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Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway
EXPERIMENTAL
CELL
RESEARCH
188,%-208
(1990)
Staurosporine Induces Dissolution of Microfilament Bundles by a Protein Kinase C-Independent Pathway K. K. HEDBERG, G. B. BIRRELL,
D. L. HABLISTON,
AND
0. H. GRIFFITH’
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403
work of nonmuscle cells is a complex, developing field. Of special interest is the apparent connection between cellular growth control and actin microfilament assembly, which was first noted in studies of cells transformed by oncogenic viruses [ 11. Further support for such a connection comes from more recent observations that the tumor promoter phorbol myristate acetate (PMA) induces rapid and dramatic microfilament alterations in certain cell types [2-51. PMA is especially useful in the study of cellular signalling pathways because it is thought to have a single primary action, the potent activation of protein kinase C (PKC) [S]. The exact relationship between PKC and control of the actin cytoskeleton remains to be determined, but current research is very suggestive of the existence of a PKC-dependent signal transduction pathway regulating microfilament assembly. One frequently used approach in the field of tumor promotion involves the use of kinase inhibitors to demonstrate a role for PKC activation in the observed effects. We have explored this approach in our investigations into the mechanism of PMA-induced cytoskeletal alterations. One of the most widely used inhibitors of PKC in recent work is staurosporine [7]. Staurosporine appears to interact with the catalytic domain of the kinase [8] and, in contrast to most other inhibitors, is effective at nanomolar rather than micromolar concentrations (Ki = 2.7 r&f for PKC [7]). However, it is clear that while staurosporine selectively inhibits PKC, it is not entirely specific for this kinase. Nanomolar inhibition of the cyclic nucleotide-dependent kinases [9], the insulin receptor tyrosine kinase [lo], and the pp60-src tyrosine kinase [9] have also been reported. Consistent with this, cellular effects of staurosporine which may not be related to inhibition of PKC have been noted [ 10-121. In this study we report that at nanomolar concentrations staurosporine rapidly induces a dramatic reorganization of the actin cytoskeleton of several cultured cell types. These effects are similar to those we previously observed with micromolar concentrations of the PKC inhibitor H-7 [ 131. The staurosporine-induced actin alterations are not identical to, nor do they prevent, those induced by the tumor promoter PMA. Furthermore, in Swiss 3T3 cells, we demonstrate that (in contrast to the
The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTKP epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate fllament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cyt&skeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKCdeficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies 0 la90 Academic PB, he. involving intact cells.
INTRODUCTION
The study of the functions and the multiple regulatory pathways of the actin-based cytoplasmic filament net1To whom correspondenceshould be addressedat Institute lecular Biology, University of Oregon, Eugene,OR 97403.
of Mo-
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effects of PMA) these effects of staurosporine are apparently not mediated by a protein kinase C-dependent pathway.
MATERIALS
AND
METHODS
Chemicaki. Staurosporine was a generous gift of Dr. Tatsuya Tamaoki of Kyowa Hakko Kogyo Co., Ltd., Japan. Staurosporine was also purchased from Calbiochem (La Jolla, CA), as was K252a. Results obtained from the use of the two different batches of staurosporine were comparable. PMA (phorbol myristate acetate) was from LC Services (Wobum, MA). All stocks were prepared in DMSO, stored as aliquots at -2O”C, and diluted into stocks in culture medium just before use. These secondary stocks were bath sonicated at least 5 min before the final dilution into the culture dish. All treatments were in culture medium containing 10% serum (see below). Cells. Swiss 3T3 and Wi38 cell cultures were from the American Type Culture Collection. PTK2 and Rat-l were gifts of Dr. Lan Bo Chen. Human foreskin fibroblast cultures were established by standard procedures. Cell culture was in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY) with 10% supplemented bovine calf serum (HyClone, Logan, UT) in the absence of antibiotics. Fluorescence microscopy. Cell cultures grown on glass coverslips were first lightly prefixed for 3 min at RT in 0.2 mg/ml dithiobis (succinimidyl propionate) (DTSP [14]; Pierce, Rockford, IL) in phosphate-buffered saline (PBS; Sigma, St. Louis, MO). They were then permeabilized with 0.4% Triton X-100 in PHEM buffer [2] (60 n&f Pipes, 25 m&f Hepes, 10 mM EGTA, 2 mM MgC12, 1 mM PMSF, pH 6.9) at 4°C for 5 min, followed by three washes in PHEM buffer. These were subsequently fixed in 2% paraformaldehyde in PHEM buffer for 5 min followed by a wash in PHEM and two washes with PBS. Equivalent results were obtained when the formaldehyde fixation procedure preceded the permeabilization step. Labeling for filamentous actin was accomplished by lo-min RT incubation with a 1:20 dilution in PBS of rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR), followed by two washes (10 min each) with excess PBS. Labeled coverslip cultures were then rinsed with water and mounted on glycerol-gelatin (Sigma) containing 0.1 M n-propyl gallate to retard photobleaching. Immunofluorescence of microtubules and intermediate filaments was as previously described [ 131. Photoelectron microscopy (PEM). Cell cultures intended for examination by PEM were grown on chromium-coated glass 5-mm-diameter round coverslips. Unlabeled cytoskeletal preparations were prefixed and permeabilized as above, except that the permeabilization time was increased to 10 min and the washing step was followed by fixation in 2% glutaraldehyde in PHEM buffer. This was followed by a distilled water wash, dehydration through a graded series of ethanol concentrations, with final dehydration accomplished by critical point drying from COz. For colloidal gold decoration of microtubules, cultures were DTSP prefixed, permeabilized in stabilization buffer (0.1 M Pipes, pH 6.9, 1 n&f EGTA, 4% PEG 8000,0.2% Triton X-100) at 37’C for 5 min, washed in buffer without detergent, and exposed to -20°C methanol for 5 min. The labeling was in three steps with intervening PBS washes: monoclonal anti-tubulin (Sigma) rhodamineconjugated goat anti-mouse Ig’s (Organon Teknika-Cappel, Malvern, PA); and protein G bound to 6 nm colloidal gold stabilized with fish gelatin as previously described for immunogold labeling [15]. The first two incubations were at 37°C for 20 min and the final was for 1 h at RT. Additional washes following the last labeling step consisted of two changes over 10 min of 0.5% fish gelatin in PBS, pH 5.5; PBS, pH 5.5, without gelatin; and then PBS, pH 7.4. After the final fixation in 2% glutaraldehyde, the labeled samples were silver enhanced [15] and then fixed and dehydrated as described above for unlabeled samples. All specimens were immediately placed in the preparation chamber of
ET AL. the photoelectron microscope and kept under vacuum until examination. The Oregon photoelectron microscope is an oil-free high-vacuum instrument that has been described previously [16,17]. The images are formed by accelerating, focusing, and recording on film the electrons emitted from the sample surface during uv-light excitation. Photoelectron microscopy has been used to image a variety of biological specimens [ 13,18-201.
