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Genes to Cells (2006) 11, 531-540. doi:10.1111/j.1365-2443.2006.00961.x
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Vimentin-Ser82 as a memory phosphorylation site in astrocytes

Takashi Oguri1, Akihito Inoko1, Hiroshi Shima2,3, Ichiro Izawa1, Nariko Arimura1,4, Tomoya Yamaguchi1, Naoyuki Inagaki5, Kozo Kaibuchi4, