Cell Motility and the Cytoskeleton 22:250-256 (1992)

Association of the p lsoform of Protein Kinase C With Vimentin Filaments Annamma Spudich, Tobias Meyer, and Lubert Stryer Department of Cell Biology, Sherman Fairchild Center, Stanford University School of Medicine, Stanford, California Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The p isoform of PKC is translocated and degraded much more rapidly than the a isoform in phorbolester-stimulated rat basophilic leukemia (RBL)cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC a and p are strikingly different in antigen-activated RBL cells. PKC p associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC a concentrates in regions of the cell periphery. This distribution of PKC p is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC p and to the intermediate filament protein vimentin are almost identical, indicating that PKC p associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC p with its substrates and thus confer specificity to the actions of this isoform. 0 1992 Wiley-Liss, Inc. Key words: cytoskeletal localization, signal transduction, intermediatefilaments, rat basophilic leukemia cells. translocation

INTRODUCTION Antigen-induced cross-linking of IgE receptors on RBL cells leads to histamine secretion (Metzger, 1986) and dramatic shape changes (Oliver et al., 1990). The morphological changes are expressed by cell spreading and by the development of deep ruffles at the cell surface. The underlying cytoskeletal reorganizations include an increase in polymerized actin (Oliver et al., 1993) and restructuring of microtubules and intermediate filaments (Sahara et al., 1990). Ruffling and shape changes are concomitant with secretion and occur within minutes after activation. These events are likely to be mediated by an increase of cytosolic calcium level and by stimulation of PKC, as indicated by the finding that secretion requires a combination of phorbol ester and calcium ionophore (Beaven et al., 1987). While calcium ionophore alone is insufficient to induce extensive 0 1992 Wiley-Liss, Inc.

changes in cell morphology and ruffling (Pfeiffer et al., 1985), phorbolesters can cause dramatic cell spreading and a restructuring of the cytoskeleton in RBL cells (A. Spudich, unpublished observations). It is therefore conceivable that the phosphorylation of cytoskeletal elements by PKC is a necessary step for antigen mediated shape changes and secretion. Cytoskeletal proteins are among the many substrates shown to be phosphorylated by PKC. For examReceived November 25, 1991; accepted February 12, 1992. Address reprint requests to Dr. Annamma Spudich at her present address at the Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305. Dr. Meyer’s present address is the Department of Cell Biology, Duke University Medical Center, Durham, NC 27710.

PKC Localization

ple, PKC phosphorylates the heavy and light chains of myosin in RBL-cells (Ludowyke et al., 1989) and myosin light chains in isolated cytoskeletons of Y- 1 adrenal cells (Papadopoulos and Hall, 1989). Purified vimentin intermediate filaments are also phosphorylated in vitro by this enzyme (Inagaki et al., 1988). It has been suggested that substrate specificity of PKC isoforms in vivo could be conferred by different subcellular localization of the isoforms (Mochly-Rosen et al., 1990). This hypothesis is consistent with the findings that PKC a colocalizes with focal contacts at the cell periphery (Jaken et al., 1989), and an as yet unidentified isoform of PKC colocalizes with actin filaments in myofibrils (Mochly-Rosen et al., 1990). Here we show that the a and p isoforms of PKC become differentially localized following antigen activation of RBL cells. While the a isoform of PKC is localized at ruffling regions of activated cells, the p isoform shows the same pattern of localization as vimentin intermediate filaments. MATERIALS AND METHODS Cell Culturing and Activation

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GIBCO-BRL), was characterized previously by MochlyRosen and coworkers (1990). Anti-peptide polyclonal antibody specific to the a isoform of PKC (Hocevar and Fields, 1991) was provided to us by Dr. Alan Fields, Case Western University. Anti-vimentin antibody (made against purified BHK cell vimentin) was a gift of Dr. Robert Goldman (Dept. of Cell Biology, Northwestern University). Secondary antibodies were purchased from Organon Technike-Cappell. Activated and unactivated cells were fixed and stained at specific times using the identical protocols. The cells were photographed using phase or epifluorescence illumination and a Zeiss 63 X objective. Preparations of Detergent Insoluble Cytoskeletons

Activated and unactivated cells were fixed and extracted simultaneously in 2% formaldehyde in Buffer A containing 5 mM EGTA, 5 mM MgCl,, 0.5 mM PMSF and 0.5% Triton X-100 for 5 minutes followed by an additional 5 min fixation in Buffer A containing 2% formaldehyde, 5 mM EGTA, 5 mM MgCl, and 0.5 mM PMSF. The extracted and fixed cells were then washed three times with PBS and stained with the antibody to PKC p using the protocol described above.