RESULTS
Untransformed fibroblasts and many epithelial cells in culture express an abundance of cytoplasmic actin filaments crosslinked into microfilament bundles (“stress fibers”). These bundles occur in roughly parallel arrays which often run along or near the cell periphery as well as traversing much of the cell interior, as illustrated by rhodamine-phalloidin fluorescence of PTK2 epithelial cells and FSB (human foreskin) fibroblasts in Figs. 1A and lE, respectively. Previous studies have shown that the phorbol ester tumor promoter PMA induces several rapid and distinctive alterations in actin organization in some epithelial cells [2] and in mouse fibroblasts [4, 51. As shown for PTK2 cells (Fig. 1B) these include loss of microfilament bundles, appearance of actin-containing aggregates in the cytoplasm, and generation of peripheral ribbon-like structures. Similar changes are seen in Swiss 3T3 fibroblasts [4]. The actin microfilaments of some nonimmortalized fibroblasts, such as the human foreskin fibroblast strain FSB shown in Figs. lE-lG, display a more subtle response during initial exposure to PMA. Rather than depletion of stress fibers and appearance of actin aggregates, fluorescence labeling for actin after 30 min of PMA exposure reveals more pronounced, “combed” microfilament bundles (Fig. 1F and Ref. [20]). Although PTKP and FSB cells provide examples of different actin cytoskeletal responses to PMA, their response to staurosporine is similar. Within 15 min of exposure to staurosporine at 20 nM (for PTKP cells, which proved to be somewhat more sensitive) or 40 r&f (for FSB fibroblasts), rhodamine-phalloidin staining reveals a decrease in numbers and a thinning of prominent microfilament bundles. By 30 min (Figs. 1C and 1G) phalloidin fluorescence reveals a cytoplasm filled with a fine, wavy, often diffuse filamentous pattern. Few cells in the culture retain a classic stress fiber array. Similar staurosporine-induced actin alterations were also noted in Wi38 (human lung fibroblasts) and Rat-l fibroblasts (not shown). These effects were also documented by immunofluorescence with an antibody specific for actin (not shown), indicating that staurosporine acts on actin filament organization rather than altering the binding of phalloidin to actin filaments. The recruitment of actin to wide ribbon-like structures and cytoplasmic aggregates, which is characteristic of PMA exposure for Swiss 3T3 and PTK2 cells, was
not a feature of any of the staurosporine-treated
cells we
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FIG. 1. Fluorescence microscopy of rhodamine-phalloidin-stained microfilament bundles in PTK2 (A-D) and FSB (E-G) cultures. Cells in A and E were controls; those in B and F were exposed for 30 min to PMA at 100 and 200 nM, respectively; those in C and G were exposed for 30 min to staurosporine at 20 and 40 n&f, respectively; and those in D were pretreated with 20 n&f staurosporine for 45 min followed by 15 min of 100 niV PMA. Arrows in B and D mark actin aggregates induced in PTK2 ceils by PMA; arrows in F indicate more prominent microfilaments induced in FSB cells by PMA, but not by staurosporine; arrows in C, D, and G point out thinning microfilament bundles in staurosporinetreated cells.
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have examined. Although actin reorganization is diflicult to quantitate, pretreatment of PTK2 or Swiss 3T3 cells with staurosporine (20-40 nM) for 45 min, followed by addition of PMA (100-200 nM) for 15 min, appeared to have additive effects. This can be seen in Fig. ID for PTK2 cells, in which both the diffuse actin pattern characteristic of staurosporine exposure and the aggregates resulting from PMA exposure appear. Thus, staurosporine in this dose range does not block PMA-induced actin alterations. Higher or longer doses of staurosporine could not be tested for their ability to prevent PMA-induced alterations because of the severe actin reorganization and pronounced morphological alterations that developed from staurosporine alone. Like PMA-induced actin reorganization, the effects of staurosporine were reversible. For example, PTK2 cells exposed to 20 nM staurosporine for 2 h became severely arborized, yet recovered fully after washing and overnight incubation in fresh medium (not shown). The overall cytoskeletal alterations induced by staurosporine were also investigated by photoelectron microscopy (PEM). This electron emission-based technique, which has been termed the electron microscope analog of fluorescence microscopy [ 181, allows direct imaging of the surfaces of uncoated, unstained samples. PEM can also be used to observe internal cellular structures, such as the cytoskeletal systems of cultured cells, provided that they have been exposed to neutral detergent to remove surface membrane. In the absence of labeling (e.g., immunogold labeling), the result is an image in which the three-dimensional structure of the exposed cytoskeletal components generates the contrast [ 191. Consequently, one application of photoelectron microscopy is to document overall alterations in cytoskeletal organization relative to other structural features within the cell. Figures 2A and 2B are photoelectron micrographs of cytoskeletal preparations of control and staurosporinetreated PTK2 cells, respectively. Untreated PTK2 cells, which grow outward from island-like confluent colonies, normally have a polygonal appearance with relatively straight boundaries between cells. PEM micrographs of unlabeled PTK2 cytoskeletal preparations show the cell-cell boundaries to have a ridgelike structure (black arrowheads, Fig. 2A, inset). The cytoplasmic region of PTK2 cells is characterized by high densities of straight semi-parallel arrays of microfilament bundles which appear in PEM micrographs as long filaments (white arrows, Fig. 2A, inset and enlargement). Cells spreading onto the substrate at the edge of the colony also have a peripheral band of fine actin filament bundles which can be seen in the upper right portion of Fig. 2A (inset and enlargement). In contrast to control cells, PTK2 cells exposed to 40 nM staurosporine for 20 min have two marked differences which are readily apparent in PEM images. One is that the cells in the colony lose their
ET AL.
