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We previously reported that vimentin, GFAP, and desmin (type III intermediate filament [IF] proteins) are mitotically phosphorylated by CDK1, Aurora-B, and Rho-kinase. This phosphorylation is critical for efficient separation of these IFs and completion of cytokinesis. Keratin 5 (K5) and K14 form a heterodimer, which constitutes IF network in basal layer cells of stratified squamous epithelia. Here, we report that the solubility of K5/K14 increased in mitosis. The in vitro assays revealed that three mitotic kinases phosphorylate K5 more than K14. We then identified Thr23/Thr144, Ser30, and Thr159 on murine K5 as major phosphorylation sites for CDK1, Aurora-B, and Rho-kinase, respectively. Using site- and phosphorylation-state-specific antibodies, we demonstrated that K5-Thr23 was phosphorylated in entire cytoplasm from prometaphase to metaphase, whereas K5-Ser30 phosphorylation occurred specifically at the cleavage furrow from anaphase to telophase. Efficient K5/K14-IF separation was impaired by K5 mutations at the sites phosphorylated by these mitotic kinases. K5-Thr23 phosphorylation was widely detected in dividing K5-positive cells of murine individuals. These results suggested that mitotic reorganization of K5/K14-IF network is governed largely through K5 phosphorylation by CDK1, Aurora-B, and Rho-kinase.

The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial–mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid–vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

The conserved serine-threonine kinase, Cdc7, plays a crucial role in initiation of DNA replication by facilitating the assembly of an initiation complex. Cdc7 is expressed at a high level and exhibits significant kinase activity not only during S-phase but also during G2/M-phases. A conserved mitotic kinase, Aurora B, is activated during M-phase by association with INCENP, forming the chromosome passenger complex with Borealin and Survivin. We show that Cdc7 phosphorylates and stimulates Aurora B kinase activity in vitro. We identified threonine-236 as a critical phosphorylation site on Aurora B that could be a target of Cdc7 or could be an autophosphorylation site stimulated by Cdc7-mediated phosphorylation elsewhere. We found that threonines at both 232 (that has been identified as an autophosphorylation site) and 236 are essential for the kinase activity of Aurora B. Cdc7 down regulation or inhibition reduced Aurora B activity in vivo and led to retarded M-phase progression. SAC imposed by paclitaxel was dramatically reversed by Cdc7 inhibition, similar to the effect of Aurora B inhibition under the similar situation. Our data show that Cdc7 contributes to M-phase progression and to spindle assembly checkpoint most likely through Aurora B activation.

SummaryThe chromosome passenger complex (CPC) must relocate from anaphase chromosomes to the cell equator for successful cytokinesis. Although this landmark event requires the mitotic kinesin MKlp2, the spatiotemporal mechanistic basis remains elusive. Here, we show that phosphoregulation of MKlp2 by the mitotic kinase Cdk1/cyclin B1 coordinates proper mitotic transition with CPC relocation. We identified multiple Cdk1/cyclin B1 phosphorylation sites within the stalk and C-terminal tail that inhibit microtubule binding and bundling, oligomerization/clustering, and chromosome targeting of MKlp2. Specifically, inhibition of these abilities by Cdk1/cyclin B1 phosphorylation is essential for proper early mitotic progression. Upon anaphase onset, however, reversal of Cdk1/cyclin B1 phosphorylation promotes MKlp2-CPC complex formation and relocates the CPC from anaphase chromosomes for successful cytokinesis. Thus, we propose that phosphoregulation of MKlp2 by Cdk1/cyclin B1 ensures that activation of MKlp2 kinesin and relocation of the CPC occur at the appropriate time and space for proper mitotic progression and genomic stability.

Background: Vimentin, an intermediate filament (IF) protein, is phosphorylated in mitosis. Results: Disruption of vimentin phosphorylation during cell division leads to chromosomal instability (CIN) and premature aging in mouse lens tissue. Conclusion: Our data document the first physiological importance of vimentin phosphorylation during mitosis for organogenesis and tissue homeostasis. Significance: Our data suggest a possible causal relationship between CIN and premature aging., Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIMSA/SA) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIMWT/SA) were indistinguishable from WT (VIMWT/WT) mice. In VIMSA/SA mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIMSA/SA, reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIMSA/SA, the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.

Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.