RBL cells were maintained as monolayer cultures as described by Ludowyke et al. (1989). Cells were harvested by trypsin treatment, diluted to 5 x lo5 cells/ml and sensitized with 0.5 p,g/ml anti-DNP IgE (Sigma). Transmission Electron Microscopy Aliquotes (75 pl) of the sensitized cells were grown on Cells were cultured on coverslips and activated glass coverslips overnight. For activation, cells were with antigen as described above. At 5 minutes after acwashed with buffer A [135 mM NaC1,5 mM KCI, 2 mM tivation, cells were fixed with 2% glutaraldehyde in MgCl,, 1.8 mM CaCl,, 5 mM glucose and 20 mM buffer A and postfixed with 1% aqueous OsO,. FollowHepes-NaOH(pH 7.4)] and activated at 37°C with 100 ing standard protocols cells were washed with distilled ng/ml antigen (DNP-BSA, Sigma) diluted in buffer A water, dehydrated through an alcohol series and embewith 0.1 mg/ml BSA. ded in Epon. Sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and viewed lmmunocytochemistry with a Philips 200 electron microscope. Activated and unactivated cells were rinsed free of medium and fixed in 2% formaldehyde in buffer A with RESULTS 5 mM EGTA, 5 mM MgCI, and 0.5 mM PMSF for 10 min at 22°C. Cells were then rinsed with 0.1 M glycine Figure la,b shows immunofluorescence images of in phosphate-buffered saline (PBS) to quench nonspe- unstimulated and antigen-stimulated RBL cells stained cific fluorescence and permeabilized for 30 seconds in with anti-PKC p antibody (CK 1.3, Mochly-Rosen et cold acetone (-10°C). After three washes in PBS at a1., 1990) and rhodamine labeled secondary antibody. In 22"C, cells were stained with antibodies for 12 hours at most unactivated cells (50-85%), PKC p staining was 4°C in PBS in the presence of 0.1 % BSA and 0.2% essentially uniform (Fig. la). Detergent-extracted unacsaponin (P/B/S). The unbound primary antibody was re- tivated cells showed little staining (data not shown). The moved by three washes in PBS at 22°C and the cells were numbers as well as the intensity of linear PKC structures stained for 1-3 hours at 22°C with rhodamine-labelled increased after activation in more than 80% of the cells secondary antibody which was previously adsorbed with (Fig. lb). Linear staining appeared within 1 minute folfixed RBL cells to reduce non-specific labelling and di- lowing the addition of antigen and was sustained for luted in P/B/S to suitable concentration. Unbound sec- about 10 minutes. The linear PKC staining seen in some ondary antibody was removed by five washes with PBS unactivated cells may have arisen from cell-cycle depenand the coverslips were mounted with Mowiol. Mono- dent translocation or from inadvertent activation during clonal antibody to PKC p (CK 1.3, purchased from the experimental manipulations. RBL cells also undergo

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Fig. 1. Immunofluorescence images of unactivated and antigen-activated RBL cells stained 5 minutes after activation with PKC p isoform specific antibody and rhodamine labelled goat anti-mouse secondary antibody. a: Unactivated RBL cells with diffuse PKC p staining show the preactivation morphology. b: Five minutes after activation with 100 ng/ml antigen, cells exhibit increased linear staining patterns and a more spread cell morphology. Bar = 10 pm.