polygonal appearance (Fig. 2B). The cell-cell boundaries retain their thick, ridgelike structure but become wavy and curved (black arrowheads, Fig. 2B, inset and enlargement). The second is that there is a drastic decrease in the population of microfilament bundles in staurosporine-treated compared with that in control cells (Figs. 2B and 2A, respectively). Cells along the edge of the colony lose their peripheral band of actin filaments (not shown), and all of the cells in the staurosporine-treated culture lose the classic stress fiber array of normal PTK2 cells. Despite these major alterations in actin patterns, immunofluorescence microscopy of parallel cultures revealed that the intermediate filament and microtubule systems remain intact and spread in staurosporinetreated cells (not shown). Although microtubules are not well-preserved by the detergent treatment used to prepare the PEM samples of Figs. 2A and 2B, intermediate filaments remain intact. These are particularly noticeable in the staurosporine-treated cells of Fig. 2B (white arrowheads), where actin bundles are not present to obscure them. In order to employ photoelectron microscopy to selectively observe the effects of staurosporine on microtubules, control and staurosporine-treated cultures were also prepared by a procedure optimal for microtubule stabilization. These samples were labeled with specific antibodies followed by protein G bound to 6 nm colloidal gold (subsequently silver enhanced) and are shown in Figs. 3A and 3B. In photoelectron images, the silver-enhanced gold label is more photoemissive than cellular material, resulting in bright labeled structures against a darker background. Labeled microtubules appear in both control and staurosporine-treated cells (Figs. 3A and 3B, respectively) as an abundance of filaments heavily decorated with fine bright dots. We noted some distortions in the microtubule patterns in staurosporine-treated cells (Fig. 3B). This may possibly be due to a decreased stability of microtubules dehydrated in the absence of microfilament bundles. Visible in the background of Figs. 3A and 3B and indicated by fat white arrows is the ridge of the cell-cell boundary, which is characteristically wavy after staurosporine treatment (Fig. 3B). The involvement of PKC in staurosporine-induced actin reorganization was investigated by asking whether staurosporine also alters actin microfilaments in cells made deficient in PKC. To “down-regulate” PKC, quiescent Swiss 3T3 cells were exposed to 200 nM PMA in spent culture medium for 30 h (Figs. 4D-4F), or to 300 nM PMA for 72 h (not shown). Controls were exposed for the same period of time to the equivalent amount of DMSO solvent in spent culture medium (Figs. 4A-4C. Thirty hours of exposure to 200 nM PMA is reportedly sufficient to reduce Swiss 3T3 protein kinase C levels below the level of detection by kinase assay [21]. To verify this for our cultures of Swiss 3T3, in a previous study
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Photoelectron micrographs of (A) control and (B) staurosporine-treated (40 nM for 20 min) PTK2 cells after use of neutral ,2 ,nt expose cytoskeletal elements. Lower magnification overviews are inset. Arrows indicate intact microfilament handlet I in the control (-4 which are absent in the treated culture (B); black arrowheads indicate cell-cell boundaries which become way Y after staurc lsporine mt ntermediate filaments (IF) remain intact and are visible in the staurosporine-treated samples (B).
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F‘IG. 3. Photoelectron micrographs of (A) control and (B) staurosporine-treated (40 nM for 20 min) PTK2 cytoskeletal preparai tions in whi ch microtubules (MT) were decorated with antitubulin antibodies and Protein G bound to 6 nm colloidal gold. The microtubule ren lain8 spread after staurosporine treatment although minor distortions are visible. Fat white arrows delineate the boundaries betweer 1 cells.
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actin microfilaments in quiescent Swiss 3T3 (:ells befo re (A-C) and FIG. 84. Fluore scence microscopy of rhodamine-phalloidin-stained after (D- -F) PMA e,xposure sufficient to down-regulate (deplete) protein kinase C. A and D are controls; B and E were cl ‘nged W ,ith 200InM PMA for .30 min; C and F were challenged with 20 nhf staurosporine for 30 min.
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we monitored the PMA-stimulated phosphorylation of the 80K putative PKC substrate protein during prolonged exposure of 3T3 cultures to PMA [ 131. Phosphorylation of the 80K protein has been described as a reliable marker of PKC activation in Swiss 3T3 cells [22]; thus, observation of PMA-stimulated phosphorylation of the 80K protein serves as a means of monitoring depletion of PKC from the cultures. We found that after 30 h exposure to 200 nM PMA, no 80K phosphorylation could be observed after addition of fresh PMA, indicating effective PKC down-regulation. Swiss 3T3 cells initially experience major actin cytoskeletal alterations after addition of PMA to culture medium, but these changes are transient. After several hours, microfilament bundles reform and are abundant by 30 h, as shown by fluorescence with rhodamine-phalloidin (Fig. 4D). The only notable difference between control and long-term PMA-treated cultures is that after chronic PMA treatment the “cobblestone” morphology that is characteristic of confluent, contact-inhibited 3T3 cells is greatly reduced, and many crisscrossed cells appear (Fig. 4D). Challenge with fresh 200 nM PMA for 30 min generates no significant actin disruption in down-regulated cells (Fig. 4E), while typical PMA-induced alterations are seen in controls (Fig. 4B). This is consistent with current thinking that PMA induces actin reorganization by a protein kinase C-dependent pathway. In the absence of PKC, freshly added PMA fails to alter actin patterns in Swiss 3T3 cells. This is not true of added staurosporine. Figures 4C and 4F compare control and down-regulated cells with respect to the actin alterations induced by 1 h of 20 nM staurosporine. Disruption of microfilament bundles and distorted cellcell boundaries are pronounced effects in both cultures. This was also true for cultures exposed to 300 nM PMA for 72 h (not shown), which is a more stringent condition for generating PKC-deficient cells. These results indicate that interaction with protein kinase C is not a plausible mechanism for the actin reorganization induced by staurosporine. DISCUSSION One of the major signal transduction pathways thought to be involved in cellular proliferation and differentiation is mediated by protein kinase C [23]. This kinase exists in a variety of mammalian and other cells as several different isoenzymes, all with an apparent molecular weight of approximately 80 kDa. PKC is activated as part of a quaternary complex containing, in addition to the kinase, diacylglycerol, phospholipid, and Ca2+. After tryptic digestion of native PKC, a 30-kDa regulatory domain and a 50 kDa catalytic domain have been isolated [24, 251. The regulatory domain is capable of binding phospholipid, diacylglycerol, and phorbol es-
ET AL.
