Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments.

To analyze the cell cycle-dependent desmin phosphorylation by Rho kinase, we developed antibodies specifically recognizing the kinase-dependent phosphorylation of desmin at Thr-16, Thr-75, and Thr-76. With these antibodies, phosphorylation of desmin was observed specifically at the cleavage furrow in late mitotic Saos-2 cells. We then found that treatment of the interphase cells with calyculin A revealed phosphorylation at all the three sites of desmin. We also found that an antibody, which specifically recognizes vimentin phosphorylated at Ser-71 by Rho kinase, became immunoreactive after calyculin A treatment. This calyculin A-induced interphase phosphorylation of vimentin at Ser-71 was blocked by Rho kinase inhibitor or by expression of the dominant-negative Rho kinase. Taken together, our results indicate that Rho kinase is activated not only in mitotic cells but also interphase ones, and phosphorylates intermediate filament proteins, although the apparent phosphorylation level is diminished to an undetectable level due to the constitutive action of type 1 protein phosphatase. The balance between intermediate filament protein phosphorylation by Rho kinase and dephosphorylation by type 1 protein phosphatase may affect the continuous exchange of intermediate filament subunits between a soluble pool and polymerized intermediate filaments.

Like other of the Ras superfamily of small GTPases, Rho acts as a molecular switch to control a variety of cellular processes: it regulates signal transduction pathways linking extracellular stimuli to the assembly of actin stress fibers and focal adhesion complexes; it is required for G 1 progression and activates serum response factor transcription factor when quiescent fibroblasts are stimulated to grow; and it plays a role in cell cycle during cytokinesis (for reviews, see Refs. [1][2][3]. Much effort has been directed toward identifying target proteins for Rho that mediate the various biological activities of the protein. Several target proteins that interact only with active, GTP-bound Rho have been identified, including protein kinase N (4,5), Rho kinase/ROK␣/ROCKII (6 -8), citron kinase (9), rhophilin (5), rhotekin (10), and mDia (11). Among them, Rho kinase has been reported to be involved in several of the cellular processes mentioned above: regulation of myosin phosphorylation (12)(13)(14), formation of stress fibers and focal adhesions (15)(16)(17), neurite retraction (18 -21), and cytokinesis (22)(23)(24).
Intermediate filaments (IFs) 1 constitute major components of the cytoskeleton and the nuclear envelope in most cell types (for a review, see Ref. 25). Although IFs were thought to be relatively stable as compared with other cytoskeletons such as actin filaments and microtubules, intensive in vitro investigations revealed that site-specific phosphorylation by several kinases, such as protein kinase A, protein kinase C (PKC), Ca 2ϩ / calmodulin kinase II (CaMKII), and cdc2 kinase, dynamically alters their filament structure (26) (for reviews, see Refs. [27][28][29]. Thereafter, some of the above kinases were found to be in vivo IF kinases, using site-and phosphorylation state-specific antibodies that recognize a phosphorylated Ser/Thr residue and its flanking sequence; cdc2 kinase is activated in early mitotic cells, PKC is activated from metaphase to anaphase, and CaMKII is activated in response to receptor-mediated phosphoinositide hydrolysis (30 -32). Rho kinase also has been identified as an in vivo IF protein kinase, which site-specifically phosphorylates glial fibrillary acidic protein and vimentin at a cleavage furrow during cytokinesis (22)(23). We have shown that Rho kinase plays an essential role in efficient segregation of glial filaments during cytokinesis because mutations in Rho kinase phosphorylation sites impaired segregation of glial filaments into daughter cells and consequently formed an unusually long bridge-like cytoplasmic structure between the daughter cells (24). We clarified that desmin, another type III IF protein, restrictedly expressed in smooth, cardiac, and skeletal muscles, also serves as a substrate for Rho kinase and identified Thr-16, Thr-75, and Thr-76 as the major phosphorylation sites in vitro (33). The intracellular localization of Rho kinase remains to be determined. In nonmuscle cells, this kinase, once activated, was found to translocate from the cytosol to plasma membrane (7). It was also reported that Rho kinase is colocalized with the vimentin filament in serumstarved fibroblasts, and when activated, it translocates to cell peripheral regions (34). Therefore, Rho kinase may be active at cell-cell and cell-substrate contact regions in interphase cells. This hypothesis is supported by several lines of experiments showing the Rho kinase-mediated regulation of ezrin, radixin, and moesin proteins (35,36), adducin (37), and focal complexes (15)(16)(17).
