutant PKC{gamma}, we determined nuclear ERK2-GFP expression levels of cells coexpressing either wild-type PKC{gamma} or SCA14 mutant PKC{gamma} using western blotting. No increased nuclear ERK2-GFP expression was observed in cells expressing SCA14 mutant PKC{gamma} compared with that cells expressing wild-type PKC{gamma} (data not shown). As nuclear accumulation is associated with ERK phosphorylation (Costa et al., 2006Go), we also determined the phosphorylation profile of endogenous ERK in cells upon PKC activation. Notably, no significant basal phosphorylation of ERK was observed in unstimulated cells overexpressing wild-type PKC{gamma} or any of the SCA14 mutants. In accordance with the translocation assay, reduced levels of ERK phosphorylation were observed in cells expressing SCA14 mutant PKC{gamma} when compared with cells expressing wild-type PKC{gamma} (Fig. 7D,E).


Figure 7
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  		Fig. 7. SCA14 mutant PKC{gamma} reduces ERK2 nuclear accumulation and phosphorylation. (A) Confocal images of HeLa cells transiently transfected with GFP-ERK2 and cotransfected with either wild-type PKC{gamma}-RFP or PKC{gamma} G118D-RFP (V138E and C142S mutants confocal data not shown). Activation of PKC{gamma} with PMA induced GFP-ERK2 nuclear accumulation in time. Images shown are recorded 250 and 500 seconds after addition of 400 nM PMA. Scale bar: 10 µm. (B) Translocation analysis shows that GFP-ERK2 nuclear translocation was reduced in cells coexpressing SCA14 mutant PKC{gamma} when compared with cells that express only GFP-ERK2 or coexpress wild-type PKC{gamma}-RFP. (C) Bars represent the ratio of nuclear/cytosolic ERK fluorescence in the absence and presence of wild-type PKC{gamma} and SCA14 mutant PKC{gamma} (unpaired t-test, **P<0.01; ***P<0.001). (D) Western blot analysis showed that endogenous ERK1 and ERK2 are phosphorylated after PKC{gamma} activation with 400 nM PMA for 10 minutes. Reduced ERK2 phosphorylation was observed in the presence of SCA14 mutant PKC{gamma} (G118D, V138E and C142S) compared with cells expressing wild-type PKC{gamma}. Furthermore, equal ERK and PKC{gamma} levels were observed using anti-ERK and anti-PKC antibodies. (E) Quantification of the band intensity. Bar graph represents the phosphorylation of ERK normalized to the amount of ERK and PKC{gamma} and related to the value obtained for cells expressing wild type PKC{gamma}. Data in C and E represent mean ± s.e.m.

 
Together these data show that SCA14 mutations located in the C1B subdomain affect PKC{gamma} kinase activity, lead to lower ERK phosphorylation levels and accordingly reduce nuclear ERK translocation. Both events will reduce MAPK signaling, and might therefore lead to reduced expression of target genes affecting neurite outgrowth and survival.


   Discussion
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 Summary
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 Results
 Discussion
Materials and Methods
 References
 
In resting cells, the accessibility of the C1 domain for phorbol ester is well regulated by masking of the C1 domain, which prevents exposure of the phorbol binding sites. This `closed' protein conformation is necessary to keep PKC inactive. Although two SCA14 mutations, V138E and C142S, completely abolish translocation of the isolated C1B subdomain to the plasma membrane upon phorbol ester stimulation (Fig. 2A,B), the G118D mutation does not seem to affect C1B PMA-induced kinetics. However, when present in the full-length PKC{gamma} protein, all three SCA14 mutations enhanced PKC{gamma} translocation to the plasma membrane upon phorbol ester stimulation (Fig. 3A,B). These finding suggest that SCA14 mutations not only affect the regulatory function of C1B subdomain, but also affect the C1A domain. This possibility would reconcile our findings with previous studies showing that the C1A and C1B subdomains of PKC{gamma} have similar affinities for phorbol ester in vitro (Ananthanarayanan et al., 2003Go). They thus have redundant roles in PKC{gamma} phorbol ester activation, because even the SCA14 mutant PKC{gamma} lacking a functional C1B subdomain showed enhanced PMA-induced translocation. Furthermore, the residues V138 and C142 have already been shown to be crucial for phorbol ester binding (Kazanietz et al., 1995Go), and modelling of the crystal structure of the C1B subdomain also demonstrated that the mutated residues indeed led to a dysfunctional phorbol ester binding site (our unpublished data). Interestingly, despite proper C1B subdomain activation upon phorbol ester stimulation, the G118D mutation also enhanced the translocation kinetics of the full-length PKC{gamma} protein, similarly to the other mutated proteins. Apparently, the SCA14 mutations affect PKC{gamma} activation via a different mechanism but with a similar outcome.