ters, while the catalytic domain binds ATP and substrate protein. Much recent work has dealt with the question of whether or not particular signal transduction processes are mediated through PKC. Two approaches have been widely used to answer this question: (1) Down-regulation of protein kinase C, and (2) the use of inhibitors of protein kinase C. Down-regulation of PKC refers to the disappearance of kinase activity and immunoreactivity as a result of prolonged exposure to active phorbol esters (e.g., PMA or phorbol 12,13dibutyrate) and has been observed in many cell types. In Swiss 3T3 cells, for example, continuous exposure to 200 nM active phorbol 12,13-dibutyrate for 24 h reduces PKC activity to less than 10% of that in untreated cells and for 40 h results in the virtually complete disappearance of PKC activity [21]. The down-regulation process does not occur to the same extent in all cell types [26] and is not yet fully understood. However, PKC translocates from the cytosol to the plasma membrane in cells exposed to phorbol esters, and there is evidence to support the possibility that PKC is then proteolytically degraded by specific proteases present in the plasma membrane [27, 281. In cell systems where down-regulation has been shown to result in the complete depletion of protein kinase C, processes occurring in response to agonists (including hormones, drugs, growth factors, or neurotransmitters) are generally considered to occur via PKC-independent pathways. In an alternate approach, a major effort has gone into the development of specific inhibitors of PKC. Inhibitors which interact predominantly with either the regulatory domain (chlorpromazine [29], tamoxifen [30], palmitoyl carnitine [31], sphingosine [32]) or the catalytic domain (H-7 [33], staurosporine [ 71, sangivamycin [34], the K252 compounds [35]) have been described. Numerous studies have made use of inhibitors to examine the role of PKC in the regulation of various cellular functions. A significant problem with the inhibitors of PKC reported to date has been a lack of specificity with many of these compounds in in vitro kinase inhibition studies. This has been particularly well-documented with the inhibitors which bind to the catalytic domain of PKC, where inhibition of cyclic AMP- and cyclic GMPdependent kinases as well as myosin light chain kinase has been demonstrated [9, 33, 351. Similar selectivity problems have been noted with inhibitors which affect the regulatory domain [36,37]. This nonspecificity of inhibition, coupled with the fact that the cell does not provide the same defined environment as the assay system, may be responsible for a variety of anomalous results reported in studies on intact cells. In the signal transduction field, there are multiple examples in which “protein kinase C inhibitors” have effects which are not replicated by or are opposite to those induced by other PKC inhibitors. Some of these
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include: Inhibition of PMA-induced platelet aggregation at sphingosine concentrations lo-fold lower than those required to inhibit PKC in platelets [37]; cyclic nucleotide- and PKC-independent effects of H-7 on murine T cell hybridoma proliferation [38]; staurosporine mimicry of tumor promoter effects with respect to induction of ornithine decarboxylase [12], murine epidermal cell differentiation [ 111, and morphological changes in astrocytes [39]; and our observation of actin microfilament disruption induced by H-7, even in PKC-deficient cells [13]. In this study we have compared the effects on the actin cytoskeleton of cultured cells due to one of the most potent inhibitors of PKC known, staurosporine, with those of the tumor promoter and widely used activator of protein kinase C, PMA. The effects resulting from treatment with PMA and staurosporine are similar, though not identical: both compounds cause a rapid and dramatic disruption of actin stress fibers. However, the actin-labeling patterns of staurosporine-treated cells are distinguishable from those of PMA-treated cells (i.e., Fig. l), and strongly resemble those induced by the kinase inhibitor H-7 [13]. The actin patterns in cells exposed to both PMA and either H-7 or staurosporine are suggestive of an additive effect (Fig. 1D). Given the opposing effects of staurosporine and PMA on PKC activity, it would seem reasonable that these agents would alter actin organization by different pathways. To clarify whether PKC is involved in the staurosporine-induced alterations, we used the well-documented process of phorbol ester down-regulation to make Swiss 3T3 cells deficient in PKC. The initial exposure to PMA results in actin reorganization, but stress fibers reform within 12 h and do not change significantly in appearance after that. Consistent with PKC downregulation, addition of fresh PMA to these cells after 30 h generates no notable actin microfilament disruption. Staurosporine (20 nM), on the other hand, results in substantial disruption of the actin stress fiber network in both control and PMA-treated cells. Because of the nearly identical effects of staurosporine on cells containing active PKC and those made deficient in PKC, we conclude that staurosporine-induced disruption of the actin cytoskeleton occurs through a PKC-independent mechanism. Both the types of cytoskeletal changes induced and the PKC-independence of the observed effects described here for staurosporine are comparable to those described in our previous study of H-7 [13]. Nevertheless, alterations in the actin cytoskeleton of cultured cells are not a feature of all kinase inhibitors. We have previously observed that 45 min exposure to 200 ~Mofeither HA I004 or the relatively specific PKC inhibitor sangivamycin does not have this effect [13]. The kinase inhibitor K252a, however, has a significant effect on PTK2 actin after 30 min at 1 pM (unpublished observations, this lab-
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oratory). This may reflect the structural similarity between K252a and staurosporine. In contrast, the similarities in H-7 and staurosporine-induced effects on actin reorganization are somewhat surprising in view of the very different structures of the two compounds (staurosporine is a microbial alkaloid [7], whereas H-7 is an isoquinolinesulfonamide derivative [33]). Consistent with these structural differences are recent studies in which different effects resulting from the use of these two compounds have been reported [ 11,121. In the case of reorganization of the actin cytoskeleton, however, not only are the alterations induced by H-7 and staurosporine very similar, but the concentration range in which significant effects first appear reflects their kinase inhibition constants. For example, staurosporine and H-7 inhibit PKC with reported Ki values of 2.7 nM [7] and 6 pM [33], respectively, and we observed actin alterations with 2050 nM staurosporine and 15-60 pM H-7. The role of kinases in eukaryotic cells is far from being completely understood, but there is some evidence for kinase-mediated control of the actin cytoskeleton. The best-known case is that of myosin light chain kinase, which maintains actin microfilament integrity through phosphorylation of myosin light chain [40]. Cyclic AMP-dependent protein kinase, in turn, can indirectly cause dissociation of actin microfilaments by phosphorylating and inhibiting myosin light chain kinase [40,41]. The complexity of phosphorylation pathways makes it difficult to sort out the exact mechanisms of action of staurosporine and H-7. Although the question remains open as to whether inhibition of kinases could be involved, PKC has essentially been ruled out as a mediator for the actin-associated effects of staurosporine and H-7. We are grateful to Dr. Taksuya Tamaoki for the generous gift of staurosporine and thank Amy Hatch for expert photographic assistance. This study was made possible by Grant CA 11695 from the National Cancer Institute. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
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S., Patel, Y., and Kakkar, V. V. (1989) Biochim. Biophys. Acta 1010,258-264. Love, J. T., Jr., Padula, S. J., Lingenheld, E. G., Amin, J. K., Sgroi, D. C., Wong, R. L., Sha’afi, R. I., and Clark, R. B. (1989) Biochem. Biophys. Res. Comm. 162,138-143.
