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PKC ε is associated with myosin IIA and actin in fibroblasts
Cellular Signalling 14 (2002) 529 – 536 www.elsevier.com/locate/cellsig
PKC e is associated with myosin IIA and actin in fibroblasts Karen England*, David Ashford, Daniel Kidd, Martin Rumsby Department of Biology, University of York, York YO10 5DD, UK Received 2 September 2001; accepted 13 November 2001
Abstract Proteins coimmunoprecipitating with protein kinase C (PKC) e in fibroblasts were identified through matrix-assisted laser desorption/ ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC e immunoprecipitates, as well as known PKC e binding proteins, actin, b’Cop and cytokeratin. Myosin is not a substrate for PKC e. Immunofluorescence analysis showed that PKC e is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC e – actin – myosin complex in cell spreading. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Actin; PKC; Actomyosin stress fibres; 3T3 cells
1. Introduction The protein kinase C (PKC) family of related phospholipid-dependent serine/threonine kinases are involved in the control of many cellular processes, including cell growth and differentiation [1,2]. To date, there are at least 10 isotypes: the conventional PKCs (a, bI, bII, g) are regulated by calcium and diacylglycerol (DAG), the novel PKCs (d, e, h, q) are calcium independent but dependent upon DAG and the atypical PKCs (l/i, z) are both DAG and calcium independent. Three PRKs form a fourth subgroup [3]. PKC e is the only isotype that has oncogenic potential [4,5], perhaps mediated through interactions with Raf 1 kinase [6,7]. PKC e is also unique in having actin [8,9] and Golgi binding domains [10 – 12]. Regulation of PKC localisation through interaction with isotype-specific binding partners (RACKS) is important in modulating PKC activity [13], and such PKC binding partners have been identified using a number of different approaches [14]. Many PKC binding proteins are localised
Abbreviations: BDM, butanedione monoxime; DAG, diacylglycerol; MALDI TOF m/s, matrix-assisted laser desorption/ionisation time of flight mass spectrometry; PAS, protein A sepharose; PKC, protein kinase C; TBS, Tris-buffered saline * Corresponding author. Department of Biochemistry, University College Cork, Cork, Ireland. Tel.: +353-21-4904128; fax: +353-21-4904259. E-mail address: [email protected] (K. England).
to sites of interaction between membranes and the cytoskeleton, e.g. MARCKS [15]. PKC e associates with b’Cop, actin, cytokeratins and cardiac cell myofibrils [10,15]. Some of these binding proteins are putative PKC substrates (STICKS) although many are not phosphorylated by PKC and so may serve as anchoring proteins or to regulate substrate phosphorylation, thereby allowing integration of PKC into other signaling pathways [13 – 15]. To investigate the role of PKC e in fibroblasts we have used matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s) fingerprinting to identify proteins that coimmunoprecipitate with PKC e. Identification of proteins through matching peptide fingerprints to databases is a reliable way of identifying proteins [16,17]. The use of MALDI TOF m/s fingerprinting to identify PKC-associated proteins has not been previously reported. Here, we describe the identification of myosin IIA in PKC e immunoprecipitates, as well as known PKC e binding proteins. We also suggest a role for myosin II, actin and PKC e in cell spreading after passage.
2. Materials and methods 2.1. Materials Swiss 3T3 cells were from the European Collection of Animal Cultures (Porton Down, UK) and the William Dunn
0898-6568/01/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 8 - 6 5 6 8 ( 0 1 ) 0 0 2 7 7 - 7
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Cell Bank (Oxford, UK). Cell culture plastic was from Gibco BRL (Life Technologies, Paisley, UK). Cell culture reagents were from Gibco BRL (Life Technologies) except for the serum which, was from PAA Laboratories (Linz, Austria). Chemicals were from Sigma (Poole, UK), unless otherwise stated. Nitrocellulose membrane (Hybond C) and [g-32P]-ATP were from Amersham, Little Chalfont, UK. Dried milk powder was Marvel (Premier Beverages, Stafford, UK). BCA reagents were from Pierce (Luton, UK). 2.1.1. Antibodies The polyclonal PKC e antibody used for Western blotting and immunoprecipitations was as previously described [18]. The polyclonal antibody to PKC e for immunofluorescence studies and monoclonal antibodies to b’Cop and actin were from Sigma. The monoclonal pan myosin antibody was from BAbCO (Richmond, CA, USA) and the polyclonal myosin II specific antibody was from Biogenesis (Poole, UK). The myosin IIA and myosin IIB specific antibodies were a gift from Dr. Peckham, Leeds. The antibody directed to the N-terminal sequence of PKC e was from Transduction Laboratories. The cytokeratin antibody was from Santa Cruz (California, USA). Peroxidase- and FITC-conjugated secondary antibodies were from Sigma. The Cy3-conjugated secondary antibody was from Jackson Laboratories (Luton, UK). 2.1.2. Cell culture Swiss 3T3 and 3T6 cells were maintained in DMEM supplemented with 10% foetal calf serum in a humidified incubator at 5% CO2 and were allowed to grow to confluency and quiescence as described previously [19]. Newly passaged cells were allowed to settle for 15 min before harvesting. Cell spreading after passage, in the presence or absence of 1 mM chelerythrine (Calbiochem), 250 nM cytochalasin D (Calbiochem) or 20 mM butanedione monoxime (BDM) (Sigma) was assessed by microscopy using a Nikon Labophot at various time points. 2.1.3. Immunofluorescence Cells were grown on glass coverslips or Permanox multiwell slides (Nunc, Gibco BRL). Immunofluoresence was carried out as described previously [19]. 2.2. Immunoprecipitation Cells were harvested in 500-ml immunoprecipitation buffer (10 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 0.5% SDS, 1% TX-100, 1% deoxycholic acid, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 2 mM AEBSF, 50 mM NaF, 5 mM Na pyrophosphate, 10 mM sodium orthovanadate pH 7.4). Samples were precleared with protein A sepharose (PAS) at 4 C for 1 h on a rotary stirrer. PAS beads were removed by centrifugation in a bench top centrifuge at full speed. Equal amounts of cell protein were incubated with anti-PKC e antibody overnight (1:50), and this was recov-
