Protein Kinase C Associates with Intermediate Filaments and Stress Fibers K. GOPAL MURTI,’ KULJEET KAUR, AND RAKESH M. GOORHA De~~rt~nt

of Virology & ~#~ec~~~ Biology, St. Jude ~~~~dre~~ Research 332 hrorth Lauderdate, P.O. Box 318, Memphis, Tennessee 38101

secretion, and signal transduction [I]. Hormones and growth factors involved in the growth and differentiation of cells bind t.o surface receptors coupled to phospholipase C and generate diacylglycerol, a regulator of PKC [2]. Phorbol esters also activate PKC by binding to the regulatory domain of PKC [3]. It is now established that PKC exists as six isozymes a, p, y, 6, t, and l [l]. Immunocytochemical studies of rat brain with a series of monoclonal antibodies (MAbs) have revealed three distinct patterns of distribution of PKC, i.e., astroglial elements, neural cells, and thalamic neurons [4]. Similarly, studies with isozyme-specific antibodies and cDNA probes have demonstrated tissue-specific expression of PKC isozymes in the cerebellum, spleen, retina, leukemic cells, pancreatic endocrine cells, and glioma cells (for references see 151). Cell fractionation studies have shown that PKC exists in cytosol, membranes, and cytoskeletal fractions and that the relative distribution of PKC changes with its activation [6-111. Immunofluorescence studies have shown that the cy-isozyme (also referred to as the type 3 kinase) associates with focal contacts in rat embryo fibroblasts [12] and that it exhibits a diffuse cytoplasmic staining pattern in NIH 3T3 cells [131. In the latter cell line, the a-isozyme has been shown to associate with the nucleus upon activation with phorbol esters [ 131.All these studies suggest that the different isozymes of PKC may have tissueand cell-specific localization and that they phosphorylate different substrates to regulate the diverse biological functions. The objective of this study is to identify the cytoskeletal networks to which PKC binds. To this end, we have conducted immunofluorescence studies with three MAbs that were prepared and characterized by MochlyRosen and Koshland [4,14,15] and Hidaka et al. [5,16]. The 1.9 MAb was shown to recognize an epitope in the catalytic domain of PKC [4, 14, 151 while the 1.3 MAb was believed to be directed against an isozyme of PKC 14, 8, 14, 151. The third antibody, MC-2a [5, 16], was demonstrated to be specific for the /3-isozyme (or type II PKC) by immunoblot analyses. The immunofluorescence studies with the above antibodies were performed on baby hamster kidney (BHK) cells, two clones of SW13 human adrenal carcinoma cells (vimentin~ and

The subcellular distribution of protein kinase C (PKC) was determined by immunofluorescenee using anti-PKC monoelonal antibodies (MAbs). The antibodies used were: (1) 1.9 MAb that is directed against an epitope in the catalytic domain of PKC, (2) 1.3 MAb that recognizes an isozyme of PKC (Mochly-Rosen, D., and Koshland, D. E., 1987, J- BioE. Chem. 262, 22912297; Mochly-Rosen, D., et al. I987 Proc. ~~~~. Acad. Sei. USA 84, 4660-4664) and (3) MC-2a MAb that is directed against the /3-isozyme of PKC (Usuda, N., et al. 1991, J. Cell Biol. 112, 1241-1247). The cells used in this study were baby hamster kidney cells, vimentin” and vimentinclones of SW13 (a human adrenal carcinoma cell line), GEM (a human T cell line), U937 (a histioeytic myeloid cell line), and HL60 (a promyelocytie leukemia cell line). The 1.9 MAb was found to recognize a variety of subcellular components, viz., nucleus (nucleoplasm and nucleolus), cytoplasm, vimentin-type intermediate filaments (IF), stress fibers, and cell membrane. Among these components the ~-isozyme-specific MAbs (1.3 and MC-2a) recognized only the IF network, stress fibers, and edges of the cell membrane. Experiments with vimentin+ and vimentinmutants of SW13 cells, double indirect immunofluorescence studies with anti-vimentin and anti-PKC antibodies, and drug studies confirmed that the IF network is the predominant cytoskeletal network labeled with all anti-PKC MAbs. Immunoblotting studies with the MC-2a MAb revealed that the observed staining of the IF network was not due to a cross-reaction of the MAb with IF proteins and that the MAb specifically recognizes PKC. These studies, while identifying the diverse cell components to which PKC binds, have demonstrated, for the first time, that PKC associates with the IF network in a variety of cell types. Additionally, the studies have confirmed the studies by others concerning the association of PKC with stress fibers. 0 1992 Academic Press, Inc.