Enhanced PKC{gamma} translocation upon phorbol ester stimulation was also observed when the V1-domain containing the pseudoinhibitory substrate was deleted in PKC{gamma}. This study showed that removal of the V1-domain increased the accessibility of the C1 domain for phorbol ester (Oancea and Meyer, 1998Go). SCA14 mutations located in the C1B subdomain apparently affect PKC{gamma} kinetics in a similar way, by altering the conformation of the protein, leading to unmasking of the C1 domain as was shown in Fig. 3C. The changed conformation of the SCA14 mutants also became obvious from our FRET studies (Fig. 5). We showed that all three SCA14 mutations led to an open PKC{gamma} protein conformation, which may be similar to that of PKC{gamma} with mutations in the V1 domain ({Delta}V and A24E), because all three variants showed enhanced PMA-induced kinetics.

Although the SCA14 mutations in the C1B subdomain showed similar kinetics and conformational changes as modification or deletion of the upstream V1-domain, they could also affect functioning of the other regulatory domain, the C2 domain (Oancea and Meyer, 1998Go; Slater et al., 2002Go; Stahelin et al., 2005Go; Stensman et al., 2004Go). However, we did not observe any effect of the SCA14 mutations on C2 functioning as measured by PKC{gamma} calcium sensitivity (Fig. 4E). Furthermore, the SCA14 mutations located in the C1B subdomain did not impair PKC{gamma} membrane dissociation as shown by the reversible histamine translocation. However, SCA14 mutant PKC did show increased membrane binding upon histamine stimulation, demonstrating that SCA14 mutant PKC{gamma} has increased affinity for the plasma membrane under physiological conditions (Fig. 4A-D).

As enhanced PMA kinetics and open PKC protein conformations are always associated with active PKC, we hypothesized that SCA14 mutant PKC{gamma} would increase signaling pathways upon activation. To our surprise, the SCA14 mutations located in the C1B subdomain reduced PKC{gamma} kinase activity in living cells as demonstrated using the fluorescent membrane-bound reporter MyrPalm-CKAR (Fig. 6). Moreover, both MARCKS and a more downstream target ERK2 showed reduced PKC{gamma}-mediated phosphorylation, which led to reduced MARCKS and MAPK activation in living cells (Fig. 6C and Fig. 7). Importantly, we did not observe enhanced basal phosphorylation of CKAR, MARCKS or ERK as a result of overexpression of SCA14 mutants. These results suggest that if the SCA14 mutants have increased basal activity, this activity is effectively counteracted in cells by phosphate activity.

PKC has been shown to regulate the structure and function of the cell by phosphorylating various cytoskeletal or functional proteins, such as MARCKS or annexins (Keenan and Kelleher, 1998Go). MARCKS has been implicated in regulating cell attachment, membrane ruffling and spreading (Myat et al., 1997Go). Furthermore, its activity is important for the reorganization of cytoskeletal structures, which are crucial events in growth cone formation and dendrite outgrowth (Geddis and Rehder, 2003Go; Matsuoka et al., 1998Go). Although there may be several possibilities that may explain the SCA14 disease pathology, defects in MARCKS-dependent cell adhesion or spreading may explain the neurodegenerative phenotype. The role of ERK signaling in neurodegenerative disorders is more complex, because both protective and deleterious roles for ERK activation in neuronal cells have been described (Apostol et al., 2006Go; Colucci-D'Amato et al., 2003Go; Lievens et al., 2005Go). The diversity of ERK activation is not surprising, because the pathway integrates various signals depending on the cellular context, including cell proliferation, differentiation, migration and survival (Bhalla and Iyengar, 1999Go; Chuderland and Seger, 2005Go; Hetman and Gozdz, 2004Go). This diversity is also mediated by several factors, such as cell or tissue type, the duration of activation, as well as the subcellular localization (Ebisuya et al., 2005Go). However, our results suggest that reduced ERK signaling underlies SCA14 disease.