ered with PAS. After washing three times with immunoprecipitation buffer, the PAS pellet was resuspended in 10% SDS and Laemmli loading buffer and was boiled for 5 min, unless otherwise stated. PAS only minus antibody controls were analysed similarly. 2.2.1. MALDI TOF analysis PKC e was immunoprecipitated as above from quiescent or newly passaged 3T3 and 3T6 cells and immunoprecipitates resolved by SDS – PAGE on 7% or 10% gels. Proteins were visualised with either Silver Staining Plus (BioRad) or Colloidal Coomassie blue for MALDI TOF m/s, and protein bands were excised and digested with 0.1 mg/ml trypsin (Boehringer Manheim, Lewes, UK) according to the method of Rosenfeld et al. [20] and as we have described previously [19]. Mass spectrometry was performed in reflector mode, and multipoint mass calibration was performed. Peptides were analysed using a Voyager-DE STR Biospectrometry Work Station and Voyager and Grams software from Perspective Biosystems. Spectra obtained were matched through ExPasy PeptIdent hhttp://www.expasy.chi and Protein Prospector MS-Fit hhttp://prospector.ucsf.edui software. 2.2.2. Western blotting Samples, usually immunoprecipitates, or 30 mg protein, in Laemmli loading buffer, were resolved by SDS – PAGE, transferred to nitrocellulose and probed for PKC e or potential PKC binding proteins as described previously [19]. 2.2.3. Gel overlay assay PKC e and myosin immunoprecipitates or whole cell lysates were resolved by SDS –PAGE and transferred to nitrocellulose and blocked with 5% milk protein in Trisbuffered saline (TBS) pH 7.4. Blots were incubated with 5 mg/ml PKC e (Calbiochem) in 1% milk protein in TBS and then washed in TBS, fixed in 0.5% formaldehyde and neutralised in 2% glycine. The blots were then washed in TBS and processed for Western blotting with a PKC e antibody as above. Controls not incubated with PKC e protein were also carried out. 2.2.4. PKC activity assay PKC e (2.5 ng; Calbiochem) was incubated with 10 mg histone, actin or chicken gizzard myosin (Sigma) in 0.5 M Tris, 25 mM MgAc, 2.5 mM EDTA, 20 mg phosphatidyl serine and 0.4 mg diolein in a final volume of 200 ml. The reaction was started with the addition of 10 ml 2.5 mM ATP containing 1.86 kBq [g-32P]-ATP and was incubated at 30 C for 30 min. Protein was precipitated by the addition of 1 ml 25%TCA and 100 ml BSA (1 mg/ml) and left on ice for 1 h. Samples were washed onto Whatman GF/B filter circles and rinsed with 15% TCA and 80% ethanol before air drying. Filters were then added to Ultima Gold scintillant and radioactivity was measured using a Packard 1900c TriCarb scintillation counter. Samples were assayed in triplicate. For one set of samples, the reaction was stopped with
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the addition of Laemmli sample buffer before resolving by SDS –PAGE. Reactions were carried out ± PKC inhibitor peptide (RFAEKGSLRQKNV). For immunoprecipitates, the experiment was carried out as above except that immunoprecipitates resuspended in assay buffer were used instead of substrate. The reaction was stopped by the addition of Laemmli sample buffer, resolved by SDS – PAGE and the dried gel exposed to Molecular Dynamics SI Phosphorimager. All data shown is typical of at least three independent experiments.
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3. Results 3.1. Identification of PKC-e-associated proteins by MALDI TOF m/s fingerprinting Proteins that coimmunoprecipitated with PKC e were visualised with Coomassie staining after SDS – PAGE (Fig. 1A). These bands were excised from gels, trypsindigested and subjected to analysis with MALDI TOF m/s. The resulting peptide fingerprints were analysed using ExPasy PeptIdent and Protein Prospector m/s fit software.
Fig. 1. PKC e coimmunoprecipitation experiments. A: (i) Negative control-PAS was added to whole cell lysates. (ii) PKC e was immunoprecipitated from 3T3 cells, and the resulting immunoprecipitate was resolved by SDS – PAGE on a 10% gel. The gel was stained with Colloidal Coomassie blue, and protein bands were excised for MALDI TOF m/s analysis are indicated. Markers in kilodaltons are indicated. B: PKC e was immunoprecipitated from 3T3 cells, and the resulting immunoprecipitate was resolved by SDS – PAGE on a 10% gel. After transfer, nitrocellulose blots were probed with antibodies to myosin II A, b’Cop, cytokeratin and actin as indicated and visualised by ECL. Markers in kilodaltons are indicated. C: Myosin II, actin, b’Cop and cytokeratin were immunoprecipitated from quiescent and newly passaged 3T3 cells. The resulting immunoprecipitates were resolved by SDS – PAGE on a 10% gel. After transfer, nitrocellulose blots were probed with an antibody to PKC e and visualised by ECL. Markers in kilodaltons are indicated. Lane 1, quiescent 3T3 cells, myosin II immunoprecipitate; lane 2, newly passaged 3T3 cells, myosin II immunoprecipitate; lane 3, quiescent 3T3 cells, actin immunoprecipitate; lane 4, newly passaged 3T3 cells, actin immunoprecipitate; lane 5, quiescent 3T3 cells, b’Cop immunoprecipitate; lane 6, newly passaged 3T3 cells, b’Cop immunoprecipitate; lane 7, quiescent 3T3 cells, cytokeratin immunoprecipitate; 8, newly passaged 3T3 cells, cytokeratin immunoprecipitate. D: PKC d and PKC z were immunoprecipitated from 3T3 cells, and the resulting immunoprecipitate was resolved by SDS – PAGE on a 10% gel. After transfer, nitrocellulose blots were probed with an antibody to myosin II and visualised by ECL. Markers in kilodaltons are indicated. E: Myosin was immunoprecipitated from 3T3 cells and resolved by SDS – PAGE and transferred to nitrocellulose. (i) The myosin region was excised and incubated with 5 mg/ml PKC e protein. The blot was then probed for PKC e as before. The PKC e immunoreactive band at 230 kDa is indicated. (ii) Negative control—the equivalent region of blot was not incubated with PKC e prior to probing for PKC e.