INYXODUCTION Protein kinase C (PKC) is an important enzyme involved in regulating cell morphology, cell contractility, 1 To whom




0014.4827/92 $5.00 Copyright 0 1992 by Academic Press, A11 rights of reproduction in any form

be addressed. 36

Inc. reserved.










vimentin-), IJ937 cells, HE60 cells, and CEM cells. The results show that PKC associates with vimentin-type intermediate filaments (IF) of diverse cell types. The results also confirm earlier observations that PKC associates with microfilaments [8f. MATERIALS



Cells and antibodies. The clones of the human adenocarcinoma cell line (SW13/V+ and SW13/V) were a kind gift of Drs. Robert Evans and M. W. Klymkowsky (University of Colorado, Boulder, CO). The SW13 cell line and BHK cells were grown as monolayers in tissue culture flasks with Eagle’s MEM with Hank’s salts and 10% fetal calf serum. The CEM (a human T cell line), U937 (a histiocytic myeloid cell Line), and HL6O (a promyelocytic leukemia cell line) cells were grown in suspension cultures in RPM1 1640 medium supplemented with 10% heat-inactivated fetal calf serum. Two of the MAbs to protein kinase C used here were prepared and characterized by Drs. D. Mochly-Rosen and D. E. Koshland, Jr. [14, 151. These antibodies were purchased from GIBCO-BRL (Gaithersburg, MD). The two MAbs were prepared against highly purified rat brain PKC containing both membrane-associated and cytosolic PKC and were tested to ensure that they are PKC-specific. Both antibodies are removed by immobilized purified PKC. Additionally, the 1.9 MAb inhibited the activity of PKC but not that of either CAMP-dependent kinase or calcium/calmodulin-dependent kinase and it recognized an epitope in the catalytic domain of PKC. The evidence that the 1.3 MAb is directed against an isozyme of PKC is derived from the following studies. The cDNAs for the a-; p-, and y-isozymes were transiently expressed in R.4T-1 cells and the binding of different PKC MAbs to these cells was examined [S]. The binding of the 1.3 MAb was observed only in cells expressing the cDNA for the P-isozyme, suggesting that the 1.3 MAb is directed against the fi-isozyme. The third MAb, MC-2a, was prepared and characterized by Hidaka et al. [5,16]. The MAb was raised against purified rat brain PKC and reacts specifical!y with chromatographically resolved type II PKC (or p-isozyme) in immunoblot experiments. The antibody (05-152) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal anti-vimentin antibodies (814318) were bought from Boehringer-Mannheim (Indianapolis, IN). Fluoresceinand rhodamine-conjugated secondary antibodies were purchased from ICN Immunobiologicals (Lisle, IL) and Sigma Chemical Co. (St. Louis, MO). Indirect immunofiuorescence. For immunofluorescence, the cells were plated on coverslips (BHK cells and SW13 cells) or spun onto g!ass slides (CEM, HL60, and U937 cells). The cells were processed for immunofluorescence using fixation with formaldehyde and permeabilization with a variety of agents including Triton X-100, Tween 20, saponin, and acetone or simultaneous fixation and permeabilization with cold methanol. Although all these methods gave the same results, the preservation of cell structure and the penetration of antibodies were better with the fixation and permeabilization procedure described by Lazarides [17]. The procedure consisted of fixing the cells with 3.7% formaldehyde followed by permeabilization with cold acetone. With the exception of the MC-2a MAb, all primary and secondary antibodies were used at dilutions ranging from 1:20 to 1:50 and the incubations were carried out at 37°C for 1 h. The MC-2a MAb was used either at full strength or diluted lo-fold and the incubation was carried out for 3 h at 37°C. The coversiips were mounted with p-phenyldiamene and the cells were viewed in a Zeiss IM35 photomicroscope equipped with epifluorescence optics. The photographs were taken on Kodak Tri-X pan film. A variety of controls was maintained to check the specificity of the antibodies used, These include coverslips that were treated with normal sera or heterologous antibodies as the primary antibodies and coverslips treated with secondary antibodies alone; none of these showed fluorescence. Western blot analysis. Rabbit brain cytosol, cell lysate from S13/ V+ cells, and purified vimentin were separated on SDS-polyacryl-