changes in their overall morphology within minutes after activation. Spreading and flattening of attached RBLcells have been reported elsewhere (Pfeiffer et al., 1985; Spudich and Wrenn , in preparation). The PKC-staining pattern in antigen-stimulated cells was prominent in the perinuclear region and extended from the perinuclear region to the cell periphery. The perinuclear staining could be resolved as a network of interconnecting filaments (Fig. 2). These stained filamentous structures were largely resistant to detergent extraction (Fig. 2) indicating that they are cytoskeletal. Figure 3a,b shows the distribution of microtubules and actin filaments in activated RBL-cells, stained with a monoclonal antibody against p tubulin or rhodaminelabelled phalloidin, respectively. The microtubules extend from the center of the cell towards the cell periphery. Phalloidin staining is concentrated at the edges and at the center of activated cells and appears to be in the regions with deep ruffles that are formed at the cell surface in response to activation (Pfeiffer et al., 1985). The pattern of PKC p staining is distinctly different from that of either actin or tubulin and closely resembles that of the intermediate filament protein vimentin (Fig. 3c), as revealed by staining RBL cells with antibodies to vimentin (a kind gift of Dr. Robert Goldman, Northwestern University). In activated cells, both the vimentin intermediate filaments and the PKC p surround the nuclei and extend from the nuclei to the plasma membrane.

Fig. 2. The linear structures stained by protein kinase p antibodies are retained in detergent extracted cytoskeletons prepared from cells at 5 minutes after activation. A cage-like staining in the perinuclear area can be seen in the cell located in the upper right corner. Bar = 10 pm.

The organization of intermediate filaments seen by immunofluorescence is reminiscent of the distribution of 100 A filaments observed in thin section electron microscopy of activated RBL cells. Bundles of intermediate filaments run parallel to the nuclear membrane and extend from the nuclear membrane and the plasma membrane (Fig. 4a,b).

PKC Localization

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Fig. 3. Organization of microtubules, actin filaments, and intermediate filaments in antigen-activated RBL cells. a: Microtubules were stained with a monoclonal antibody to p tubulin. b: Actin filaments were stained with rhodamine labelled phalloidin. c: Vimentin containing intermediate filaments were stained with a polyclonal antibody against vimentin. Bar = 10 pm.

Fig. 4. Thin section electron micrographs show (a) the orientation of a bundle of intermediate filaments in an RBL cell at 5 min after activation. Bar = 2 Fm. b: At higher magnification, the bundle of filaments extends from the plasma membrane to the nucleus. Bar = 0.2 pm.

To confirm the coincidence of PKC p and vimentin intermediate filaments, cells were double stained with primary antibodies to PKC p and to vimentin, followed

by rhodamine and fluorescein labelled secondary antibodies, respectively. The pattern of staining by vimentin and PKC p antibodies was virtually coincident (Fig.

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5a,b), suggesting that the p isoform of PKC associates with vimentin intermediate filaments after activation. The onset for antigen induced translocation of the PKC p isoform is consistent with the kinetics reported previously for histamine secretion and cell shape changes (Pfeiffer et al., 1985). For comparison, the staining pattern of the a isoform of PKC in antigen stimulated RBL cells show most prominent labeling in regions of the cell periphery (Fig. 6). Unactivated cells had a nearly uniform staining throughout the cytoplasm (data not shown). Thus the 01 isoform of PKC, in contrast to the p isoform, translocates from the cytosol to regions near the plasma membrane, as suggested previously (Ganesan et al., 1990 and references therein). These peripheral areas with concentrated PKC 01 staining are likely to be the sites of ruffling activity, seen at the edges of many cells by video microscopy following antigen activation (Spudich, A., and Wrenn, J.T., manuscript in preparation).

DISCUSSION Different PKC isoforms are differentially expressed in specific tissues and two or more isoforms are often found in the same cell type (Nishizuka, 1986; Huang et al., 1987; Moshly-Rosen et al., 1987; Nishizuka, 1988). Threonine and serine residues of a broad spectrum of substrates are phosphorylated by these isoforms in vitro (Nishizuka, 1988). Different isoforms, however, may choose their substrates more selectively in their native cellular environment. A growing number of reports relate activation of cells to PKC stimulation and to cytoskeletal involvements in cell behaviors (Schliwa et al., 1984; Jaken et al., 1989; Papadopoulos and Hall, 1989; Mochly-Rosen et al., 1990; Bershadsky et al., 1990; Apgar, 1991). The stimulation of PKC by diacylglycerol and calcium or by phorbolesters is thought to be coincident with a translocation of PKC from a cytosolic to a particulate fraction (Kraft and Anderson, 1983). While earlier work suggested that PKC associates with the plasma membrane after activation of cells (for example, see It0 et al., 1988), more recent work indicates that specific isoforms may translocate to different and specific docking sites (Jaken et al., 1989; Leach et al., 1989; Mochly-Rosen et al., 1990; Hocevar and Fields, 1991). In some cases these docking sites seem to be components of the cytoskeleton. Jaken et al. (1989) found that a substantial fraction of PKC 01 in rat embryo fibroblasts colocalizes with vinculin and talin in focal contacts of cells attached to a substratum. Mochly-Rosen and coworkers (1990) reported the translocation of an as yet unidentified isoform of PKC (neither ct nor p) to actin filaments in cardiac myocytes and fibroblasts. Our study now reports