Cytokinesis is regulated by several protein kinases, such as Aurora-B and Rho-kinase/ROCK. We have indicated that these two kinases are the cleavage furrow (CF) kinases that accumulate at the cleavage furrow and phosphorylate several intermediate filament (IF) proteins into two daughter cells. It has been reported that Aurora-B phosphorylates MgcRacGAP to functionally convert to a RhoGAP during cytokinesis. Therefore, we investigated here the relationship between Aurora-B and Rho-kinase/ROCK in cytokinesis, by using small interfering RNA (siRNA) technique. Aurora-B depletion did not alter the cleavage furrow-specific localization of Rho-kinase/ROCK and vice versa. Treatment of Aurora-B or Rho-kinase/ROCK siRNA increased multinucleate cells, and the effect of double depletion was additive. Aurora-B depletion induced the reduction of cleavage furrow-specific phosphorylation of vimentin at Ser72 but not vimentin at Ser71, myosin light chain (MLC) at Ser19, and myosin binding subunit of myosin phosphatase (MBS) at Ser852. In contrast, Rho-kinase/ROCK depletion led to the reduction of cleavage furrow-specific phosphorylation of MLC at Ser19, MBS at Ser852, and vimentin at Ser71 but not vimentin at Ser72. Cleavage furrow-specific ezrin/radixin/moesin (ERM) phosphorylation was not altered in the Aurora-B- and/or Rho-kinase/ROCK-depleted cells. In addition, C3 or toxin B treatment did not abolish ERM phosphorylation at the cleavage furrow in cells attaining cytokinesis. These results suggest that Aurora-B and Rho-kinase/ROCK regulate the progression of cytokinesis without communicating to each other, and there may exist a novel protein kinase which phosphorylates ERM at the cleavage furrow.

Aurora-B is a protein kinase required for chromosome segregation and the progression of cytokinesis during the cell cycle. We report here that Aurora-B phosphorylates GFAP and desmin in vitro, and this phosphorylation leads to a reduction in filament forming ability. The sites phosphorylated by Aurora-B; Thr-7/Ser-13/Ser-38 of GFAP, and Thr-16 of desmin are common with those related to Rho-associated kinase (Rho-kinase), which has been reported to phosphorylate GFAP and desmin at cleavage furrow during cytokinesis. We identified Ser-59 of desmin to be a specific site phosphorylated by Aurora-B in vitro. Use of an antibody that specifically recognized desmin phosphorylated at Ser-59 led to the finding that the site is also phosphorylated specifically at the cleavage furrow during cytokinesis in Saos-2 cells. Desmin mutants, in which in vitro phosphorylation sites by Aurora-B and/or Rho-kinase are changed to Ala or Gly, cause dramatic defects in filament separation between daughter cells in cytokinesis. The results presented here suggest the possibility that Aurora-B may regulate cleavage furrow-specific phosphorylation and segregation of type III IFs coordinatedly with Rho-kinase during cytokinesis.

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.

Background: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments. Results: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization. Conclusion: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation.

BACKGROUND: Histone H3 (H3) phosphorylation plays important roles in mitotic chromosome condensation. We reported that H3 phosphorylation occurs at Ser28, as well as at Ser10 during mitosis, at least in mammals. Aurora B was recently demonstrated to be responsible for Ser10 phosphorylation in S. cerevisiae, C. elegans, Drosophila and Xenopus egg extract. RESULTS: We compared the distribution of Aurora-B with that of H3 phosphorylation. Aurora-B was primarily localized in the heterochromatin of late G2 phase cells, where only Ser10 phosphorylation was observed. The treatment of such cells with calyculin A induced Ser28 phosphorylation in the Aurora-B-localized area. During prophase to metaphase, Aurora-B was distributed in condensing chromosomes where Ser10 and Ser28 were phosphorylated. Aurora-B can phosphorylate H3-Ser10 and -Ser28 in nucleosomes in vitro. Transfection of a dominant-negative mutant of Aurora-B resulted in a reduction of H3 phosphorylation, not only at Ser10 but also Ser28, during mitosis. CONCLUSIONS: With regard to mitotic chromosome condensation, Aurora-B directly phosphorylated H3, not only at Ser10 but also at Ser28. The level of Ser28 phosphorylation is diminished to undetectable levels by PP1 phosphatase prior to entry into mitosis.