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Table 1 MALDI TOF m/s fingerprinting of PKC-e-associated proteins Protein
Best match-Swissprot accession number
MOWSE score
1 2 3 4 5 6
nonmuscle myosin heavy chain A (U31463) b’Cop (P35606) PKC e (P16054) cytokeratin (P50446) IgG heavy chain actin (X13055)
6.13e + 006 4.16e + 003 not done 1.93e + 007 not done 2.45e + 006
Proteins that coimmunoprecipitated with PKC e were analysed by MALDI TOF m/s. The resulting fingerprints were analysed by Protein Prospector and PeptIdent software, both gave similar results. Best matches shown here are from Protein Prospector along with the MOWSE score, a measure of the significance of the match.
A summary of proteins detected and best matches is shown in Table 1. MOWSE scores as a measure of the significance of the match are given (Protein Prospector MS-Fit hhttp:// prospector.ucsf.edui software). This approach identified two
major proteins in the PKC e immunoprecipitates. One protein was identified as actin, a known PKC e binding protein. The other was identified as myosin IIA heavy chain, a nonmuscle myosin. The closest sequence match is to the rat form of myosin IIA as the mouse form is not in the database. Both PeptIdent and Protein Prospector identified myosin IIA as a better match than myosin IIB. MALDI TOF m/s analysis of tryptic peptides also identified peptides of masses that corresponded to peptides that are conserved in myosin IIA between species but differ in myosin IIB. b’Cop and cytokeratin, both known to associate with PKC e, as well as IgG heavy and light chains, were also detected. Immunoprecipitations were also carried out in the presence or absence of detergents and using antibodies to the Nand C-terminal regions of PKC e; similar protein profiles were seen (data not shown). PAS beads in the absence of antibody did not bind any of the same proteins detected in the immunoprecipitates (Fig. 1A). 2D Gel electrophoresis was
Fig. 2. Myosin II and actin are not PKC e substrates. A: Phosphorylation of myosin, actin and histone by PKC e in the presence of DAG and PS, ± PKC inhibitor peptide, was measured as mean DPM per GF/B filter circle ± S.D. (n = 3). B: PKC e immunoprecipitates were incubated with PKC e in the presence of DAG and PS and [32P]ATP. The immunoprecipitates were resolved by SDS – PAGE and phosphate incorporation monitored using the Molecular Dynamics SI Phosphorimager. Incorporation of phosphate into a 95-kDa band (PKC e) is shown).
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also carried out to separate any potential PKC-e-associated proteins, which comigrated with the IgG or other protein bands; no further proteins were detected (data not shown). We detected no differences in the protein profiles of PKC e immunoprecipitates from nuclear and cytoplasmic fractions of quiescent and newly passaged cells (data not shown). The association of PKC e with myosin IIA, actin, cytokeratin and b’Cop was confirmed by analysing PKC e immunoprecipitates by Western blotting with antibodies to actin, myosin IIA, cytokeratin and b’Cop (Fig. 1B). (A myosin IIB specific antibody did not show any immunoreactivity with PKC e immunoprecipitates—data not shown.) The reverse approach showed that myosin II, actin, cytokeratin and b’Cop immunoprecipitates contained PKC e (Fig. 1C). Interestingly, screening with other PKC isoform specific antibodies showed that PKC d and z were also present
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in myosin immunoprecipitates and vice versa (Fig. 1D). However, PKC a, g and bII immunoprecipitates did not contain myosin IIA showing that there is isoform selectivity in this interaction (data not shown). PKC e95, the hyperphosphorylated form of PKC e [19], was the major species coimmunoprecipitating with antibodies to actin, myosin II and b’Cop (Fig. 1C). Protein extraction was performed in the presence and absence of 2 mM ATP to ensure that the association between PKC e and myosin was not an artifact due to myosin – actin interactions caused by low ATP levels [21]; myosin IIA was still associated with PKC e immunoprecipitates in the presence of ATP (data not shown). Blots of PKC e and myosin immunoprecipitates incubated with PKC e showed positive immunoreactivity for PKC e, suggesting that PKC e can bind directly to myosin IIA (Fig. 1E). Gel overlay assays on whole cell lysates also
Fig. 3. Colocalisation of PKC e, myosin and actin in 3T3 cells. A: Immunofluorescence analysis of 3T3 cells labelled with antibodies to myosin II (polyclonal), pan myosin (monoclonal), actin (monoclonal) and PKC e (polyclonal); (i) pan myosin, 70% confluent; (ii) PKC e, 70% confluent; (iii) myosin II, 70% confluent; (iv) actin, 70% confluent; (v) pan myosin, quiescent; (vi) PKC e, quiescent. Scale bar represents 10 mm. B: Immunofluorescence analysis with antibodies to pan myosin and PKC e after 15 min of treatment of 3T3 cells with 250 nM cytochalasin D; (i) pan myosin; (ii) PKC e. Scale bar represents 10 mm.