39.
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CELL
RESEARCH
188,%-208
(1990)
Staurosporine Induces Dissolution of Microfilament Bundles by a Protein Kinase C-Independent Pathway K. K. HEDBERG, G. B. BIRRELL,
D. L. HABLISTON,
AND
0. H. GRIFFITH’
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403
work of nonmuscle cells is a complex, developing field. Of special interest is the apparent connection between cellular growth control and actin microfilament assembly, which was first noted in studies of cells transformed by oncogenic viruses [ 11. Further support for such a connection comes from more recent observations that the tumor promoter phorbol myristate acetate (PMA) induces rapid and dramatic microfilament alterations in certain cell types [2-51. PMA is especially useful in the study of cellular signalling pathways because it is thought to have a single primary action, the potent activation of protein kinase C (PKC) [S]. The exact relationship between PKC and control of the actin cytoskeleton remains to be determined, but current research is very suggestive of the existence of a PKC-dependent signal transduction pathway regulating microfilament assembly. One frequently used approach in the field of tumor promotion involves the use of kinase inhibitors to demonstrate a role for PKC activation in the observed effects. We have explored this approach in our investigations into the mechanism of PMA-induced cytoskeletal alterations. One of the most widely used inhibitors of PKC in recent work is staurosporine [7]. Staurosporine appears to interact with the catalytic domain of the kinase [8] and, in contrast to most other inhibitors, is effective at nanomolar rather than micromolar concentrations (Ki = 2.7 r&f for PKC [7]). However, it is clear that while staurosporine selectively inhibits PKC, it is not entirely specific for this kinase. Nanomolar inhibition of the cyclic nucleotide-dependent kinases [9], the insulin receptor tyrosine kinase [lo], and the pp60-src tyrosine kinase [9] have also been reported. Consistent with this, cellular effects of staurosporine which may not be related to inhibition of PKC have been noted [ 10-121. In this study we report that at nanomolar concentrations staurosporine rapidly induces a dramatic reorganization of the actin cytoskeleton of several cultured cell types. These effects are similar to those we previously observed with micromolar concentrations of the PKC inhibitor H-7 [ 131. The staurosporine-induced actin alterations are not identical to, nor do they prevent, those induced by the tumor promoter PMA. Furthermore, in Swiss 3T3 cells, we demonstrate that (in contrast to the
The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTKP epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate fllament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cyt&skeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKCdeficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies 0 la90 Academic PB, he. involving intact cells.
INTRODUCTION
The study of the functions and the multiple regulatory pathways of the actin-based cytoplasmic filament net1To whom correspondenceshould be addressedat Institute lecular Biology, University of Oregon, Eugene,OR 97403.
of Mo-
199
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All
$3.00
opyright 0 1990 by Academic Press, Inc. rights of reproductionin any form reserved. c
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effects of PMA) these effects of staurosporine are apparently not mediated by a protein kinase C-dependent pathway.
MATERIALS
AND
METHODS
Chemicaki. Staurosporine was a generous gift of Dr. Tatsuya Tamaoki of Kyowa Hakko Kogyo Co., Ltd., Japan. Staurosporine was also purchased from Calbiochem (La Jolla, CA), as was K252a. Results obtained from the use of the two different batches of staurosporine were comparable. PMA (phorbol myristate acetate) was from LC Services (Wobum, MA). All stocks were prepared in DMSO, stored as aliquots at -2O”C, and diluted into stocks in culture medium just before use. These secondary stocks were bath sonicated at least 5 min before the final dilution into the culture dish. All treatments were in culture medium containing 10% serum (see below). Cells. Swiss 3T3 and Wi38 cell cultures were from the American Type Culture Collection. PTK2 and Rat-l were gifts of Dr. Lan Bo Chen. Human foreskin fibroblast cultures were established by standard procedures. Cell culture was in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY) with 10% supplemented bovine calf serum (HyClone, Logan, UT) in the absence of antibiotics. Fluorescence microscopy. Cell cultures grown on glass coverslips were first lightly prefixed for 3 min at RT in 0.2 mg/ml dithiobis (succinimidyl propionate) (DTSP [14]; Pierce, Rockford, IL) in phosphate-buffered saline (PBS; Sigma, St. Louis, MO). They were then permeabilized with 0.4% Triton X-100 in PHEM buffer [2] (60 n&f Pipes, 25 m&f Hepes, 10 mM EGTA, 2 mM MgC12, 1 mM PMSF, pH 6.9) at 4°C for 5 min, followed by three washes in PHEM buffer. These were subsequently fixed in 2% paraformaldehyde in PHEM buffer for 5 min followed by a wash in PHEM and two washes with PBS. Equivalent results were obtained when the formaldehyde fixation procedure preceded the permeabilization step. Labeling for filamentous actin was accomplished by lo-min RT incubation with a 1:20 dilution in PBS of rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR), followed by two washes (10 min each) with excess PBS. Labeled coverslip cultures were then rinsed with water and mounted on glycerol-gelatin (Sigma) containing 0.1 M n-propyl gallate to retard photobleaching. Immunofluorescence of microtubules and intermediate filaments was as previously described [ 131. Photoelectron microscopy (PEM). Cell cultures intended for examination by PEM were grown on chromium-coated glass 5-mm-diameter round coverslips. Unlabeled cytoskeletal preparations were prefixed and permeabilized as above, except that the permeabilization time was increased to 10 min and the washing step was followed by fixation in 2% glutaraldehyde in PHEM buffer. This was followed by a distilled water wash, dehydration through a graded series of ethanol concentrations, with final dehydration accomplished by critical point drying from COz. For colloidal gold decoration of microtubules, cultures were DTSP prefixed, permeabilized in stabilization buffer (0.1 M Pipes, pH 6.9, 1 n&f EGTA, 4% PEG 8000,0.2% Triton X-100) at 37’C for 5 min, washed in buffer without detergent, and exposed to -20°C methanol for 5 min. The labeling was in three steps with intervening PBS washes: monoclonal anti-tubulin (Sigma) rhodamineconjugated goat anti-mouse Ig’s (Organon Teknika-Cappel, Malvern, PA); and protein G bound to 6 nm colloidal gold stabilized with fish gelatin as previously described for immunogold labeling [15]. The first two incubations were at 37°C for 20 min and the final was for 1 h at RT. Additional washes following the last labeling step consisted of two changes over 10 min of 0.5% fish gelatin in PBS, pH 5.5; PBS, pH 5.5, without gelatin; and then PBS, pH 7.4. After the final fixation in 2% glutaraldehyde, the labeled samples were silver enhanced [15] and then fixed and dehydrated as described above for unlabeled samples. All specimens were immediately placed in the preparation chamber of
ET AL. the photoelectron microscope and kept under vacuum until examination. The Oregon photoelectron microscope is an oil-free high-vacuum instrument that has been described previously [16,17]. The images are formed by accelerating, focusing, and recording on film the electrons emitted from the sample surface during uv-light excitation. Photoelectron microscopy has been used to image a variety of biological specimens [ 13,18-201.