Background Vimentin, which is one of the intermediate filaments, is the major cytoskeletal component in developing neurones or neuroblastoma cells. Rho-associated kinase (Rho-kinase), is rich in neurones and is found downstream of Rho. It is involved in the agonist-induced neurite retraction of neuronal cells, and phosphorylates vimentin at Ser-38 and Ser-71 resulting in in vitro disassembly of the filaments. Results We have investigated the distribution of vimentin phosphorylated by Rho-kinase in N2a neuroblastoma cells using site-specific phosphorylation-dependent antibodies. TM71 immunoreactivity, which specifically indicates Ser-71 phosphorylation on vimentin, was found in some neurites of dibutyryl cAMP-differentiated N2a cells. Transfection of the constitutively active form of Rho-kinase, CAT, significantly elevated TM71 immunoreactivity, and induced neurite retraction or cell rounding. Conversely, transfection of the dominant negative form of Rho-kinase, RB/PH(TT), or treatment of 10 μM Y-27632, a Rho-kinase specific inhibitor, abolished TM71 immuno-reactivity, and induced irregular neurite outgrowth. In contrast, 20 n M okadaic acid (OA) induced neurite retraction and specifically elevated TM71 immunoreactivity. In the OA-induced neurite retraction, tubulin disappeared in retracting neurites, where vimentin and actin remained co-localized. Furthermore, the OA-induced elevation of TM71 immunoreactivity and neurite retraction were completely blocked by pretreatment with 10 μM Y-27632, or by the ectopic expression of RB/PH(TT). Conclusions This study suggests that the localized phosphorylation of vimentin by Rho-kinase in neurites was closely related with the cellular morphology of N2a cells, and that the Rho-kinase activity towards vimentin was balanced with OA-sensitive phosphatases.

p21-activated kinase (PAK) and Rho-associated kinase (Rho-kinase) have been shown to induce Ca(2+)-independent contraction of smooth muscle. PAK-induced contraction of Triton-skinned smooth muscle correlates with increased phosphorylation of caldesmon and desmin, although the role of desmin phosphorylation has remained obscure. Here we report that desmin serves as an excellent substrate for PAK in vitro. PAK phosphorylated desmin in a GTP. Cdc42/Rac-dependent manner. Phosphorylation of desmin by PAK dramatically inhibited its filament-forming ability. PAK phosphorylated mainly serine residues of the head domain of desmin, and the major phosphorylation sites differed from those for Rho-kinase. These results suggest that different site-specific phosphorylation of desmin via two divergent protein kinases downstream of Rho family GTPases would seem to increase the regulatory potential for organization of desmin filaments.

Intermediate filaments (IFs), which form the structural framework of cytoskeleton, have been found to be dramatically reorganized during mitosis. Some protein kinases activated in mitosis are thought to control spatial and temporal IF reorganization through phosphorylation of IF proteins. Rho-associated kinase (Rho-kinase), one of the putative targets of the small GTPase Rho, does phosphorylate IF proteins, specifically at the cleavage furrow during cytokinesis. This cleavage furrow-specific phosphorylation plays an important role in the local IF breakdown and efficient separation of IF networks. Recent studies on Rho signaling pathways have introduced new models about the molecular mechanism of rearrangements of cytoskeletons including IFs during cytokinesis.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.

We found that vimentin, the most widely expressed intermediate filament protein, served as an excellent substrate for Rho-associated kinase (Rho-kinase) and that vimentin phosphorylated by Rho-kinase lost its ability to form filaments in vitro. Two amino-terminal sites on vimentin, Ser38 and Ser71, were identified as the major phosphorylation sites for Rho-kinase, and Ser71 was the most favored and unique phosphorylation site for Rho-kinase in vitro. To analyze the vimentin phosphorylation by Rho-kinase in vivo, we prepared an antibody GK71 that specifically recognizes the phosphorylation of vimentin-Ser71. Ectopic expression of constitutively active Rho-kinase in COS-7 cells induced phosphorylation of vimentin at Ser71, followed by the reorganization of vimentin filament networks. During the cell cycle, the phosphorylation of vimentin-Ser71 occurred only at the cleavage furrow in late mitotic cells but not in interphase or early mitotic cells. This cleavage furrow-specific phosphorylation of vimentin-Ser71 was observed in the various types of cells we examined. All these accumulating observations increase the possibility that Rho-kinase may have a definite role in governing regulatory processes in assembly-disassembly and turnover of vimentin filaments at the cleavage furrow during cytokinesis.