In the present work, we newly developed site-and phosphorylation state-specific antibodies for the three Rho kinase phosphorylation sites of desmin in order to analyze the physiological significance of desmin phosphorylation by Rho kinase. Using these antibodies, we found that all the three sites were phosphorylated specifically at the cleavage furrow during cytokinesis. This evidence strongly supports our proposal that Rho kinase acts as a cleavage furrow kinase for IF proteins.
Although various kinases are activated spatiotemporally during the cell cycle and phosphorylate IF proteins, it has been suggested that protein phosphatase is also important for maintenance of the filament structure and plasticity because the IF structure is immediately altered when the cells are treated with protein phosphatase inhibitors (38 -41).
We also investigated novel functional aspects of Rho kinase on desmin and vimentin in interphase cells, using the phosphatase inhibitors calyculin A (CA) and ocadaic acid (OA). Rho kinase is active to some extent in interphase cells, as well as mitotic cells, although the phosphorylation is apparently masked due to effects of type 1 protein phosphatase (PP1). Our data allow for a new proposal regarding dynamic exchange of type III IF subunits between a soluble pool and the polymerized IFs: Rho kinase-and PP1-mediataed IF protein phosphorylation and deposphorylation, respectively, may influence steady state equilibrium.
Cell Culture and Transfection-Human osteosarcoma Saos2 cells (a gift from Dr. H. Saya, Kumamoto University, Kumamoto, Japan), MDCK cells and NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin, and streptomycin in an atmosphere of 5% CO 2 . Saos2 cells were seeded on coverslips in six-well plates at 1 ϫ 10 5 cells/well, and the next day, cells were transfected with pEF-BOS-Myc-RhoK-CAT, using Lipo-fectAMINE Plus™ (Life Technologies, Inc.). Forty-eight h after transfection, the cells were fixed for immunocytochemistry. In some experi-ments, Saos-2, MDCK, and NIH3T3 cells were seeded on 100-mm plates at 1 ϫ 10 6 cells/plate or 24-well plates at 1 ϫ 10 4 cells/well; the next day, the cells were treated with various concentrations of CA or OA, and then cells were harvested for immunoblot analysis or were fixed for immunocytochemistry.
Immunocytochemistry and Immunoblotting-Cells fixed with 3.7% formaldehyde/phosphate-buffered saline for 10 min, followed by treatment with methanol for 10 min at Ϫ20°C, were stained with rabbit polyclonal antibodies (␣-PD16, ␣-PD75, ␣-PD76, ␣-desmin, ␣-Rho kinase, and GK71) and mouse monoclonal antibodies (9E10 and ␣-vimentin) for 2 h at 37°C. The immunoreactivities were visualized by incubation with Alexa TM 488 goat anti-rabbit antibody (Molecular Probes) and with FluoroLink TM Cy TM 2 labeled goat anti-mouse antibody (Amersham Pharmacia Biotech) for 1 h at 37°C, and then the samples were examined under a confocal microscope (Olympus, LSM-GB200). For immunoblotting, lysate of 1 ϫ 10 5 CA-or OA-treated cells were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylide difluoride membranes (Immobilon-P, Millipore). The membranes were then incubated with polyclonal and monoclonal antibodies, as described above, for 2 h at room temperature, and visualized by making use of horseradish peroxidase-conjugated anti-rabbit or antimouse antibodies (Amersham Pharmacia Biotech) and the ECL immunoblotting detection system (Amersham Pharmacia Biotech). In some experiments, quantification of the phosphorylation level was done using laser densitometry, and the level was expressed in arbitrary optical density units. In cell suspension experiments, NIH3T3 cells were detached from culture dishes by trypsin treatment. Cells were then once washed by centrifugation, resuspended in the medium, and treated with various concentrations of CA or OA followed by immunoblot and immunocytochemical analyses.