RESULTS
Untransformed fibroblasts and many epithelial cells in culture express an abundance of cytoplasmic actin filaments crosslinked into microfilament bundles (“stress fibers”). These bundles occur in roughly parallel arrays which often run along or near the cell periphery as well as traversing much of the cell interior, as illustrated by rhodamine-phalloidin fluorescence of PTK2 epithelial cells and FSB (human foreskin) fibroblasts in Figs. 1A and lE, respectively. Previous studies have shown that the phorbol ester tumor promoter PMA induces several rapid and distinctive alterations in actin organization in some epithelial cells [2] and in mouse fibroblasts [4, 51. As shown for PTK2 cells (Fig. 1B) these include loss of microfilament bundles, appearance of actin-containing aggregates in the cytoplasm, and generation of peripheral ribbon-like structures. Similar changes are seen in Swiss 3T3 fibroblasts [4]. The actin microfilaments of some nonimmortalized fibroblasts, such as the human foreskin fibroblast strain FSB shown in Figs. lE-lG, display a more subtle response during initial exposure to PMA. Rather than depletion of stress fibers and appearance of actin aggregates, fluorescence labeling for actin after 30 min of PMA exposure reveals more pronounced, “combed” microfilament bundles (Fig. 1F and Ref. [20]). Although PTKP and FSB cells provide examples of different actin cytoskeletal responses to PMA, their response to staurosporine is similar. Within 15 min of exposure to staurosporine at 20 nM (for PTKP cells, which proved to be somewhat more sensitive) or 40 r&f (for FSB fibroblasts), rhodamine-phalloidin staining reveals a decrease in numbers and a thinning of prominent microfilament bundles. By 30 min (Figs. 1C and 1G) phalloidin fluorescence reveals a cytoplasm filled with a fine, wavy, often diffuse filamentous pattern. Few cells in the culture retain a classic stress fiber array. Similar staurosporine-induced actin alterations were also noted in Wi38 (human lung fibroblasts) and Rat-l fibroblasts (not shown). These effects were also documented by immunofluorescence with an antibody specific for actin (not shown), indicating that staurosporine acts on actin filament organization rather than altering the binding of phalloidin to actin filaments. The recruitment of actin to wide ribbon-like structures and cytoplasmic aggregates, which is characteristic of PMA exposure for Swiss 3T3 and PTK2 cells, was
not a feature of any of the staurosporine-treated
cells we
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FIG. 1. Fluorescence microscopy of rhodamine-phalloidin-stained microfilament bundles in PTK2 (A-D) and FSB (E-G) cultures. Cells in A and E were controls; those in B and F were exposed for 30 min to PMA at 100 and 200 nM, respectively; those in C and G were exposed for 30 min to staurosporine at 20 and 40 n&f, respectively; and those in D were pretreated with 20 n&f staurosporine for 45 min followed by 15 min of 100 niV PMA. Arrows in B and D mark actin aggregates induced in PTK2 ceils by PMA; arrows in F indicate more prominent microfilaments induced in FSB cells by PMA, but not by staurosporine; arrows in C, D, and G point out thinning microfilament bundles in staurosporinetreated cells.
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have examined. Although actin reorganization is diflicult to quantitate, pretreatment of PTK2 or Swiss 3T3 cells with staurosporine (20-40 nM) for 45 min, followed by addition of PMA (100-200 nM) for 15 min, appeared to have additive effects. This can be seen in Fig. ID for PTK2 cells, in which both the diffuse actin pattern characteristic of staurosporine exposure and the aggregates resulting from PMA exposure appear. Thus, staurosporine in this dose range does not block PMA-induced actin alterations. Higher or longer doses of staurosporine could not be tested for their ability to prevent PMA-induced alterations because of the severe actin reorganization and pronounced morphological alterations that developed from staurosporine alone. Like PMA-induced actin reorganization, the effects of staurosporine were reversible. For example, PTK2 cells exposed to 20 nM staurosporine for 2 h became severely arborized, yet recovered fully after washing and overnight incubation in fresh medium (not shown). The overall cytoskeletal alterations induced by staurosporine were also investigated by photoelectron microscopy (PEM). This electron emission-based technique, which has been termed the electron microscope analog of fluorescence microscopy [ 181, allows direct imaging of the surfaces of uncoated, unstained samples. PEM can also be used to observe internal cellular structures, such as the cytoskeletal systems of cultured cells, provided that they have been exposed to neutral detergent to remove surface membrane. In the absence of labeling (e.g., immunogold labeling), the result is an image in which the three-dimensional structure of the exposed cytoskeletal components generates the contrast [ 191. Consequently, one application of photoelectron microscopy is to document overall alterations in cytoskeletal organization relative to other structural features within the cell. Figures 2A and 2B are photoelectron micrographs of cytoskeletal preparations of control and staurosporinetreated PTK2 cells, respectively. Untreated PTK2 cells, which grow outward from island-like confluent colonies, normally have a polygonal appearance with relatively straight boundaries between cells. PEM micrographs of unlabeled PTK2 cytoskeletal preparations show the cell-cell boundaries to have a ridgelike structure (black arrowheads, Fig. 2A, inset). The cytoplasmic region of PTK2 cells is characterized by high densities of straight semi-parallel arrays of microfilament bundles which appear in PEM micrographs as long filaments (white arrows, Fig. 2A, inset and enlargement). Cells spreading onto the substrate at the edge of the colony also have a peripheral band of fine actin filament bundles which can be seen in the upper right portion of Fig. 2A (inset and enlargement). In contrast to control cells, PTK2 cells exposed to 40 nM staurosporine for 20 min have two marked differences which are readily apparent in PEM images. One is that the cells in the colony lose their
ET AL.