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showed evidence of PKC e interaction with a 230-kDa protein supporting this; similar assays with BSA showed no PKC e interactions (data not shown). 3.2. Actin and myosin are not substrates for PKC e phosphorylation The ability of PKC e to phosphorylate actin and myosin was determined by measuring the incorporation of [32P] into actin and myosin using a standard PKC activity assay. Chicken gizzard muscle was used as it is closely related to myosin IIA and is commercially available. No incorporation of radiolabel into myosin or actin was detected, but the PKC substrate histone was phosphorylated (Fig. 2A). These findings suggest that PKC e does not phosphorylate myosin heavy chains. To identify PKC e substrates present in PKCe immunoprecipitates assays were carried out as above in the presence of 2.5 ng PKC e to ensure catalytic activity. After resolution by SDS – PAGE, incorporation of [32P] was visualised on a phosphorimager (Fig. 2B). The only [32P] detected was associated with a protein of approximately 97 kDa. This comigrated with PKC e and shows autophosphorylation of PKC e. No [32P] was incorporated into any other protein band. This showed that myosin IIA, actin, b’Cop and cytokeratin are not PKC e substrates. 3.3. Immunofluorescence studies of PKC e, myosin IIA and actin in 3T3 and 3T6 cells Immunofluorescence staining showed that PKC e colocalised with actin and myosin in some but not all 3T3 and 3T6 cells (Fig. 3A). In about 10% of cells, which were very spread out, PKC e was localised to filaments, which also stained positively for actin and myosin II (Fig. 3A). In quiescent cells, both PKC e and myosin were localised to the perinuclear, possibly the Golgi region (Fig. 3A). PKC e has been reported to have a Golgi localisation through interactions with its RACK, b’Cop [10]. As reported, actin microfilaments were not detected in quiescent cells [22]. Cytochalasin D, an actin microfilament disrupting agent, disrupted myosin II and PKC e localisation on filaments (Fig. 3B), suggesting that these filaments are actomyosin stress fibres. 3.4. Treatment of cells with PKC inhibitors blocks cell spreading after passage Passage of cells in the presence of the PKC inhibitor chelerythrine, the microfilament disrupting drug cytochalasin D, or the myosin ATPase inhibiting drug BDM, inhibited cell spreading as visualised by phase contrast microscopy (Fig. 4A). Immunofluorescence analysis of PKC e, actin and myosin II in these conditions showed that all three proteins are normally localised to membrane ruffles at the edge of passaged cells. However, this localisation is disrupted by
Fig. 4. Cell spreading after passage is inhibited by chelerythrine, cytochalasin D and BDM. A: 3T3 cells were passaged and allowed to settle for 4 h. Cells were fixed for immunofluorescence and phase contrast micrographs were taken: (i) medium containing DMSO solvent control; (ii) cells passaged into medium containing 1 mM chelerythrine; (iii) cells passaged into medium containing 250 nM cytochalasin D; (iv) cells passaged into medium containing 20 mM BDM. Scale bar represents 10 mm. B: Immunofluorescence analysis with antibodies to pan myosin, actin and PKC e after 3T3 cells were passaged and allowed to settle for 4 h: (i) pan myosin, solvent control; (ii) PKC e, solvent control; (iii) actin, solvent control; (iv) pan myosin, cells passaged into 1 mM chelerythrine; (v) PKC e, cells passaged into 1 mM chelerythrine; (vi) actin, cells passaged into 1 mM chelerythrine. Scale bar represents 10 mm.
treatment with chelerythrine, cytochalasin D and BDM (Fig. 4B, cytochalasin D and BDM data not shown).
4. Discussion We present evidence here for the association of PKC e with actomyosin stress fibers in fibroblasts. These fibers are important in cell adhesion in nonmuscle cells. Both PKC and myosin II have been implicated in the regulation of postmitotic cell spreading [23 – 28]. We have previously shown that as cells are passaged, PKC e changes from a 95-kDa form to an 87-kDa form due to the loss of a phosphate at Ser729 in the hydrophobic region [19]. This is associated with a change in the localisation of PKC e from the perinuclear region to the cytoplasm [19]. Activation of PKC is associated with its subsequent dephosphorylation,
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and it is therefore likely that this dephosphorylation represents activation of PKC e upon passage [29,30]. Our results show that actin and myosin are predominantly associated with PKC e95, the hyperphosphorylated form of the protein. The apparent activation of PKC e upon cell passage that we have previously reported [19] may implicate PKC e in cell spreading after passage, especially since PKC e has been shown to be important in HeLa cell spreading onto gelatin through interactions with actin [23,24]. Myosin II has also been implicated in cell spreading [26 – 28]. Our results show that if cells are passaged in the presence of inhibitors of myosin ATPase or PKC or actin filament disrupting agents, cell spreading is inhibited, confirming a need for myosin, PKC and actin in the spreading process. Our identification of myosin II A in PKC e immunoprecipitates and vice versa, as well as their colocalisation, suggests that both proteins are associated within the cell. This may be a direct association, as suggested by the gel overlay assay, but the possibility that this interaction is mediated through actin cannot be ruled out. The results described here demonstrate that MALDI TOF m/s is a quick, reliable and convenient method to identify PKC binding proteins. Using this approach with coimmunoprecipitation and immunofluorescence, we report for the first time an association between PKC e and myosin II A heavy chains. Our approach also confirmed that PKC e is associated with actin, cytokeratin and b’Cop [8,9,12,14,15]. Our results are in agreement with other reports demonstrating the importance of interactions between PKC isotypes and the cytoskeleton [14,15]. Database screening has shown that myosin heavy chains contain PKC phosphorylation motifs, and there is some evidence in the literature for a role for PKC in the phosphorylation of myosin light and heavy chains [31 – 33]. However, our results indicate that PKC e does not phosphorylate myosin IIA showing that this isoform is not directly involved in the regulation of myosin function in fibroblasts by phosphorylation. It is, however, possible that PKC e can modulate myosin activity noncatalytically or that myosin may regulate PKC e activity, either through modulation of its catalytic activity or through determining substrate interactions. Other reports have indicated an association between PKC e and myosin. For example, it has been shown that arachidonic acid treatment of cardiac cells causes PKC e translocation to the Z-line where actin filaments are anchored near myofilaments [13,34 – 36]. It has been shown in vitro that interaction between PKC e and actin activates PKC e [8,9]. This site of interaction on PKC e has been mapped to amino acids 223– 228, and when this site is deleted, PKC e loses its actin binding ability and its association with adhesion sites [8,9]. Both PKC e and myosin II colocalise with actin filaments in spreading fibroblasts; these probably represent actomyosin stress fibres. Treatment of cells with cytochalasin D destroys both PKC e and myosin II localisation with these filaments,
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suggesting that actin filaments are important for localisation of both proteins. We also identified b’Cop, the PKC e RACK as a PKC-eassociated protein in 3T3 and 3T6 cells. b’Cop is a Golgiassociated protein, and PKC e has been reported to have a role in Golgi function through interactions with b’Cop [12]. Our immunofluorescence results demonstrate that in quiescent cells, PKC e and myosin II are localised to the perinuclear region; we have previously reported that this perinuclear staining colocalises with Golgi staining [19]. It has previously been reported that both actin and nonmuscle myosin II are associated with the Golgi and play an as yet undefined role in vesicular trafficking [37,38]. It is feasible that PKC e, actin and nonmuscle myosin IIA together coordinate some regulatory aspect of Golgi function or vesicle transport. We propose that in 3T3 and 3T6 fibroblasts, PKC e is part of an actin – myosin complex that functions in the control of cell spreading and adhesion after passage. The role of PKC e in such a complex requires further investigation and will no doubt be elucidated in part by analysis of PKC binding proteins and ligands in a variety of cell types.
Acknowledgments KE was funded by the BBSRC Integration of Cellular Responses Initiative. Peter Ashton and Rachael Curwen are thanked for their help with 2D gel electrophoresis and MALDI TOF m/s.