Treatment with Rho Kinase Inhibitors and Microinjection of a Dominant Negative Rho Kinase Expression
Vector-NIH3T3 cells were seeded on 100-mm plates at 1 ϫ 10 6 cells/plate or 24 well plates at 1 ϫ 10 4 cells/well; next day, the cells were preincubated with various concentrations of HA1077 or Y-27632 for 30 min, and then CA was added (final concentration, 20 nM). After treatment with CA for for 15 min, the cells were harvested for immunoblot analysis or were fixed for immunocytochemistry. For microinjection, NIH3T3 cell were seeded on 13-mm glass coverslips at 1 ϫ 10 4 cells/slip. The next day, pEF-BOS-Myc-RB/PH(TT) (0.1 mg/ml) or pEF-BOS-Myc-COIL (0.1 mg/ml) was microinjected into the nuclei of the cells. Twenty-four h after injection, the cells were treated with 20 nM CA for 15 min and were fixed for immunocytochemical analysis.
Phosphorylation of Desmin-Thr-16, Thr-75 and Thr-76 by Active Rho Kinase in Saos-2 Cells-To investigate the phospho-rylation of desmin by Rho kinase in cells, we asked whether Rho kinase could phosphorylate Thr-16, Thr-75, and Thr-76 of desmin in Saos-2 human osteoblast cells ectopically expressing the constitutively active version of Rho kinase (Fig. 2). Cells were transiently transfected with pEF-BOS-Myc mammalian expression vector encoding the constitutively activated catalytic domain of bovine Rho kinase (RhoK-CAT). The cells were double-stained with monoclonal antibody 9E10 for the Myc epitope-tagged RhoK-CAT and ␣-Des ( Fig. 2A), ␣-PD16 (Fig.  2B), ␣-PD75 (Fig. 2C), or ␣-PD76 (Fig. 2D). Immunocytochemical analyses revealed that phosphorylation of Thr-16, Thr-75, and Thr-76 of desmin occurred in cells expressing RhoK-CAT (Fig. 2, B-D). As is the case with vimentin (23), phosphorylation of desmin by Rho kinase induces collapse, a dynamic change of desmin-IF organization (Fig. 2, A-D). This phosphorylation was not detected in the cells expressing the catalytic domain mutated at the ATP-binding site (CAT-KD; K121G) (data not shown), indicating that the kinase domain is essential for the desmin phosphorylation we observed. These results show that the constitutive active form of Rho-kinse phosphorylates all three in vitro Rho kinase phosphorylation sites of desmin in cells.
Specific Phosphorylation of Desmin-Thr-16, Thr-75, and Thr-76 at Cleavage Furrow during the Cell Cycle-In the next set of experiments, based on the foregoing biochemical and immunocytochemical observation that Thr-16, Thr-75, and Thr-76 of desmin are in vivo phosphorylation sites by Rho kinase, the spatial and temporal distribution of the three phosphorylated sites in Saos-2 cells was analyzed using ␣-PD16, ␣-PD75, and ␣-PD76. Under the conditions used, all the immunoreactivity of ␣-PD16, ␣-PD75, and ␣-PD76 was detected only in late mitotic cells and specifically at the cleavage furrow ( Fig.  3) but not in interphase cells (Fig. 4A) or in early mitotic cells, such as prometaphase or metaphase (data not shown). When desmin was stained with ␣-Des, which reacts with phosphorylated and unphosphorylated desmin, the filamentous structures were observed in mitotic daughter cells (Fig. 3) and in interphase cells (Fig. 4A). In late mitotic Saos-2 cells, Rho kinase also accumulates specifically at the cleavage furrow, as illustrated in Fig. 3, findings that strongly suggest a direct interaction of the kinase with desmin. From these results, we conclude that Rho kinase acts as desmin kinase at the cleavage furrow during cytokinesis. On the basis of the in vitro observation that phosphorylation of desmin by Rho kinase inhibits filament formation (33), we propose that desmin filaments are phosphorylated by Rho kinase and depolymerized at the cleavage furrow during cytokinesis, and hence the segregation of desmin into daughter cells is efficient.