polygonal appearance (Fig. 2B). The cell-cell boundaries retain their thick, ridgelike structure but become wavy and curved (black arrowheads, Fig. 2B, inset and enlargement). The second is that there is a drastic decrease in the population of microfilament bundles in staurosporine-treated compared with that in control cells (Figs. 2B and 2A, respectively). Cells along the edge of the colony lose their peripheral band of actin filaments (not shown), and all of the cells in the staurosporine-treated culture lose the classic stress fiber array of normal PTK2 cells. Despite these major alterations in actin patterns, immunofluorescence microscopy of parallel cultures revealed that the intermediate filament and microtubule systems remain intact and spread in staurosporinetreated cells (not shown). Although microtubules are not well-preserved by the detergent treatment used to prepare the PEM samples of Figs. 2A and 2B, intermediate filaments remain intact. These are particularly noticeable in the staurosporine-treated cells of Fig. 2B (white arrowheads), where actin bundles are not present to obscure them. In order to employ photoelectron microscopy to selectively observe the effects of staurosporine on microtubules, control and staurosporine-treated cultures were also prepared by a procedure optimal for microtubule stabilization. These samples were labeled with specific antibodies followed by protein G bound to 6 nm colloidal gold (subsequently silver enhanced) and are shown in Figs. 3A and 3B. In photoelectron images, the silver-enhanced gold label is more photoemissive than cellular material, resulting in bright labeled structures against a darker background. Labeled microtubules appear in both control and staurosporine-treated cells (Figs. 3A and 3B, respectively) as an abundance of filaments heavily decorated with fine bright dots. We noted some distortions in the microtubule patterns in staurosporine-treated cells (Fig. 3B). This may possibly be due to a decreased stability of microtubules dehydrated in the absence of microfilament bundles. Visible in the background of Figs. 3A and 3B and indicated by fat white arrows is the ridge of the cell-cell boundary, which is characteristically wavy after staurosporine treatment (Fig. 3B). The involvement of PKC in staurosporine-induced actin reorganization was investigated by asking whether staurosporine also alters actin microfilaments in cells made deficient in PKC. To “down-regulate” PKC, quiescent Swiss 3T3 cells were exposed to 200 nM PMA in spent culture medium for 30 h (Figs. 4D-4F), or to 300 nM PMA for 72 h (not shown). Controls were exposed for the same period of time to the equivalent amount of DMSO solvent in spent culture medium (Figs. 4A-4C. Thirty hours of exposure to 200 nM PMA is reportedly sufficient to reduce Swiss 3T3 protein kinase C levels below the level of detection by kinase assay [21]. To verify this for our cultures of Swiss 3T3, in a previous study
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Photoelectron micrographs of (A) control and (B) staurosporine-treated (40 nM for 20 min) PTK2 cells after use of neutral ,2 ,nt expose cytoskeletal elements. Lower magnification overviews are inset. Arrows indicate intact microfilament handlet I in the control (-4 which are absent in the treated culture (B); black arrowheads indicate cell-cell boundaries which become way Y after staurc lsporine mt ntermediate filaments (IF) remain intact and are visible in the staurosporine-treated samples (B).
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F‘IG. 3. Photoelectron micrographs of (A) control and (B) staurosporine-treated (40 nM for 20 min) PTK2 cytoskeletal preparai tions in whi ch microtubules (MT) were decorated with antitubulin antibodies and Protein G bound to 6 nm colloidal gold. The microtubule ren lain8 spread after staurosporine treatment although minor distortions are visible. Fat white arrows delineate the boundaries betweer 1 cells.
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actin microfilaments in quiescent Swiss 3T3 (:ells befo re (A-C) and FIG. 84. Fluore scence microscopy of rhodamine-phalloidin-stained after (D- -F) PMA e,xposure sufficient to down-regulate (deplete) protein kinase C. A and D are controls; B and E were cl ‘nged W ,ith 200InM PMA for .30 min; C and F were challenged with 20 nhf staurosporine for 30 min.
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we monitored the PMA-stimulated phosphorylation of the 80K putative PKC substrate protein during prolonged exposure of 3T3 cultures to PMA [ 131. Phosphorylation of the 80K protein has been described as a reliable marker of PKC activation in Swiss 3T3 cells [22]; thus, observation of PMA-stimulated phosphorylation of the 80K protein serves as a means of monitoring depletion of PKC from the cultures. We found that after 30 h exposure to 200 nM PMA, no 80K phosphorylation could be observed after addition of fresh PMA, indicating effective PKC down-regulation. Swiss 3T3 cells initially experience major actin cytoskeletal alterations after addition of PMA to culture medium, but these changes are transient. After several hours, microfilament bundles reform and are abundant by 30 h, as shown by fluorescence with rhodamine-phalloidin (Fig. 4D). The only notable difference between control and long-term PMA-treated cultures is that after chronic PMA treatment the “cobblestone” morphology that is characteristic of confluent, contact-inhibited 3T3 cells is greatly reduced, and many crisscrossed cells appear (Fig. 4D). Challenge with fresh 200 nM PMA for 30 min generates no significant actin disruption in down-regulated cells (Fig. 4E), while typical PMA-induced alterations are seen in controls (Fig. 4B). This is consistent with current thinking that PMA induces actin reorganization by a protein kinase C-dependent pathway. In the absence of PKC, freshly added PMA fails to alter actin patterns in Swiss 3T3 cells. This is not true of added staurosporine. Figures 4C and 4F compare control and down-regulated cells with respect to the actin alterations induced by 1 h of 20 nM staurosporine. Disruption of microfilament bundles and distorted cellcell boundaries are pronounced effects in both cultures. This was also true for cultures exposed to 300 nM PMA for 72 h (not shown), which is a more stringent condition for generating PKC-deficient cells. These results indicate that interaction with protein kinase C is not a plausible mechanism for the actin reorganization induced by staurosporine. DISCUSSION One of the major signal transduction pathways thought to be involved in cellular proliferation and differentiation is mediated by protein kinase C [23]. This kinase exists in a variety of mammalian and other cells as several different isoenzymes, all with an apparent molecular weight of approximately 80 kDa. PKC is activated as part of a quaternary complex containing, in addition to the kinase, diacylglycerol, phospholipid, and Ca2+. After tryptic digestion of native PKC, a 30-kDa regulatory domain and a 50 kDa catalytic domain have been isolated [24, 251. The regulatory domain is capable of binding phospholipid, diacylglycerol, and phorbol es-
ET AL.
