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

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

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

View larger version (46K): [in this window] [in a new window]   Fig. 1. Scheme and protein expression levels of wild-type and SCA14 mutant PKC. (A) Schematic representation of the primary structure of PKC, 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 and the C1B subdomain are fused to GFP and RFP. (C) Western blot for PKC, demonstrating equal expression of the different PKC 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-GFP or C1B-GFP fusion plasmid. SCA14 mutations do not alter the cellular localization and distribution of full-length PKC and C1B proteins in HeLa cells. Scale bars: 10 µm.

View larger version (52K): [in this window] [in a new window]   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.

View larger version (57K): [in this window] [in a new window]   Fig. 3. SCA14 mutations located in C1B enhanced PKC translocation kinetics upon phorbol ester stimulation. (A) Confocal images of HeLa cells cotransfected with wild-type PKC-GFP and SCA14 mutant (G118D, V138E and C142S) PKC-RFP. PMA activation induces irreversible translocation of PKC 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 proteins compared with wild-type PKC to the plasma membrane. (C) Translocation analysis of wild-type PKC-GFP, SCA14 mutant PKC-GFP (G118D, V138E and C142S) and VPKC-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.

View larger version (28K): [in this window] [in a new window]   Fig. 4. SCA14 mutant PKC shows increased membrane targeting but does not affect C2 domain functioning. (A) Translocation analysis of HeLa cells cotransfected with wild-type PKC-RFP and wild-type PKC-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 proteins fused to different fluorophores. Furthermore, wild-type PKC-RFP was cotransfected with SCA14 mutant G118D (B), V138E (C) and C142S (D) PKC-GFP. No difference was observed in membrane dissociation between wild-type and SCA14-mutant PKC. Increased translocation amplitudes were observed for SCA14 mutant PKC compared with wild-type PKC. Graphs show the translocation curve of single cells, but represent three independent experiments. (E) Quantification of the amount of PKC translocated to the plasma membrane upon histamine stimulation. Bars represent the ratio between the maximal amplitude of wild-type PKC and the maximal amplitude of SCA14-mutant PKC. 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-GFP and mutant G118D, V138E or C142S PKC-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.

View larger version (40K): [in this window] [in a new window]   Fig. 5. SCA14 mutations located in C1B open PKC 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.

View larger version (36K): [in this window] [in a new window]   Fig. 6. SCA14 mutations in C1B reduce PKC kinase activity in living cells. (A) HeLa cells were transiently transfected with either MyrPalm-CKAR alone or cotransfected with wild-type PKC-RFP or the different SCA14 mutant PKC-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 (G118D, V138E and C142S) when compared with wild-type PKC 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 and SCA14 mutant PKC (unpaired t-test, ***P<0.001). (C) Western blot analysis shows reduced MARCKS phosphorylation in whole cell lysates of SCA14-mutant PKC upon PMA stimulation (400 nM) compared with cells containing wild-type PKC using anti-phospho-MARCKS antibody. Top panel shows equal PKC 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, and related to the value obtained for cells expressing wild-type PKC. Data in B and D represent mean ± s.e.m.

View larger version (46K): [in this window] [in a new window]   Fig. 7. SCA14 mutant PKC reduces ERK2 nuclear accumulation and phosphorylation. (A) Confocal images of HeLa cells transiently transfected with GFP-ERK2 and cotransfected with either wild-type PKC-RFP or PKC G118D-RFP (V138E and C142S mutants confocal data not shown). Activation of PKC 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 when compared with cells that express only GFP-ERK2 or coexpress wild-type PKC-RFP. (C) Bars represent the ratio of nuclear/cytosolic ERK fluorescence in the absence and presence of wild-type PKC and SCA14 mutant PKC (unpaired t-test, **P<0.01; ***P<0.001). (D) Western blot analysis showed that endogenous ERK1 and ERK2 are phosphorylated after PKC activation with 400 nM PMA for 10 minutes. Reduced ERK2 phosphorylation was observed in the presence of SCA14 mutant PKC (G118D, V138E and C142S) compared with cells expressing wild-type PKC. Furthermore, equal ERK and PKC 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 and related to the value obtained for cells expressing wild type PKC. Data in C and E represent mean ± s.e.m.

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