Phosphorylation of Thr-16, Thr-75, and Thr-76 of Desmin in Interphase Cells Treated with Calyculin A-Phosphorylation of IF proteins may play a role in IF structural organization. Rho kinase was found to be co-localized with IF filament in interphase cells (34). To assess the possible function(s) of Rho kinase in IF reorganization, we next analyzed the activation state of Rho kinase on desmin in interphase cells. Intact interphase cells showed a characteristic network of desmin filaments spanning the cytoplasm, determined using an anti-desmin antibody (Fig. 4A, ␣-Des, left panel). After treatment with the phosphatase inhibitor CA (20 nM) for 20 min, cells began to round up, the organization of desmin filament was altered, and the IF network collapsed to form a desmin-containing arc near the perinuclear region (Fig. 4A, ␣-Des, right panel). All the immunoreactivity of ␣-PD16, ␣-PD75, and ␣-PD76 appeared in interphase cells treated with CA (Fig. 4A, right panels). Although the staining patterns varied, the filamentous structure was clearly disrupted and the desmin filaments had collapsed. To confirm that Thr-16, Thr-75, and Thr-76 of desmin were phosphorylated in response to CA, immunoblot analysis was performed. As shown in Fig. 4B, ␣-PD16, ␣-PD75, or ␣-PD76immunoreactive band was observed when interphase cells were treated with CA. Taken together, it is most likely that Rho kinase is to some extent activated even in interphase cells, and an unidentified CA-sensitive phosphatase causes the rapid phosphate turnover on IFs.
Phosphorylation of Vimentin by Rho Kinase in Response to Calyculin A-Because we determined that Ser-71 of vimentin is a specific site for Rho-kinse, and Ser-33, Ser-55, and Ser-82 residues of vimentin are sites specific for PKC, cdc2 kinase, and CaMKII, respectively (29), vimentin is a suitable substrate to verify that Rho kinase, rather than other kinases, phosphorylates IF proteins and regulates their structure and plasticity in interphase cells. We next examined phosphorylation states of vimentin, widely expressed in culture cells, in response to CA, using several types of site-and phosphorylation state-specific antibodies for vimentin. We found that the phosphorylation of Ser-71 appeared in the vimentin filament (Fig. 5) determined using an antibody, GK71, that recognizes the phosphorylation of vimentin-Ser-71 (23). On the other hand, the phosphorylation of Ser-33, Ser-55, and Ser-82 was not observed, and it did not increase in interphase cells treated with CA, respectively (Fig. 5), indicating that PKC and cdc2 kinase are not involved in vimentin phosphorylation in interphase Saos-2 cells. CaMKII activity was to some extent detectable but the level was less altered by CA treatment. Moreover, we found an increase in the phosphorylation level of other Rho kinase substrates, such as the myosin binding subunit of myosin phosphatase and myosin light chain (data not shown). Collectively, we concluded that Rho kinase is mainly responsible for IF protein phosphorylation in interphase Saos-2 cells.
To determine whether CA-induced vimentin phosphorylation by Rho kinase could be observed in other cells, MDCK cells and NIH3T3 cells were used (Fig. 6, B and C). When MDCK  and NIH3T3 cells were treated with 25 nM CA for 20 min and 20 nM CA for 15 min, respectively, the morphology of the vimentin filament network and phosphorylation state of vimentin-Ser-71 were analyzed. Fig. 6, B and C, shows that these cells also began to round up, and the vimentin organization was altered. GK71 reacted with vimentin in these interphase cells (Fig. 6, B and C). In Fig. 6D, the CA-induced vimentin-Ser-71 phosphorylation was also confirmed by immunoblot analysis. As in Saos-2 cells, the phosphorylation of Ser-33 and Ser-55 was not observed in MDCK and NIH3T3 cells treated with CA (data not shown). The weak phosphorylation of Ser-82 was less altered (data not shown). These results suggest that Rho kinase-mediated phosphorylation of vimentin in response to CA is a general feature in vimentin-expressing cells.
Different Dose Response Effects of Calyculin A and Okadaic Acid on the Immunoreactivity of GK71-We then used immunoblots to examine the appearance of vimentin phosphorylation by Rho kinase in NIH3T3 cells using OA, another potent inhibitor of PP1 and type 2A protein phosphatase (PP2A). There were obvious differences between the dose-response effects of CA and OA (Fig. 7). The immunoreactivity of GK71 appeared first at a dose range of 10 -20 nM in CA-treated cells, but at 3-10 M in OA-treated cells. An approximately 100-fold difference in inhibitory sensitivity would mean that OA has a 50 -100-fold weaker effect than CA on PP1. OA and CA are reported to inhibit PP2A with a similar potency (45,46). In the course of this experiment, we found that CA-treated cells became round and readily detached from culture dishes, as com-pared with findings in case of OA treatment. Thus, the GK71 immunoreactivity detected in the above experiments may reflect morphological change of the cells rather than phosphatase inhibition. To rule out this possibility, we did the same experiments using suspended and round NIH3T3 cells instead of those attached to culture dishes. Consequently, CA and OA showed comparable effects on GK71 reactivity of suspended and round NIH3T3 cells to findings in the cells attached to culture dishes (data not shown). From these data, taken together, we conclude that PP1 dephosphorylates Ser-71 of vimentin in intact interphase cells.