Rho-associated kinase (Rho-kinase), the putative target of the small GTP-binding protein Rho, phosphorylated neurofilament protein (NF-L) in vitro with approximately 1 mole phosphate per mole NF-L. Phosphorylated NF-L no longer formed the 10 nm filaments, and NF-L filaments were phosphorylated with a result of nearly complete disassembly. NF-L phosphorylated by Rho-kinase was digested with trypsin, and digested fragments were assigned by MALDI/TOF. Unique phosphorylation sites were found at Ser-26 and Ser-57 in the head domain of NF-L. These results indicate that domain- and site-specific phosphorylation by Rho-kinase may regulate the assembly-disassembly of NF-L filaments.

Protein phosphorylation plays critical roles in multiple aspects of cellular events. Site- and phosphorylation state-specific antibodies are indispensable to analyze spatially and temporally distribution of protein phosphorylation in cells. Such information provides some clues of its biological function. Here, we describe a strategy to design a phosphopeptide as an antigen for a site- and phosphorylation state-specific antibody. Importantly, this strategy is also applicable to the production of other types of antibodies, which specifically recognize the site-specific modification, such as acetylation, methylation, and proteolysis. This protocol also focuses on the screening for monoclonal version of a site- and phosphorylation state-specific antibody.

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.

Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at ∼1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

Vimentin, one of type III intermediate filament (IF) proteins, is expressed not only in mesenchymal cells but also in most types of tumor cells. In the present study, we introduced several types of vimentin mutated at putative phosphorylation sites in its amino-terminal head domain into type III IF-negative T24 cells. Site-specific mutation induced the formation of an unusually long bridge-like IF structure between the unseparated daughter cells, although these mutants formed the filament network similar to wild type in interphase cells. Together with sites phosphorylated by Rho-kinase and protein kinase C (PKC), vimentin-Ser72, which can not be phosphorylated by any known vimentin kinase, was one of the mutation sites essential for this phenotype. We further demonstrated that vimentin-Ser72 was phosphorylated specifically at the cleavage furrow during cytokinesis. These observations suggest the existence of a novel protein kinase responsible for vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. We propose that Rho-kinase, PKC, and an unidentified vimentin-Ser72 kinase may play important roles in vimentin filament separation during cytokinesis.

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

We obtained evidence that Rho-associated kinase (Rho-kinase) phosphorylates desmin, the myogenic intermediate filament protein, with approximately 2 mol phosphate per mole of desminin vitro.Desmin phosphorylated by Rho-kinase lost the potential to form 10-nm filaments. Thr-16, Thr-75, and Thr-76 on desmin proved to be the major phosphorylation sites for Rho-kinase. All these sites are located within the head domain and are different from the reported phosphorylation sites of protein kinase A, protein kinase C, and cdc2 kinase. We are entertaining the notion that Rho-kinase may regulate filament structures of desmin by site-specific phosphorylation.

Ca2+ plays a central role in cell signaling, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of Ca2+ actions. The spatial distribution of intracellular Ca2+ signaling is not homogenous, rather it is dynamically organized, and it has been speculated that spatial patterns of Ca2+ signals may function as a form of cellular information transmitted to downstream molecules. To address this issue, we studied the intracellular distributions of the signalings by CaMKII and Ca2+ in the same astrocytes. The former was visualized by monitoring site-specific phosphorylation of a cytoskeletal protein vimentin, using site- and phosphorylation-specific antibodies, while the latter was examined by fura-2-based Ca2+ microscopy. Local Ca2+ signals induced vimentin phosphorylation by CaMKII localized in the same area. On the other hand, Ca2+ waves in astrocytes induced global phosphorylation of vimentin by CaMKII. A small population of vimentin filaments highly phosphorylated by CaMKII underwent structural alteration into short filaments at electron microscopic level. These results indicate that CaMKII transmits spatial patterns of Ca2+ signals to vimentin as cellular information. The possibility is discussed that spatial patterns of vimentin phosphorylation may be important for intracellular organization of vimentin filament networks.

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.