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PKC e is associated with myosin IIA and actin in fibroblasts Karen England*, David Ashford, Daniel Kidd, Martin Rumsby Department of Biology, University of York, York YO10 5DD, UK Received 2 September 2001; accepted 13 November 2001
Abstract Proteins coimmunoprecipitating with protein kinase C (PKC) e in fibroblasts were identified through matrix-assisted laser desorption/ ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC e immunoprecipitates, as well as known PKC e binding proteins, actin, b’Cop and cytokeratin. Myosin is not a substrate for PKC e. Immunofluorescence analysis showed that PKC e is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC e – actin – myosin complex in cell spreading. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Actin; PKC; Actomyosin stress fibres; 3T3 cells
1. Introduction The protein kinase C (PKC) family of related phospholipid-dependent serine/threonine kinases are involved in the control of many cellular processes, including cell growth and differentiation [1,2]. To date, there are at least 10 isotypes: the conventional PKCs (a, bI, bII, g) are regulated by calcium and diacylglycerol (DAG), the novel PKCs (d, e, h, q) are calcium independent but dependent upon DAG and the atypical PKCs (l/i, z) are both DAG and calcium independent. Three PRKs form a fourth subgroup [3]. PKC e is the only isotype that has oncogenic potential [4,5], perhaps mediated through interactions with Raf 1 kinase [6,7]. PKC e is also unique in having actin [8,9] and Golgi binding domains [10 – 12]. Regulation of PKC localisation through interaction with isotype-specific binding partners (RACKS) is important in modulating PKC activity [13], and such PKC binding partners have been identified using a number of different approaches [14]. Many PKC binding proteins are localised
Abbreviations: BDM, butanedione monoxime; DAG, diacylglycerol; MALDI TOF m/s, matrix-assisted laser desorption/ionisation time of flight mass spectrometry; PAS, protein A sepharose; PKC, protein kinase C; TBS, Tris-buffered saline * Corresponding author. Department of Biochemistry, University College Cork, Cork, Ireland. Tel.: +353-21-4904128; fax: +353-21-4904259. E-mail address: [email protected] (K. England).
to sites of interaction between membranes and the cytoskeleton, e.g. MARCKS [15]. PKC e associates with b’Cop, actin, cytokeratins and cardiac cell myofibrils [10,15]. Some of these binding proteins are putative PKC substrates (STICKS) although many are not phosphorylated by PKC and so may serve as anchoring proteins or to regulate substrate phosphorylation, thereby allowing integration of PKC into other signaling pathways [13 – 15]. To investigate the role of PKC e in fibroblasts we have used matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s) fingerprinting to identify proteins that coimmunoprecipitate with PKC e. Identification of proteins through matching peptide fingerprints to databases is a reliable way of identifying proteins [16,17]. The use of MALDI TOF m/s fingerprinting to identify PKC-associated proteins has not been previously reported. Here, we describe the identification of myosin IIA in PKC e immunoprecipitates, as well as known PKC e binding proteins. We also suggest a role for myosin II, actin and PKC e in cell spreading after passage.
2. Materials and methods 2.1. Materials Swiss 3T3 cells were from the European Collection of Animal Cultures (Porton Down, UK) and the William Dunn
0898-6568/01/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 8 - 6 5 6 8 ( 0 1 ) 0 0 2 7 7 - 7
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Cell Bank (Oxford, UK). Cell culture plastic was from Gibco BRL (Life Technologies, Paisley, UK). Cell culture reagents were from Gibco BRL (Life Technologies) except for the serum which, was from PAA Laboratories (Linz, Austria). Chemicals were from Sigma (Poole, UK), unless otherwise stated. Nitrocellulose membrane (Hybond C) and [g-32P]-ATP were from Amersham, Little Chalfont, UK. Dried milk powder was Marvel (Premier Beverages, Stafford, UK). BCA reagents were from Pierce (Luton, UK). 2.1.1. Antibodies The polyclonal PKC e antibody used for Western blotting and immunoprecipitations was as previously described [18]. The polyclonal antibody to PKC e for immunofluorescence studies and monoclonal antibodies to b’Cop and actin were from Sigma. The monoclonal pan myosin antibody was from BAbCO (Richmond, CA, USA) and the polyclonal myosin II specific antibody was from Biogenesis (Poole, UK). The myosin IIA and myosin IIB specific antibodies were a gift from Dr. Peckham, Leeds. The antibody directed to the N-terminal sequence of PKC e was from Transduction Laboratories. The cytokeratin antibody was from Santa Cruz (California, USA). Peroxidase- and FITC-conjugated secondary antibodies were from Sigma. The Cy3-conjugated secondary antibody was from Jackson Laboratories (Luton, UK). 2.1.2. Cell culture Swiss 3T3 and 3T6 cells were maintained in DMEM supplemented with 10% foetal calf serum in a humidified incubator at 5% CO2 and were allowed to grow to confluency and quiescence as described previously [19]. Newly passaged cells were allowed to settle for 15 min before harvesting. Cell spreading after passage, in the presence or absence of 1 mM chelerythrine (Calbiochem), 250 nM cytochalasin D (Calbiochem) or 20 mM butanedione monoxime (BDM) (Sigma) was assessed by microscopy using a Nikon Labophot at various time points. 2.1.3. Immunofluorescence Cells were grown on glass coverslips or Permanox multiwell slides (Nunc, Gibco BRL). Immunofluoresence was carried out as described previously [19]. 2.2. Immunoprecipitation Cells were harvested in 500-ml immunoprecipitation buffer (10 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 0.5% SDS, 1% TX-100, 1% deoxycholic acid, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 2 mM AEBSF, 50 mM NaF, 5 mM Na pyrophosphate, 10 mM sodium orthovanadate pH 7.4). Samples were precleared with protein A sepharose (PAS) at 4 C for 1 h on a rotary stirrer. PAS beads were removed by centrifugation in a bench top centrifuge at full speed. Equal amounts of cell protein were incubated with anti-PKC e antibody overnight (1:50), and this was recov-