Effects of Rho Kinase Inhibitors and Expression of the Dominant Negative Form of Rho Kinase on Vimentin Phosphorylation in Response to Calculin A-Recently
, it has been reported that chemical compounds such as HA1077 and Y-27632 selectively inhibit the activity of Rho kinase (47)(48)(49). To confirm that Ser-71 of vimentin is phosphorylated by Rho kinase and not other kinases in interphase cells treated with CA, we investigated the effects of the two Rho kinase inhibitors. NIH3T3 cells were preincubated with these inhibitors (10 M) for 30 min and then treated with CA and double-stained with ␣-vimentin and GK71. When cells were preincubated with the inhibitors, CA-induced immunoreactivity of GK71 was completely blocked ( Fig. 8A). Immunoblot analyses also revealed that GK71-immunoreactivity was inhibited by both of HA1077 and Y-27632 (Fig. 8B). Furthermore, a dominant-negative form of Rho kinase, RB/PH(TT), was used to confirm that the Ser-71 was phsophorylated by Rho kinase. Twenty-four hours after the microinjection of RB/PH(TT) expression plasmid into nuclei of NIH3T3 cells, the cells were treated with 20 nM CA for 15 min and analyzed using 9E10 for the Myc epitope-tagged RB/ PH(TT) and GK71. When cells were microinjected with pEF-BOS-COIL, which contains the coil domain of Rho kinase, as a control, GK71-immunoreactivity was observed both in COILexpressing cells (Fig. 9A, arrow) and nonexpressing cells (Fig.  9A, arrowhead). On the other hand, in cells expressing RB/ PH(TT) the GK71 immunoreactivity was not observed after CA treatment (Fig. 9B, arrow). Under these conditions, Ser-71 phosphorylation was detected in cells not expressing of RB/ PH(TT) (Fig. 9B, arrowhead). We conclude that Rho kinase phosphorylates vimentin at Ser-71, in response to CA in interphase cells. Our results strongly suggest that the constitutive phosphorylation of IF proteins by Rho kinase occurs significantly, whereas a more extensive phosphate turnover occurs on IFs by PP1 in interphase cells. DISCUSSION We earlier clarified that desmin can serve as a good substrate for Rho kinase in vitro (33). In the present study, using newly developed site-and phosphorylation state-specific antibodies for Rho kinase phosphorylation sites of desmin, Rho kinase is found to phosphorylate desmin specifically at the cleavage furrow during cytokinesis, as is the case with other type III IF proteins, glial fibrillary acidic protein, and vimentin (22,23). We have found that Rho kinase itself accumulates at the cleavage furrow in Saos-2 human osteoblast cells, as was noted in U251 human glioma cells (42). Thus, it is a common phenomenon for all type III IF proteins that Rho kinase acts as a cleavage furrow kinase, and it is most likely that Rho kinase regulates the organization of these IFs during cytokinesis via a common molecular mechanism, which ensures effective segregation of IFs into daughter cells. Citron kinase, another Rho target protein with a kinase domain homologous to that of Rho kinase, was also found to localize to the cleavage furrow and the midbody of Hela cells (50). Citron kinase is thought to work in the contractile process rather than actin reorganization (50). The different and redundant functions of Rho kinase and citron kinase in cytokinetic process remain to be elucidated. These two kinases may participate in different steps during cytokinesis because citron kinase does not phosphorylate any type III IF proteins in the conditions under which Rho kinase does. 2 To date, little is known of precise molecular mechanisms of spatiotemporal Rho kinase activation in cytokinesis. It is possible that Rho kinase is recruited to the cleavage furrow and is activated during cytokinesis. Another possibility is that accumulation of Rho kinase with basal activity at the cleavage furrow in late mitotic cells, but not local activation of the kinase at the cleavage furrow, is sufficient for exerting its role in cytokinesis. Regardless, in both cases, when net Rho kinase activity becomes dominant over phosphatase activity due to accumulation and/or activation at the cleavage furrow, Rho kinase phosphorylates various substrates (such as IF proteins, myosin light chain, and ezrin, radixin, and moesin) and facilitates cytokinesis.