To address the question of how increased PKC{gamma} kinetics resulting from an open conformation corresponds to reduced activity, we are currently studying the maturation process of SCA14 mutant PKC{gamma} and performing membrane-binding studies to determine maximal PKC activity upon membrane interactions. We suggest that an open PKC{gamma} protein conformation at resting state affects its maturation process via PDPK1. The maturation of PKCs after synthesis involves the phosphorylation of the enzyme at multiple serine/threonine sites. Phosphorylation by PDPK1 is the first phosphorylation event in this maturation process and acts as an on-off switch for PKC activity. As PDPK1 preferably interacts and covalently modifies substrates when they are in open conformations (Sonnenburg et al., 2001Go), we hypothesize that SCA14 mutations affect the proper maturation process of PKC{gamma} leading to reduced PKC kinase activity.

Interestingly, the neurological phenotype of SCA14 patients (heterozygous mutations) is more severe than that of the PKC{gamma}-knockout mice (Chen et al., 1995Go). Therefore, the increase in SCA14 mutant PKC{gamma} kinetics but decrease in activity may explain the dominant-negative effect of the mutated PKC{gamma} protein, because it might compete with wild-type PKC{gamma} for its ligands at the plasma membrane. A similar competition between PKC isoforms for phorbol ester binding has been observed previously (Lenz et al., 2002Go), where agonist stimulation induced selective membrane recruitment of classical and/or novel PKC isoforms. This phenomenon may also explain why even less ERK phosphorylation was observed in cell lines overexpressing SCA14 mutant PKC{gamma} compared with cells that did not overexpress PKC{gamma} (Fig. 7D).

Although SCA14 is caused by mutations in the PKC{gamma} gene, other SCA types that show comparable disease phenotypes are caused by mutations in genes encoding other signaling proteins. These proteins include tau-tubulin-associated kinase 2 (SCA11), {alpha}1 subunit of the P/Q-type calcium channel (SCA6), regulatory subunit of the protein phosphatase PP2A (SCA12) and fibroblast growth factor 14 (SCA27) (Holmes et al., 2001Go; Houlden et al., 2007Go; van de Leemput et al., 2007Go; van Swieten et al., 2003Go; Zhuchenko et al., 1997Go). Future research is required to determine whether these different proteins are components of shared signaling routes or even are linked within one common signaling cascade that underlies SCA disease in general. Furthermore, FGF signaling, Ca2+ influx and phosphatase activity also regulate PKC functioning, and place PKC{gamma} at an important position in neuronal signaling pathways. Therefore, it is not surprising that PKC is involved in various diseases, such as cancer, cardiac and lung diseases, diabetes and neurodegenerative disorders, such as SCA14. To select PKC as a target for therapeutic approaches, we need to know more about PKC isoform differences to generate selective inhibitors or activators. Nevertheless investigating `naturally occurring' PKC mutations may help us to understand the mechanisms of PKC activation and regulation and the subsequent effects on downstream signaling.

In conclusion, we show that SCA14 mutations located in the C1B subdomain of PKC{gamma} enhance PMA-induced kinetics and an open PKC{gamma}s protein conformation. This results in increased membrane targeting upon physiological stimuli but leads to decreased kinase activity and downstream MAPK signaling. We propose that SCA14 mutations unmask the C1 domain, expose the phorbol binding sites by opening of the PKC{gamma} protein conformation, which subsequently results in increased accessibility of the whole C1 domain for phorbol ester. The increased accessibility subsequently leads to increased PMA translocation kinetics. Unexpectedly, these events corresponded with decreased kinase activity, as shown by the reduced phosphorylation capacity of PKC{gamma} in living cells. As a consequence, SCA14 mutant PKC{gamma} reduced ERK activation, which highlights the MAPK signaling pathway for possible therapeutic intervention in SCA14 disease.