ters, while the catalytic domain binds ATP and substrate protein. Much recent work has dealt with the question of whether or not particular signal transduction processes are mediated through PKC. Two approaches have been widely used to answer this question: (1) Down-regulation of protein kinase C, and (2) the use of inhibitors of protein kinase C. Down-regulation of PKC refers to the disappearance of kinase activity and immunoreactivity as a result of prolonged exposure to active phorbol esters (e.g., PMA or phorbol 12,13dibutyrate) and has been observed in many cell types. In Swiss 3T3 cells, for example, continuous exposure to 200 nM active phorbol 12,13-dibutyrate for 24 h reduces PKC activity to less than 10% of that in untreated cells and for 40 h results in the virtually complete disappearance of PKC activity [21]. The down-regulation process does not occur to the same extent in all cell types [26] and is not yet fully understood. However, PKC translocates from the cytosol to the plasma membrane in cells exposed to phorbol esters, and there is evidence to support the possibility that PKC is then proteolytically degraded by specific proteases present in the plasma membrane [27, 281. In cell systems where down-regulation has been shown to result in the complete depletion of protein kinase C, processes occurring in response to agonists (including hormones, drugs, growth factors, or neurotransmitters) are generally considered to occur via PKC-independent pathways. In an alternate approach, a major effort has gone into the development of specific inhibitors of PKC. Inhibitors which interact predominantly with either the regulatory domain (chlorpromazine [29], tamoxifen [30], palmitoyl carnitine [31], sphingosine [32]) or the catalytic domain (H-7 [33], staurosporine [ 71, sangivamycin [34], the K252 compounds [35]) have been described. Numerous studies have made use of inhibitors to examine the role of PKC in the regulation of various cellular functions. A significant problem with the inhibitors of PKC reported to date has been a lack of specificity with many of these compounds in in vitro kinase inhibition studies. This has been particularly well-documented with the inhibitors which bind to the catalytic domain of PKC, where inhibition of cyclic AMP- and cyclic GMPdependent kinases as well as myosin light chain kinase has been demonstrated [9, 33, 351. Similar selectivity problems have been noted with inhibitors which affect the regulatory domain [36,37]. This nonspecificity of inhibition, coupled with the fact that the cell does not provide the same defined environment as the assay system, may be responsible for a variety of anomalous results reported in studies on intact cells. In the signal transduction field, there are multiple examples in which “protein kinase C inhibitors” have effects which are not replicated by or are opposite to those induced by other PKC inhibitors. Some of these
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include: Inhibition of PMA-induced platelet aggregation at sphingosine concentrations lo-fold lower than those required to inhibit PKC in platelets [37]; cyclic nucleotide- and PKC-independent effects of H-7 on murine T cell hybridoma proliferation [38]; staurosporine mimicry of tumor promoter effects with respect to induction of ornithine decarboxylase [12], murine epidermal cell differentiation [ 111, and morphological changes in astrocytes [39]; and our observation of actin microfilament disruption induced by H-7, even in PKC-deficient cells [13]. In this study we have compared the effects on the actin cytoskeleton of cultured cells due to one of the most potent inhibitors of PKC known, staurosporine, with those of the tumor promoter and widely used activator of protein kinase C, PMA. The effects resulting from treatment with PMA and staurosporine are similar, though not identical: both compounds cause a rapid and dramatic disruption of actin stress fibers. However, the actin-labeling patterns of staurosporine-treated cells are distinguishable from those of PMA-treated cells (i.e., Fig. l), and strongly resemble those induced by the kinase inhibitor H-7 [13]. The actin patterns in cells exposed to both PMA and either H-7 or staurosporine are suggestive of an additive effect (Fig. 1D). Given the opposing effects of staurosporine and PMA on PKC activity, it would seem reasonable that these agents would alter actin organization by different pathways. To clarify whether PKC is involved in the staurosporine-induced alterations, we used the well-documented process of phorbol ester down-regulation to make Swiss 3T3 cells deficient in PKC. The initial exposure to PMA results in actin reorganization, but stress fibers reform within 12 h and do not change significantly in appearance after that. Consistent with PKC downregulation, addition of fresh PMA to these cells after 30 h generates no notable actin microfilament disruption. Staurosporine (20 nM), on the other hand, results in substantial disruption of the actin stress fiber network in both control and PMA-treated cells. Because of the nearly identical effects of staurosporine on cells containing active PKC and those made deficient in PKC, we conclude that staurosporine-induced disruption of the actin cytoskeleton occurs through a PKC-independent mechanism. Both the types of cytoskeletal changes induced and the PKC-independence of the observed effects described here for staurosporine are comparable to those described in our previous study of H-7 [13]. Nevertheless, alterations in the actin cytoskeleton of cultured cells are not a feature of all kinase inhibitors. We have previously observed that 45 min exposure to 200 ~Mofeither HA I004 or the relatively specific PKC inhibitor sangivamycin does not have this effect [13]. The kinase inhibitor K252a, however, has a significant effect on PTK2 actin after 30 min at 1 pM (unpublished observations, this lab-
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oratory). This may reflect the structural similarity between K252a and staurosporine. In contrast, the similarities in H-7 and staurosporine-induced effects on actin reorganization are somewhat surprising in view of the very different structures of the two compounds (staurosporine is a microbial alkaloid [7], whereas H-7 is an isoquinolinesulfonamide derivative [33]). Consistent with these structural differences are recent studies in which different effects resulting from the use of these two compounds have been reported [ 11,121. In the case of reorganization of the actin cytoskeleton, however, not only are the alterations induced by H-7 and staurosporine very similar, but the concentration range in which significant effects first appear reflects their kinase inhibition constants. For example, staurosporine and H-7 inhibit PKC with reported Ki values of 2.7 nM [7] and 6 pM [33], respectively, and we observed actin alterations with 2050 nM staurosporine and 15-60 pM H-7. The role of kinases in eukaryotic cells is far from being completely understood, but there is some evidence for kinase-mediated control of the actin cytoskeleton. The best-known case is that of myosin light chain kinase, which maintains actin microfilament integrity through phosphorylation of myosin light chain [40]. Cyclic AMP-dependent protein kinase, in turn, can indirectly cause dissociation of actin microfilaments by phosphorylating and inhibiting myosin light chain kinase [40,41]. The complexity of phosphorylation pathways makes it difficult to sort out the exact mechanisms of action of staurosporine and H-7. Although the question remains open as to whether inhibition of kinases could be involved, PKC has essentially been ruled out as a mediator for the actin-associated effects of staurosporine and H-7. We are grateful to Dr. Taksuya Tamaoki for the generous gift of staurosporine and thank Amy Hatch for expert photographic assistance. This study was made possible by Grant CA 11695 from the National Cancer Institute. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
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