It has been reported that phosphatases play an essential role for the maintenance and structural integrity of IFs in interphase cells, determined using the protein phosphatase inhibitors CA and OA. CA and OA are potent inhibitors for PP1 and PP2A and are often used to demonstrate the involvement of PP1 and PP2A in a biological cellular processes. CA and OA inhibit PP2A with a similar potency, whereas OA is 50 -100fold weaker than CA as a PP1 inhibitor. CA and OA are weakly sensitive and completely insensitive to other phosphatases, protein phosphatases 2B and 2C, respectively. In the course of experiments on desmin/vimentin phosphorylation with siteand phosphorylation state-specific antibodies, we investigated the effects of CA and OA and determined that PP1 functions as a desmin/vimentin phosphatase. PP1 is known to play a pivotal role in the cell cycle and is regulated by the interaction of the catalytic subunit with a variety of regulatory proteins that have as an important role to localize the enzyme and to determine substrate specificity. The involvement of PP1 in IF integrity was suggested for vimentin when CA and OA were used (38). In neuronal cells, PP2A was identified to be a neurofilament-associated phosphatase that may preserve the filamen- tous structure of neurofilament (51). As for keratin 8/18, PP1 and/or PP2A is suggested to function in disassembly and reorganization (39,40). Although protein phosphatases responsible for IF organization have been noted in some cell systems, as mentioned above, the kinase(s) that phosphorylates IFs in interphase cells is unknown. Only CaMK may be a candidate for a major role in keratin 8/18 phosphorylation in interphase cells (39). Utilizing a series of site-and phosphorylation state-specific antibodies, we have shown here that Rho kinase phosphorylates desmin and/or vimentin in interphase Saos-2, NIH3T3, and MDCK cells, but the phosphorylation was evident only after CA treatment. In comparison, vimentin phosphorylation by PKC and cdc2 kinase did not appear in NIH3T3 cells even after CA treatment, and the phosphorylation level by CaMKII was not altered by CA treatment. Furthermore, we confirmed that Rho kinase induces phosphorylation of vimentin in CAtreated interphase NIH3T3 cells, using Rho kinase inhibitors (47) and expression of the dominant-negative form of Rho kinase, RB/PH(TT) (20); the phosphorylation of the Rho-kianse specific site, Ser-71, on vimentin was completely blocked by pretreatment with Y-27632, HA1077 or the ectopic expression of RB/PH(TT). Thus, Rho kinase functions as an interphase IF kinase (although dephosphorylation activity of PP1 on desmin and vimentin is thought to be higher than the phosphorylation activity by Rho kinase in interphase cells).
The present study sheds light on the tightly balanced activity of Rho kinase and PP1 in interphase cells. As for the biological significance of IF protein phosphorylation by Rho kinase observed here, one can speculate the modulation of IF protein dynamics. Ectopically expressed or microinjected IF proteins were found to be incorporated into preexisting IFs along their entire surface (52,53). Consistent with these observations, transient expression of assembly-deficient mutant keratin in epithelial cells (54) and microinjection of peptides derived from IF sequence motifs essential for IF assembly (55) disrupted the endogenous IF system. These results clearly indicate that IFs are highly dynamic, and a continuous exchange of subunit proteins occurs on the entire filament surface between a soluble pool and the polymerized IFs, reaching a steady state equilibrium (56). Mechanisms regulating the equilibrium state are unknown. Based on the results observed in this study, we consider that Rho kinase is one candidate that, together with PP1, modulates the equilibrium between a soluble IF protein pool and a polymerized protein pool. The balance of Rho kinase and PP1 activities seems essential not only for modulating IF structure and plasticity but also for cell-substrate adhesion, cell motility, and actin reorganization in interphase cells. Further analyses are required to clarify the molecular mechanism of the concerted action of Rho kinase and PP1 in individual cellular processes, including IF protein dynamics in interphase.
As for the effects of CA and OA observed on Rho kinase activity, it must be noted that CA and OA act as strong tumor promoters. It is possible that CA, and maybe OA, causes tumor promotion by disturbing the balance between Rho kinase and PP1 activities. If such is indeed the case, increments in Rho kinase activity by diminishing PP1 may explain some characteristic features of cancer cells, including abnormal growth, invasion, and metastasis.