FIG. I. Immunofluorescence micrographs of permeabilized baby hamster kidney cells stained with the i.9 anti-PKC MAb. The antibody stained two components of the cytoskeleton, fibers that are aligned parallel to the long axis of the cell and a filamentous network within the cell.

amide gels (100 V for 4 h) and blotted onto Immobilon-P membrane (Millipore Corp.). Individual lanes were cut and sequentially incubated (each step 3 h at 21°C) with PBS containing 5% dried milk, anti-PKC (MC-2a) MAb (5 @g/ml) or anti-vimentin (814318) MAb (1:20), and ‘251-Iabeled goat anti-mouse antibodies (ICN Immunobiologicals). Between each step, the membrane strips were washed with TBS containing 0.05% Tween-20. The strips were exposed to XAR-5 X-ray film for 16 b. RESULTS

Immunofluorescence with 1.9 Anti-P When permeabilized BHK cells were ex munofluorescence with the 16.9anti-PKC component that seemed stained the most was the cytoskeleton (Fig. 1). Within the eytoskeleton, two superimposed staining patterns were evident, that of a wavy network extending throughout the cell and that of ed parallel to the long axis of the terns are typical of IF network and




stress fibers (microfilament bundles) in these cells [ 181. Focusing of the cells at different planes revealed diffuse staining of the cytoplasm and a punctate staining of the nuclei; the edges of the cell membrane were also stained (data not shown). To determine if the 1.9 MAb stains similar structures in other cell types, immunofluorescence studies were performed with other cell lines including CEM, U937, and HL60. These cell lines have rounded morphology, grow in suspension cultures, and differentiate in vitro in response to phorbol esters. Because these cells grow only in suspension cultures, they had to be spun onto glass slides (cytospin) before processing for immunofluorescence. The staining patterns of these cells were similar to those obtained with BHK cells. All cell types stained intensely with the 1.9 MAb and, as with BHK, the cell component that appeared to stain most was the cytoskeleton (Fig. 2). Because these cells were all rounded, it was generally difficult to identify the cell components that stained. However, during centrifugation for immunofluorescence, several cells at the edges of the centrifuged sample were flattened or lysed, revealing a better picture of the staining of cell components. In CEM cells the intact cells showed intense labeling of the whole cell with most of the label concentrated on a fibrous mass (Fig. 2A). Flattened or lysed cells at the edge of the centrifuged sample revealed the staining of a filamentous network and the nuclei (Fig. 2B). Within the nuclei, the nucleoplasm appears to be diffusely stained whereas the nucleoli are intensely stained (Fig. 2B). No staining of the microfilaments was seen because these cells have no detectable stress fibers as revealed by staining with rhodamine-phalloidin (data not shown). U937 cells also showed a pattern of labeling similar to that of the CEM cells; i.e., both nuclei and cytoskeleton were intensely stained (Figs. 2C-2E). As with CEM cells the nucleoli showed intense staining whereas the nucleoplasm revealed diffuse staining (Fig. 2E). The staining pattern of the cytoskeleton observed in all these cells resembled that of the IF network. To verify if this was so, U937 cells were labeled with antivimentin and examined (Fig. 2F); the labeling pattern of cytoskeleton with anti-PKC and that of the IF network with anti-vimentin were similar (compare Figs. 2D and 2F). The labeling pattern of HL60 cells with 1.9 MAb was similar to that of CEM and U937 cell lines except that the IF network was less pronounced in HL60 cells (data not shown). Together, the results with all these cell types suggest that the 1.9 MAb that recognizes an epitope in the catalytic domain of PKC identifies several distinct sites of PKC distribution and these include the nucleoplasm, the nucleoli, the cytoskeleton, the cytoplasm, and the membrane at the cell periphery. The Major Cytoskeletal Network that Stains with the 1.9 MAb Is the IF Network It appears from the above results that the major component that stained with the 1.9 MAb was the cytoskele-