that antigen activated RBL cells exhibit a dual translocation of PKC a to the cell periphery and of PKC p to vimentin filaments. Electron micrographs and immunofluorescence images of RBL-cells show that bundles of intermediate filaments form a web around the nucleus and extend from the nucleus to the cell periphery. The colocalization of PKC p and vimentin intermediate filaments reported here may explain the finding by Hocevar and Fields (1991) that PKC PI1 associates with an isolated nuclear membrane fraction following bryostatin I treatment of HL60 cells. Our findings may also be useful to understand the observations of Sahara et al. (1990) showing an activation-induced change in intermediate filament organization in RBL cells. There is evidence that phosphorylation regulates the assembly state of intermediate filaments in vitro (for review, see Robson, 1989). It is possible that PKC p translocation to vimentin filaments may initiate the reorganization of those filaments in vivo. Our findings may also be useful to test the hypothesis of Bershadsky et al. (1990) that PMA-induced cell shape changes in mouse fibroblasts are caused by interactions between intermediate filaments and the actin cytoskeleton. The PKC p colocalization with vimentin filaments described here could be either a result of direct binding of PKC p to vimentin or of binding of PKC p to another factor(s) associated with vimentin filaments. The inherently low resolution of light microscopy does not allow us to exclude the possibility that the colocalization between intermediate filaments and PKC p is mediated by other factors. Further biochemical experiments will be required to demonstrate that this colocalization is a specific interaction between intermediate filaments and the kinase . PKC p mediated phosphorylation of vimentin or other factors associated with intermediate filaments could regulate the spatial organization of intermediate filaments. Independent of whether the binding of the different PKC isoforms to vimentin filaments, actin filaments, talin or other cytoskeletal components is a direct or an indirect one, such cytoskeletal arrays could serve as specific docking sites at which PKC isoforms encounter their respective substrates. The localization of kinase isoforms and their substrates to such arrays could be a general mechanism to confer specificity to signal transduction pathways.

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Fig. 5 . Double immunofluorescence staining of activated RBL cells 3.5 min after antigen addition labelled with antibodies against vimentin and PKC p. a: FITC-labelled goat anti-rabbit antibody stains vimentin and is shown in green. b: Rhodamine labelled goat anti-mouse antibody stains PKC p and is shown in red. Bar = 5 km.

Fig. 5 .