   Materials and Methods
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 Summary
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 Results
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 Materials and Methods
References
 
DNA constructs
The wild-type and SCA14 mutant Gly118Asp PKC{gamma}-GFP constructs were described previously (Verbeek et al., 2005Go). The Val138Glu and the Cys142Ser mutations were introduced into PRKCG cDNA with the Quikchange II Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). For the Val138Glu and the Cys142Ser mutations the following primers were used: Val138Glu forward, 5'-GTC GCG AGA TGA ACG AGC ACC GGC GCT GTG TG-3'; Val138Glu reverse, 5'-CAC ACA GCG CCG GTG CTC GTT CAT CTC GCA GC-3'; Cys142Ser forward, 5'-CGT GCA CCG GCG CTC TGT GCG TAG CGT GC-3'; and Cys142Ser reverse, 5'-GCA CGC TAC GCA CAG AGC GCC GGT GCA CG-3'. The CIB cDNAs were amplified from the corresponding PKC{gamma}-GFP constructs by PCR using the following primers: forward, 5'-GCC GAA TTC ACC ATG CAC AAG TTC CGC CTG CAT AG-3' and reverse, 5'-CGG GGA TCC CGG CAC AGG GAG GGC ACG CT-3' generating an EcoRI site at the 5' end and a BamHI site at the 3' end. This facilitated cloning into the EGFP-N1 plasmid. In addition, the EGFP-N1 plasmid (Clontech) was replaced by a RFP-N1 expression vector (Campbell et al., 2002Go). The MyrPalm-CKAR construct and YFP-PKC{delta}-CFP were kindly provided by A. Newton (University of California at San Diego, La Jolla, CA) and by B. Blumberg (National Cancer Institute NIH, Bethesda, MD), respectively. The EYFP-PKC{delta}-ECFP construct was used as template to generate the EYFP-PKC{gamma}-ECFP and the EYFP-PKC{gamma}-SCFP3A constructs. PKC{delta} was removed from the EYFP-ECFP vector using XhoI and BamHI. Wild-type and SCA14-mutant PKC{gamma} inserts flanked with XhoI and BamHI site were amplified from the corresponding PKC{gamma}-GFP plasmids using the following primer: forward, 5'-GCC CTC GAG ACC ATG GCT GGT CTG GGC CCC-3' and reverse, 5'-CGG GGA TCC CGC ATG ACG GGC ACA GGC AC-3' creating XhoI and BamHI sites on the 3' end and 5' end, respectively. The ECFP was subsequently replaced with SCFP3A, a previously described brighter, monomeric variant of CFP (Kremers et al., 2006Go), using the flanking BamHI and NotI restriction sites. The GFP-MARCKS construct was kindly provided by H. Mogami (Hamamatsu University School of Medicine, Hamamatsu, Japan) and the GFP-ERK2 was cloned by P. Stork (Oregon Health & Science University, Portland, OR). All constructs were verified by sequencing.

Cell culture and transfection
HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM); high glucose supplemented with 10% fetal bovine serum and 5% penicillin (100 U/ml), streptomycin (100 mg/ml) and glutamate (100 mg/ml). The cultures were maintained at 37°C in an atmosphere of 10% CO2. The cells were dissociated using trypsinization and plated (0.3x10–6 cells) onto glass coverslips (24 mm; Fisher Scientific, Braunsweg, Germany) in a six-well plate and transfected after 1 day. Each well of cells was treated with a mixture of 1 µg DNA and 2 µl Fugene 6.0 transfection reagent in serum-free medium (100 µl transfection per well; Roche Diagnostics, Indianapolis, IN). After transfection, the cells were cultured at 37°C for at least 24 hours. HEK 293T cells were cultured in Iscove's modified Dulbecco's medium (IMDM); high glucose supplemented with 10% fetal bovine serum and 5% penicillin (100 U/ml), streptomycin (100 mg/ml) and glutamate (100 mg/ml) in an atmosphere of 5% CO2. One day before transfection, 0.1x10–6 cells were seeded into 12-well plates and transiently transfected with 0.5 µg DNA using Fugene reagent. After transfection, the cells were cultured at 37°C for 48 hours.