ton. Additionally, among the cytoskeletal components stained, the predominant structure appears to be the vimentin IF network with less pronounced staining of stress fibers (microfilament bundles). To further verify whether the major network that stained was the IF network, the following experiments were performed. First, the immunofluorescence study with the 1.9 MAb was performed with clones of human adrenal carcinoma cell line SW13 which either contain (SW13/Vf) or lack (SWl3/V) detectable vimentin IFS; both these clones, however, contain normal microfilaments and microtubules [ 191. This cell line provided a unique opportunity to examine not only the IF-specific staining of the 1.9 MAb but also to recognize what other cell components stain with the 1.9 MAb in a cell clone that lacks IF network. Both SW13/V+ and SW13/Vcells were first stained with an anti-vimentin antibody to check for the presence or absence of the IF network. The results shown in Figs. 3A and 3B confirm that the SWI3/V+ cells contain the IF network whereas the SW13/Vcells lack such a network. When stained with the 1.9 MAb the SW13/Vf cells exhibited intense staining of the IF network that obscured the staining of all other cell components (Fig. 3C). In clones of SW13/V-, the staining pattern was quite different. No staining pattern corresponding to the IF network was evident nor was there a pattern typical of microtubles; the only recognizable cytoskeletal elements stained were a few stress fibers. As with other cell types examined, the nuclei showed punctate staining, the cytoplasm showed diffuse staining, and the periphery of the cell showed intense staining (Fig. 3D). Second, the SW13/V+ cells were treated with colchicine and then examined by immunofluorescence with the 1.9 MAb. The depolymerization of microtubules with colchicine was known to collapse the IF network into a ring around the nucleus or a tight knot near the nucleus [20, 211. If the 1.9 MAb labels the IF network, then it should label the collapsed mass of IF. The SW13 V+ cells were treated with colchicine (10 pg/ ml) for 2 h at 37°C and then stained with either anti-vimentin antibody (Fig. 4A) or 1.9 MAb (Fig. 4B) or both (Figs. 4C and 4D). When stained with the anti-vimentin antibody, the cells showed the labeling of the collapsed masses of IFS near the nucleus (Fig. 4A). Staining of these cells with 1.9 MAb revealed the intense labeling of a collapsed masses of filaments near the nucleus as well as stress fibers, cytoplasm, and nuclei (Fig. 4B). That the 1.9 MAb identifies the collapsed IF network was further verified by double indirect immunofluorescence. In this experiment the cells were first incubated with polyclonal (goat) anti-vimentin antibody (fluorescein) followed by 1.9 MAbs (rhodamine). The results showed a coincidence of both IF and 1.9 MAb labeling patterns over the collapsed mass of IF network (Figs. 4C and 4D). Third, BHK cells were double-labeled for immunofluorescence with 1.9 MAb and anti-vimentin antibody as









FIG. 2. Immunoffuores~ence micrographs of CEM (A, Bf and U937 cells (Cl I?, E) stained wit.h 1.9 anti-PKC MAII, and U937 cells stained with the anti-vimentin antibody. In (A) the permeabilized CEM cells show intense labeling of the whole cell. Qccasionall~ the cells show the staining of a filamentous network (arrow). The cells at edge of the coverslip flatten and lyse (B) to reveal the stainhzg of cytnskeleton and the nuclei (especially the nucleoli). The U937 cells also reveal the staining of a filamentous network in intact (C) and lysed cells (D). The nuclei released from lysed cells (E) show staining particularly in the nucleoli. (P) shows the staining of the intermediate filaments of IJ937 cells with a monoclonal anti-vimentin antibody. Nu, nucleoli; N, nucleus.