ered with PAS. After washing three times with immunoprecipitation buffer, the PAS pellet was resuspended in 10% SDS and Laemmli loading buffer and was boiled for 5 min, unless otherwise stated. PAS only minus antibody controls were analysed similarly. 2.2.1. MALDI TOF analysis PKC e was immunoprecipitated as above from quiescent or newly passaged 3T3 and 3T6 cells and immunoprecipitates resolved by SDS – PAGE on 7% or 10% gels. Proteins were visualised with either Silver Staining Plus (BioRad) or Colloidal Coomassie blue for MALDI TOF m/s, and protein bands were excised and digested with 0.1 mg/ml trypsin (Boehringer Manheim, Lewes, UK) according to the method of Rosenfeld et al. [20] and as we have described previously [19]. Mass spectrometry was performed in reflector mode, and multipoint mass calibration was performed. Peptides were analysed using a Voyager-DE STR Biospectrometry Work Station and Voyager and Grams software from Perspective Biosystems. Spectra obtained were matched through ExPasy PeptIdent hhttp://www.expasy.chi and Protein Prospector MS-Fit hhttp://prospector.ucsf.edui software. 2.2.2. Western blotting Samples, usually immunoprecipitates, or 30 mg protein, in Laemmli loading buffer, were resolved by SDS – PAGE, transferred to nitrocellulose and probed for PKC e or potential PKC binding proteins as described previously [19]. 2.2.3. Gel overlay assay PKC e and myosin immunoprecipitates or whole cell lysates were resolved by SDS –PAGE and transferred to nitrocellulose and blocked with 5% milk protein in Trisbuffered saline (TBS) pH 7.4. Blots were incubated with 5 mg/ml PKC e (Calbiochem) in 1% milk protein in TBS and then washed in TBS, fixed in 0.5% formaldehyde and neutralised in 2% glycine. The blots were then washed in TBS and processed for Western blotting with a PKC e antibody as above. Controls not incubated with PKC e protein were also carried out. 2.2.4. PKC activity assay PKC e (2.5 ng; Calbiochem) was incubated with 10 mg histone, actin or chicken gizzard myosin (Sigma) in 0.5 M Tris, 25 mM MgAc, 2.5 mM EDTA, 20 mg phosphatidyl serine and 0.4 mg diolein in a final volume of 200 ml. The reaction was started with the addition of 10 ml 2.5 mM ATP containing 1.86 kBq [g-32P]-ATP and was incubated at 30 C for 30 min. Protein was precipitated by the addition of 1 ml 25%TCA and 100 ml BSA (1 mg/ml) and left on ice for 1 h. Samples were washed onto Whatman GF/B filter circles and rinsed with 15% TCA and 80% ethanol before air drying. Filters were then added to Ultima Gold scintillant and radioactivity was measured using a Packard 1900c TriCarb scintillation counter. Samples were assayed in triplicate. For one set of samples, the reaction was stopped with
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the addition of Laemmli sample buffer before resolving by SDS –PAGE. Reactions were carried out ± PKC inhibitor peptide (RFAEKGSLRQKNV). For immunoprecipitates, the experiment was carried out as above except that immunoprecipitates resuspended in assay buffer were used instead of substrate. The reaction was stopped by the addition of Laemmli sample buffer, resolved by SDS – PAGE and the dried gel exposed to Molecular Dynamics SI Phosphorimager. All data shown is typical of at least three independent experiments.
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3. Results 3.1. Identification of PKC-e-associated proteins by MALDI TOF m/s fingerprinting Proteins that coimmunoprecipitated with PKC e were visualised with Coomassie staining after SDS – PAGE (Fig. 1A). These bands were excised from gels, trypsindigested and subjected to analysis with MALDI TOF m/s. The resulting peptide fingerprints were analysed using ExPasy PeptIdent and Protein Prospector m/s fit software.
Fig. 1. PKC e coimmunoprecipitation experiments. A: (i) Negative control-PAS was added to whole cell lysates. (ii) PKC e was immunoprecipitated from 3T3 cells, and the resulting immunoprecipitate was resolved by SDS – PAGE on a 10% gel. The gel was stained with Colloidal Coomassie blue, and protein bands were excised for MALDI TOF m/s analysis are indicated. Markers in kilodaltons are indicated. B: PKC e was immunoprecipitated from 3T3 cells, and the resulting immunoprecipitate was resolved by SDS – PAGE on a 10% gel. After transfer, nitrocellulose blots were probed with antibodies to myosin II A, b’Cop, cytokeratin and actin as indicated and visualised by ECL. Markers in kilodaltons are indicated. C: Myosin II, actin, b’Cop and cytokeratin were immunoprecipitated from quiescent and newly passaged 3T3 cells. The resulting immunoprecipitates were resolved by SDS – PAGE on a 10% gel. After transfer, nitrocellulose blots were probed with an antibody to PKC e and visualised by ECL. Markers in kilodaltons are indicated. Lane 1, quiescent 3T3 cells, myosin II immunoprecipitate; lane 2, newly passaged 3T3 cells, myosin II immunoprecipitate; lane 3, quiescent 3T3 cells, actin immunoprecipitate; lane 4, newly passaged 3T3 cells, actin immunoprecipitate; lane 5, quiescent 3T3 cells, b’Cop immunoprecipitate; lane 6, newly passaged 3T3 cells, b’Cop immunoprecipitate; lane 7, quiescent 3T3 cells, cytokeratin immunoprecipitate; 8, newly passaged 3T3 cells, cytokeratin immunoprecipitate. D: PKC d and PKC z were immunoprecipitated from 3T3 cells, and the resulting immunoprecipitate was resolved by SDS – PAGE on a 10% gel. After transfer, nitrocellulose blots were probed with an antibody to myosin II and visualised by ECL. Markers in kilodaltons are indicated. E: Myosin was immunoprecipitated from 3T3 cells and resolved by SDS – PAGE and transferred to nitrocellulose. (i) The myosin region was excised and incubated with 5 mg/ml PKC e protein. The blot was then probed for PKC e as before. The PKC e immunoreactive band at 230 kDa is indicated. (ii) Negative control—the equivalent region of blot was not incubated with PKC e prior to probing for PKC e.