Live cell microscopy
Before imaging, the culture medium was replaced by Hank's balanced salt solution containing CaCl2 supplemented with 5 mM HEPES. Translocation analysis was performed by confocal microscopy (Leica Sp2) using a 63x objective at room temperature (21-25°C). Fluorescent images of GFP and RFP constructs were obtained in sequential mode. The time-lapse experiments were controlled by the manufacturer's acquisition software and the images were acquired every 12 seconds. To measure kinase activity in single living cells, the FRET reporter MyrPalm-CKAR was imaged on a Zeiss 200M inverted fluorescence microscope equipped with a 40x Plan-Neofluar (1.3 NA) oil-immersion lens (Zeiss, Oberkochen, Germany). The set-up was controlled by MetaMorph software. Excitation light from a Cairn Xenon Arc lamp was selected by a monochromator (Cairn Research, Kent, UK) (420 nm, bandwidth 30 nm) and reflected by a 455DCLP dichroic mirror. Emission was passed through 470/30 nm (CFP) and 535/30 nm (YFP) band-pass filters. Images were acquired with a Photometrics CoolSnap HQ CCD camera with 4x4 binning and an exposure time of 200 ms per image. The time interval was 4 seconds and 121 images were acquired. The average fluorescence intensity of individual cells was measured using ImageJ (http://rsb.info.nih.gov/ij/), background subtracted and normalized to the initial fluorescence. Subsequently the YFP/CFP ratio was calculated as a measure of kinase activity. FLIM experiments and subsequent image analysis were performed as described (Goedhart et al., 2007Go). A 442 nm helium-cadmium laser (125 mW, Melles-Griot, Carlsbad, CA) was intensity modulated at a frequency of 75.1 MHz. The light was reflected by a 455DCLP dichroic mirror and the CFP emission was passed through a D480/40 band-pass emission filter (Chroma Technology). A 63x objective (Plan Apochromat NA 1.4 oil) was used for all measurements. The FRET efficiency E was calculated according to: E=(1–({tau}DA/{tau}D))x100% in which {tau}DA is the fluorescence lifetime of the donor in presence of the acceptor (i.e. the PKC FRET constructs) and {tau}D is the fluorescence lifetime of the donor (i.e. unfused SCFP3A) in absence of the acceptor. Since frequency domain FLIM yields a phase lifetime and a modulation lifetime, the FRET efficiency can be calculated based on {tau}{pi} and {tau}M.

Immunoblotting
The cells were harvested 48 hours after transfection and lysed with ice-cold 2x Tris buffer (100 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM EDTA pH 8.0, 1% Triton-X100) supplemented with complete mini protease inhibitor cocktail (Roche, Palo Alto, CA) and phosphatase inhibitor cocktail (Sigma). Cell lysates were incubated for 30 minutes on ice and centrifuged at 14,000 r.p.m. for 20 minutes at 4°C. Protein concentrations in the lysates were determined with the Bradford protein assay and equal protein levels were loaded on 10% SDS-PAGE gels. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane filter (Schleicher & Schuell, Dassel, Germany) and blocked in 5% milk to prepare for western blotting. The western blots were analyzed with anti-PKC ({alpha},β and {gamma}; Upstate), anti-GFP (Santa Cruz), anti-phospho-(Ser)-PKC-substrate (Cell Signaling), anti-phosho-p44/42-MAP kinase (Thr202/Tyr204) (Cell Signaling), anti-p44/42 MAP kinase (Cell Signaling), anti-MARCKS phosphoSer152/156 (Chemicon), and anti-MARKCS (Chemicon) antibodies.


   Acknowledgments
 
We would like to thank Anastassis Perrakis (NKI, Amsterdam) for his technical assistance with the structure modelling of the C1B domain, and we thank Dorus Gadella (UvA, Amsterdam) for valuable advice.


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 Introduction
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 Discussion
 Materials and Methods
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