d~acribed above. The results showed that the IF network is decorated by bot,h ~~~ti-vi~e~tin and anti-PKC antibodies (data not shown). All of the above results strongly favor the notions that (1) the IF network labels i~t~~~el~ with the 1.9 MAb, (2) microfilaments also label but to a lesser extent, and (3) ~icrotubules show no detectable label with the 1.9 MAb,

All cell types ~xa~iued fur staining of the 1.9 &!IAb were also stained with the 1.3 MAb, an antibody that was believed to be directed against the ~-isozy~e of C fs]., In BHK cells the overall intensity of staining

by the 1.3 ~A~ was much less than the ES9 though the co~ce~t~atiou of the ~~t~b~ of inc~b~t~o~ were similar. The antib do~~~~~t~y the IF network and to a lesser extent the stress fibers (Fig. 5A). Unlike with 1.9 showed very little, if any, labeling with the I. CEM, W937, and ML60 cell lines showed sta IF network and, as with l3HK cells no nuclear staining 3/V” cells showed was observed (Fig. 5B). The S staining of the IF network and 6s stress fibers (Fig. SC). 1x1 ~W~3/Vcells, on the other hand, the major pattern observed was the sta~~i~~ of the stress fibers and to a lesser extent the cell gri~~~~y (Fig. SD). In general, the 1.3 gab stains the cells ~~~c~ less intensely






(B, D) clones of SW13 (human adrenal carcinoma) cell line FIG. 3. Immuno~uorescen~e micrographs of vimentin+ (A, C) an d vimentin[tamed with monoclonal anti-vimentin antibody (A, B) or 1.9 anti-E ‘KC MAb (C, D). The anti-vimentin antibody labels the IF netwol rk in ~imentin+ clones (A) but not in vimentin-” clones (B). The 1.9 MAb la1 )els a network in vimentin+ cells (C) that is absent in vimentincells (D). n (D), the 1.9 MAb labels stress fibers (SF), cytoplasm, and the nut leus as well.

than the 1.9 MAb. Additionally, the diffuse cytoplasmic staining and the punctate nuclear staining by 1.9 MAb is absent in cells stained with 1.3 MAb. Thus the 1.3 MAb, which is believed to be @-isozyme-specific, appears to specifically bind the IF network, stress fibers, and cell periphery. Immunofhorescence (MC-2a) MA b



The studies with the 1.3 MAb have provided the first clue that the &isozyme of PKC may associate with the cytoskeletal elements and cell periphery. To verify this further, we have repeated the experiments using an an-

tibody with proven specificity to the P-isozyme (MC-2a; see Refs. [5,16]). The staining pattern of this antibody with S13/V+ cells is shown in Figs. 5E and 5F. The MC2a MAb, just like the 1.3 MAb, predominantly stained the IF network (Fig. 5E). A weaker staining of the stress fibers was also noted, especially in cells in which the IF network is collapsed (Fig. 5F). These results confirm that the ,&isozyme of PKC associates with IF and stress fibers. Specificity

of Anti-PKC


To ensure that the binding of anti-PKC MAbs was not due to a cross-reaction of these antibodies to IFS, we








PIG. 4. Immunofluores~ance mi~rogra~hs of SW13/V+ cells (A-D) and BHK cells (E, F). The SWl3iV’ cells were treated with colchicine to collapse the IF and the cells were stained either with monoclonai anti-vimentin antibody (A) or with 1.9 anti-PKC MAb (B), or both (C, Dj. In (A) note the labeling of collapsed masses of IF near the nucleus. In (B) the 1.9 MAb labeled the collapsed masses of IF, stress fibers, nucleus, and cytoplasm. (C) and (D) show a colchicine-treated SW13/V+ cell that was double-stained with monoclonal anti-vimentin antibody (6) and 1.9 anti-PKC MAb (Dj; note the coincidence of label on a knot of II? (arrowheads) near the nucleus.