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Spudich et al. Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K . , Nishi, Y., and Sato, C. (1988): Intermediate filament reconstitution in vitro. The role of phosphorylation on the assembly-disassembly of desmin. J. Biol. Chem. 2605970-5978. Ito, T., Tanaka, T., Yoshida, T., Onoda, K., Ohta, H . , Hagiwara, M., Itoh, Y., Ogura, M., Saito, H., and Hidaka, H. (1988): Immunocytochemical evidence for translocation of protein kinase C in human megakaryoblastic leukemia cells: synergistic effects of Ca2+ and activators or protein kinase C on the plasma membrane association. J. Cell. Biol. 107:929937. Jaken, S . , Leach, K., and Klauck, T. (1989): Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J. Cell Biol. 109:697-704. Kraft, A.S., and Anderson, W.B. (1983): Phorbol esters increase the amount of Ca2 , phospholipid-dependent protein kinase associated with plasma membrane. Nature 301:621-623. Leach, K.L., Powers, E.A., Ruff, V. A,, Jaken, S . , and Kauffman, S . (1989): Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells. J. Cell Biol. 1091685-695. Ludowyke, R.I., Peleg, I., Beaven, M.A., and Adelstein, R.S. (1989): Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J. Biol. Chem. 259:8808-8814. Metzger, H., Alcaraz, G., Hohman, R.,Kinet, J-P., Pribluda, V., and Quatro, R. (1986): The receptor with high affinity for immunoglobulin E. Annu. Rev. Immunol. 4:419-470. Mochly-Rosen, D., Henrich, C.J., Cheever, L., Khaner, H., and Simpson, P.C. (1990): A protein kinase C isozyme is translocated to cytoskeletal elements on activation. Cell Regulation 1:693-706. Mochly-Rosen, D., Basbaum, A.L., and Koshland Jr., D.E. (1987): Distinct cellular and regional location of immunoreactive protein kinase C in rat brain. Proc. Natl. Acad. Sci. USA 84: 4660- 4664. Nishizuka, Y. (1986): Studies and perspectives of protein kinase C. Science 233:305-3 12. Nishizuka, Y. (1988): The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334:661665. Oliver, J.M., Seagrave, J., Stump, R.F., Pfeiffer, J.R., and Deanin, G.G. (1988): Signal transduction and cellular response in RBL2H3 mast cells. Prog. Allergy 42:185-245. Papadopoulos, V., and Hall, P.H. (1989): Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton. J. Cell Biol. 108553-567. Pfeiffer, J.R., Seagrave, J.C., Davis, B.H., Deanin, G., and Oliver, J.M. (1985): Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells. J. Cell Biol. 101:2148-2155. Robson, R.M. (1989): intermediate filaments. Cum. Opin. Cell. Biol. 1:36-43. Sahara, N., Siraganian, R.P., and Oliver, C.J. (1990): Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells. Histochem. Cytochem. 7:975983. Schliwa, J.L., Nakamura, T., Porter, K.R., and Euteneuer, U. (1984): A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells. J. Cell Biol. 99: 1045-1 059. Vikstrom, K.I., Borisy, G.G., and Goldman, R.D. (1989): Dynamic aspects of intermediate filament networks in BHK-21 cells. Proc. Natl. Acad. Sci. USA 86549-593, +

Fig. 6. PKC a staining of activated RBL cells 3.5 min after activation is concentrated in regions of the cell periphery. Cells were stained with anti-peptide antibody specific for PKC a. Bar = 10 pm.

ACKNOWLEDGMENTS

We thank Dr. Joan T. Wrenn for doing the thin section electron microscopy and Mr. Jack Horne for help with cell culturing. This work was supported by grant MH45324 to Dr. Lubert Stryer. REFERENCES Apgar, J.R. (1991): Regulation of antigen-induced F-actin response in rat basophilic leukemia cells by protein kinase C. J. Cell Biol. 112:1157-1163. Beaven, M.A., Guthrie, D.F., Rogers, J., Moore, J.P., Smith, G.A., Hesketh, T.R., and Metcalfe, J.C. (1987): Synergistic signals in the mechanism of antigen-induced exocytosis in 2H3 cells: Evidence for an unidentified signal required for histamine secretion. J. Cell Biol. 105:1129-1136. Bershadsky, A.D., Ivanowa, O.Y., Lyass, L.A., Pletyushkina, O.Y., Vasiliev, J.M., and Gelfand, I.M. (1990): Cytoskeletal reorganizations responsible for the phorbol ester-induced formation of cytoplasmic processes: Possible involvement of intermediate filaments. Proc. Natl. Acad. Sci. USA 87:1884-1888. Ganesan, S., Calle, R., Zawalich, K., Smallwood, J.I., Zawalich, W.S., and Rasmussen, H. (1990): Glucose-induced translocation of protein kinase C in rat pancreatic islets. Proc. Natl. Acad. Sci. USA 87:9893-9897. Hocevar, B.A., and Fields, A.P. (1991): Selective translocation of BII-protein kinase C to the nucleus of human promyelocytic HL60 leukemia cells. J. Biol. Chem. 26:28-33. Huang, F.L., Yoshida, Y., Cunha-Melo, J.R., Beaven, M.A., and Huang, K.P. (1989): Differential down-regulation of protein kinase C isozymes. J. Biol. Chem. 264:4238-4243. Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., and Sato, C. (1987): Site-specific phosphorylation induces disassembly of vimentin filaments in vitro. Nature 328:649-652.

Association of the beta isoform of protein kinase C with vimentin filaments.

Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Sc...
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