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Table 1 MALDI TOF m/s fingerprinting of PKC-e-associated proteins Protein
Best match-Swissprot accession number
MOWSE score
1 2 3 4 5 6
nonmuscle myosin heavy chain A (U31463) b’Cop (P35606) PKC e (P16054) cytokeratin (P50446) IgG heavy chain actin (X13055)
6.13e + 006 4.16e + 003 not done 1.93e + 007 not done 2.45e + 006
Proteins that coimmunoprecipitated with PKC e were analysed by MALDI TOF m/s. The resulting fingerprints were analysed by Protein Prospector and PeptIdent software, both gave similar results. Best matches shown here are from Protein Prospector along with the MOWSE score, a measure of the significance of the match.
A summary of proteins detected and best matches is shown in Table 1. MOWSE scores as a measure of the significance of the match are given (Protein Prospector MS-Fit hhttp:// prospector.ucsf.edui software). This approach identified two
major proteins in the PKC e immunoprecipitates. One protein was identified as actin, a known PKC e binding protein. The other was identified as myosin IIA heavy chain, a nonmuscle myosin. The closest sequence match is to the rat form of myosin IIA as the mouse form is not in the database. Both PeptIdent and Protein Prospector identified myosin IIA as a better match than myosin IIB. MALDI TOF m/s analysis of tryptic peptides also identified peptides of masses that corresponded to peptides that are conserved in myosin IIA between species but differ in myosin IIB. b’Cop and cytokeratin, both known to associate with PKC e, as well as IgG heavy and light chains, were also detected. Immunoprecipitations were also carried out in the presence or absence of detergents and using antibodies to the Nand C-terminal regions of PKC e; similar protein profiles were seen (data not shown). PAS beads in the absence of antibody did not bind any of the same proteins detected in the immunoprecipitates (Fig. 1A). 2D Gel electrophoresis was
Fig. 2. Myosin II and actin are not PKC e substrates. A: Phosphorylation of myosin, actin and histone by PKC e in the presence of DAG and PS, ± PKC inhibitor peptide, was measured as mean DPM per GF/B filter circle ± S.D. (n = 3). B: PKC e immunoprecipitates were incubated with PKC e in the presence of DAG and PS and [32P]ATP. The immunoprecipitates were resolved by SDS – PAGE and phosphate incorporation monitored using the Molecular Dynamics SI Phosphorimager. Incorporation of phosphate into a 95-kDa band (PKC e) is shown).
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also carried out to separate any potential PKC-e-associated proteins, which comigrated with the IgG or other protein bands; no further proteins were detected (data not shown). We detected no differences in the protein profiles of PKC e immunoprecipitates from nuclear and cytoplasmic fractions of quiescent and newly passaged cells (data not shown). The association of PKC e with myosin IIA, actin, cytokeratin and b’Cop was confirmed by analysing PKC e immunoprecipitates by Western blotting with antibodies to actin, myosin IIA, cytokeratin and b’Cop (Fig. 1B). (A myosin IIB specific antibody did not show any immunoreactivity with PKC e immunoprecipitates—data not shown.) The reverse approach showed that myosin II, actin, cytokeratin and b’Cop immunoprecipitates contained PKC e (Fig. 1C). Interestingly, screening with other PKC isoform specific antibodies showed that PKC d and z were also present
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in myosin immunoprecipitates and vice versa (Fig. 1D). However, PKC a, g and bII immunoprecipitates did not contain myosin IIA showing that there is isoform selectivity in this interaction (data not shown). PKC e95, the hyperphosphorylated form of PKC e [19], was the major species coimmunoprecipitating with antibodies to actin, myosin II and b’Cop (Fig. 1C). Protein extraction was performed in the presence and absence of 2 mM ATP to ensure that the association between PKC e and myosin was not an artifact due to myosin – actin interactions caused by low ATP levels [21]; myosin IIA was still associated with PKC e immunoprecipitates in the presence of ATP (data not shown). Blots of PKC e and myosin immunoprecipitates incubated with PKC e showed positive immunoreactivity for PKC e, suggesting that PKC e can bind directly to myosin IIA (Fig. 1E). Gel overlay assays on whole cell lysates also
Fig. 3. Colocalisation of PKC e, myosin and actin in 3T3 cells. A: Immunofluorescence analysis of 3T3 cells labelled with antibodies to myosin II (polyclonal), pan myosin (monoclonal), actin (monoclonal) and PKC e (polyclonal); (i) pan myosin, 70% confluent; (ii) PKC e, 70% confluent; (iii) myosin II, 70% confluent; (iv) actin, 70% confluent; (v) pan myosin, quiescent; (vi) PKC e, quiescent. Scale bar represents 10 mm. B: Immunofluorescence analysis with antibodies to pan myosin and PKC e after 15 min of treatment of 3T3 cells with 250 nM cytochalasin D; (i) pan myosin; (ii) PKC e. Scale bar represents 10 mm.