performed Western blot analysis with one of the antiPKC MAbs (Fig. 6). The proteins from the cytoplasmic extracts of S13/V’ cells (lanes 1, 2, and 3) and rabbit brain cytosol (lane 4) were separated on a 7.5% SDSpolyaerylamide gel. To some samples, purified vimentin (2 pg) was added to the cell extracts (lanes 2 and 4). Western blot analysis &owed that the anti-vimenti~ MAb readily detected vimentin in S13/V’ cell extracts with (lane I) or without (lane 2) the added vimentin. The anti-PKC MAb (MC-Za) did not cross-react with vime~tin in S13/V+ cell extracts (lane 3) nor didit recognize PKC in these extracts. Apparently the S13/V+ cells do not contain amounts of PKC sufficient for detection by Western blotting. The anti-PKC MAb specifically reeognized the PKC in brain cytosol that was mixed with purified vimentin (lane 4). These results suggest that the anti-PKC MAb used does not cross-react with IF ~~o~e~ns and that it is specific for PKC.

Protein kinase C is an ~~~orta~t enzyme involved in a variety of cell sanctions i~cl~di~~ maintenance of cell morphology, cell contractility, secretion, cell growth and differentiation, and signal transduction (for references see IS]). Several earlier studies have shown that the enzyme exists in six isozymes and that cells contain more than one isozyme [I, 41. The functions of these isozymes are not known but they are thought to exist in ~ffe~ent cell compartments where they phos~hory~ate

specific substrates. Previous b~o~be~~c~~ studies have shown that PKC ~bospbory~ates s~~str~~e~ in cytosol, membranes ~ytos~e~eto~, and nucleus [l-3, $I] and that it translocates from cytosol to the dete~~e~t-~es~s~a~~ cell fraction or to the membrane fraction upon activation by a number of agents including phorbol esters, cytokines, and hormones. previous ~~~~~o~~orescence studies have shown the association of PKC isozymes with the focal contracts of rat embryo fibrobiasts [X2], the nuclei o~~~o~bol~~~tivated NIM 3T3 cells 1131, and the ~c~o~~arne~ts of ~horbo~-activated cardiac fibroblasts [8]. The current ava~~abi~~~y of ~~~C-s~ec~~~ MAbs that recognize the catalytic omakn (common to all isozymes) or the i~d~v~~~a~ isoz es ~~o~~ted us to c an a variety examine the sub~e~~~~ar d~s~~ib~t~o~ of of cell types. The contributions of this stu y co~~e~nil~g the PKC ~strib~tion are as follows. First, using the I.9 is directed against the catalytic domain of PKC [14,16] and the immunofiuorescence method, we h.ave defined the following subcellular ~ornpa~~e~ts In which PKC is present, viz., ~~~leop~as~, nucleoli, v~rne~t~~ IF network, rn~~ro~~a~e~t bundles, ~yto~~asrn~ eeti membrane, and focal contacts. The fact that this antibody recognized all of the cell ~orn~o~e~ts that were previously d~s~r~~ed as ~o~ta~~ing P C strongly suggests that this antibody is indeed d~~e~~e~ against the eatalytic domain of PKC and cross-reacts with all PKC isozymes. Second, we have, for the first time, identified the asso~~~t~o~ of P C witb v~~e~~~~-t~~ IF network and



EPIG. 5. Immunofluorescence micrograph of cells the 1.3 MAb stained stress fibers as well as IF (A). In in !SW13/Vc cells the IF network and stress fibers mbrane are also visible (D). In SW13/V+ cells the li”b”, !rs (F).

that were stained with the 1.3 anti-PKC MAb (A-D) or CEM cells (that are flattened during centrifugation) the show labeling (Cf. In SW13/Vcells the stress fibers MC-2a MAb predominantly stained the IF network (E)




MC-2a MAb (E, IF show intense and labeling of and, to a lesser

F). In BHK (:ells staining (B) and the edges of the extent, the st ress

FIG. 6. Anti-PKC (MC-2a) MAb does not cross-react with vimentin. Proteins in the S13/Vc cell extracts (lanes 1, 2, and 3) and rabbit brain cytosol (lane 4) were separated on SDS-polyacrylamide gels. Some of these lysates (lanes 2 and 4) were mixed with purified vimentin. The proteins were blotted onto Immobilon-P membranes and incubated with anti-v~ment~n MAb (lanes 1 and 2) or anti-PKC MAb (lanes 3 and 4) foIlowe~ by “5f-labeled goat anti-mouse antibodies The membrane strips were exposed to X-ray film. Lane 1, S13/V+ cell extract incubated with ante-virne~t~~ M.&b; lane 2, S13IV+ cell extract mixed with purified vimentin and incubated with anti-vimentin MAb; lane 3, S13/V” cell extract incubated with anti-PKC (MC2a) MAb; iane 4, rabbit brain cytosol mixed with purified vimentin and incubated with anti-PKC (MC-2a) MAb. The bands in lanes 1 and 2 correspond t,o vimentin (Mz N 55000) and the band in lane 4 by their location corresponds to PKC (MF = 80000) as determined relative to the molecuiar weight markers (not shown in the figure).

~on~rrn~d the previous observations concerning the presence of PKC in mi~ro~~aments [8]. The fact that none of the PKC isozymes bind to the microtubule network (as identified by staining of cells with the 1.9 MAb) suggests that the association of PKC with the cytoskeleton is specific. Third, we have found that two p-isozyme-specific antibodies from different sources, 1.3 Ab and MC-Za, bind predominantly to the IF network. The nesters blot studies performed with the MC-2a MAb showed that the binding of this antibody to the IF network was not due to a cross-reaction of the MAb with IF proteins. The significance of the association of PKC with cytoskeletal components remains to be understood. It is possible that the cytoskeletal structures recognized by the anti-PKC MAbs simply represent cell compartments in which PKC is stored; upon activation, the enzyme may move from one ~ompartme~t to the other, phosphory-

lating substrates at different locations. A~~.g~nat~v~~y, it is possible that PKC associates with specific eytoskeletal structures beeause it ~~os~h~ryiate~ these structures and regulates their function. For example, vimentin is known to be an excellent su trate in uitro for PKC [X2], cA~~-de~e~de~~ kinase 2, 231, and p34cdc” kinase [24]. Each of these ~~~ase~ is known to plms~borylate distinct sites in the vime~ti.~ molecule and ~~osphorylatio~ of v~rne~t~~ is known to ~~~t~~l the assembly and ~sass~mbly of IFs. In this context, it is also noteworthy tha t another kinase, creatine phosphokinase, was foun to be associated with IFs of certain mammalian tis culture cells [27]. Stress fibers contain myosin light chain, which is also a substrate for PKC [IO]. Since myosin of the stress fibers is believed to produce the isometric tension which in turn helps to bind the cell to the s~bst~a~~m [ZS], ~hos~bory~at~o~ of myos~n light chain may repulate call shape and cell ~ttacbmen~. T ~-is~zyme of PKC, which is associated with vimentin IFS and stress fi.bers, may regulate cell functions that are carried out by but Talin, a protein of the focal contacts of cultured cells, is also known to be an 1n vitro substrate flor PKC [%I]. Previous ~~~~~o~~o~esce~ce studies have shown that the cw-isozyme of PKC colocalizes with talin in the focal contacts in rat embryo ~b~oblasts [X2]. It is possible that the a-isozyme may regulate the a~~o~~a~i~~of mi~~~~b~a~e~ cro~lam~nt bundles wit e plasrm thereby rnod~~at~~~ cell rn~~~~~~o~y. Thus, the subeellular location of both (Y- and ~-i~~z~rn~s is consistent with their role in cytoskeletal functions that regulate and cell 2ttachme~t~ cell rno~~holo~~ cell contractility, The authors thank Ms. Peggy Brown, Ms. Josie .Harris-Chambers, and Ms. Ramona Tirey for expert technical assistance and Ms. Glenith White for typing the manuscript. This work was supported by American Cancer Society Research Grant CD-253, Cancer Center Support Grant CA-21765 from the National Institutes of Health, and the American Lebanese Syrian Associated Charities of St. Jude Cbildren’s Research Hospital.

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Protein kinase C associates with intermediate filaments and stress fibers.

The subcellular distribution of protein kinase C (PKC) was determined by immunofluorescence using anti-PKC monoclonal antibodies (MAbs). The antibodie...
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