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showed evidence of PKC e interaction with a 230-kDa protein supporting this; similar assays with BSA showed no PKC e interactions (data not shown). 3.2. Actin and myosin are not substrates for PKC e phosphorylation The ability of PKC e to phosphorylate actin and myosin was determined by measuring the incorporation of [32P] into actin and myosin using a standard PKC activity assay. Chicken gizzard muscle was used as it is closely related to myosin IIA and is commercially available. No incorporation of radiolabel into myosin or actin was detected, but the PKC substrate histone was phosphorylated (Fig. 2A). These findings suggest that PKC e does not phosphorylate myosin heavy chains. To identify PKC e substrates present in PKCe immunoprecipitates assays were carried out as above in the presence of 2.5 ng PKC e to ensure catalytic activity. After resolution by SDS – PAGE, incorporation of [32P] was visualised on a phosphorimager (Fig. 2B). The only [32P] detected was associated with a protein of approximately 97 kDa. This comigrated with PKC e and shows autophosphorylation of PKC e. No [32P] was incorporated into any other protein band. This showed that myosin IIA, actin, b’Cop and cytokeratin are not PKC e substrates. 3.3. Immunofluorescence studies of PKC e, myosin IIA and actin in 3T3 and 3T6 cells Immunofluorescence staining showed that PKC e colocalised with actin and myosin in some but not all 3T3 and 3T6 cells (Fig. 3A). In about 10% of cells, which were very spread out, PKC e was localised to filaments, which also stained positively for actin and myosin II (Fig. 3A). In quiescent cells, both PKC e and myosin were localised to the perinuclear, possibly the Golgi region (Fig. 3A). PKC e has been reported to have a Golgi localisation through interactions with its RACK, b’Cop [10]. As reported, actin microfilaments were not detected in quiescent cells [22]. Cytochalasin D, an actin microfilament disrupting agent, disrupted myosin II and PKC e localisation on filaments (Fig. 3B), suggesting that these filaments are actomyosin stress fibres. 3.4. Treatment of cells with PKC inhibitors blocks cell spreading after passage Passage of cells in the presence of the PKC inhibitor chelerythrine, the microfilament disrupting drug cytochalasin D, or the myosin ATPase inhibiting drug BDM, inhibited cell spreading as visualised by phase contrast microscopy (Fig. 4A). Immunofluorescence analysis of PKC e, actin and myosin II in these conditions showed that all three proteins are normally localised to membrane ruffles at the edge of passaged cells. However, this localisation is disrupted by
Fig. 4. Cell spreading after passage is inhibited by chelerythrine, cytochalasin D and BDM. A: 3T3 cells were passaged and allowed to settle for 4 h. Cells were fixed for immunofluorescence and phase contrast micrographs were taken: (i) medium containing DMSO solvent control; (ii) cells passaged into medium containing 1 mM chelerythrine; (iii) cells passaged into medium containing 250 nM cytochalasin D; (iv) cells passaged into medium containing 20 mM BDM. Scale bar represents 10 mm. B: Immunofluorescence analysis with antibodies to pan myosin, actin and PKC e after 3T3 cells were passaged and allowed to settle for 4 h: (i) pan myosin, solvent control; (ii) PKC e, solvent control; (iii) actin, solvent control; (iv) pan myosin, cells passaged into 1 mM chelerythrine; (v) PKC e, cells passaged into 1 mM chelerythrine; (vi) actin, cells passaged into 1 mM chelerythrine. Scale bar represents 10 mm.
treatment with chelerythrine, cytochalasin D and BDM (Fig. 4B, cytochalasin D and BDM data not shown).
4. Discussion We present evidence here for the association of PKC e with actomyosin stress fibers in fibroblasts. These fibers are important in cell adhesion in nonmuscle cells. Both PKC and myosin II have been implicated in the regulation of postmitotic cell spreading [23 – 28]. We have previously shown that as cells are passaged, PKC e changes from a 95-kDa form to an 87-kDa form due to the loss of a phosphate at Ser729 in the hydrophobic region [19]. This is associated with a change in the localisation of PKC e from the perinuclear region to the cytoplasm [19]. Activation of PKC is associated with its subsequent dephosphorylation,
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and it is therefore likely that this dephosphorylation represents activation of PKC e upon passage [29,30]. Our results show that actin and myosin are predominantly associated with PKC e95, the hyperphosphorylated form of the protein. The apparent activation of PKC e upon cell passage that we have previously reported [19] may implicate PKC e in cell spreading after passage, especially since PKC e has been shown to be important in HeLa cell spreading onto gelatin through interactions with actin [23,24]. Myosin II has also been implicated in cell spreading [26 – 28]. Our results show that if cells are passaged in the presence of inhibitors of myosin ATPase or PKC or actin filament disrupting agents, cell spreading is inhibited, confirming a need for myosin, PKC and actin in the spreading process. Our identification of myosin II A in PKC e immunoprecipitates and vice versa, as well as their colocalisation, suggests that both proteins are associated within the cell. This may be a direct association, as suggested by the gel overlay assay, but the possibility that this interaction is mediated through actin cannot be ruled out. The results described here demonstrate that MALDI TOF m/s is a quick, reliable and convenient method to identify PKC binding proteins. Using this approach with coimmunoprecipitation and immunofluorescence, we report for the first time an association between PKC e and myosin II A heavy chains. Our approach also confirmed that PKC e is associated with actin, cytokeratin and b’Cop [8,9,12,14,15]. Our results are in agreement with other reports demonstrating the importance of interactions between PKC isotypes and the cytoskeleton [14,15]. Database screening has shown that myosin heavy chains contain PKC phosphorylation motifs, and there is some evidence in the literature for a role for PKC in the phosphorylation of myosin light and heavy chains [31 – 33]. However, our results indicate that PKC e does not phosphorylate myosin IIA showing that this isoform is not directly involved in the regulation of myosin function in fibroblasts by phosphorylation. It is, however, possible that PKC e can modulate myosin activity noncatalytically or that myosin may regulate PKC e activity, either through modulation of its catalytic activity or through determining substrate interactions. Other reports have indicated an association between PKC e and myosin. For example, it has been shown that arachidonic acid treatment of cardiac cells causes PKC e translocation to the Z-line where actin filaments are anchored near myofilaments [13,34 – 36]. It has been shown in vitro that interaction between PKC e and actin activates PKC e [8,9]. This site of interaction on PKC e has been mapped to amino acids 223– 228, and when this site is deleted, PKC e loses its actin binding ability and its association with adhesion sites [8,9]. Both PKC e and myosin II colocalise with actin filaments in spreading fibroblasts; these probably represent actomyosin stress fibres. Treatment of cells with cytochalasin D destroys both PKC e and myosin II localisation with these filaments,
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suggesting that actin filaments are important for localisation of both proteins. We also identified b’Cop, the PKC e RACK as a PKC-eassociated protein in 3T3 and 3T6 cells. b’Cop is a Golgiassociated protein, and PKC e has been reported to have a role in Golgi function through interactions with b’Cop [12]. Our immunofluorescence results demonstrate that in quiescent cells, PKC e and myosin II are localised to the perinuclear region; we have previously reported that this perinuclear staining colocalises with Golgi staining [19]. It has previously been reported that both actin and nonmuscle myosin II are associated with the Golgi and play an as yet undefined role in vesicular trafficking [37,38]. It is feasible that PKC e, actin and nonmuscle myosin IIA together coordinate some regulatory aspect of Golgi function or vesicle transport. We propose that in 3T3 and 3T6 fibroblasts, PKC e is part of an actin – myosin complex that functions in the control of cell spreading and adhesion after passage. The role of PKC e in such a complex requires further investigation and will no doubt be elucidated in part by analysis of PKC binding proteins and ligands in a variety of cell types.
Acknowledgments KE was funded by the BBSRC Integration of Cellular Responses Initiative. Peter Ashton and Rachael Curwen are thanked for their help with 2D gel electrophoresis and MALDI TOF m/s.
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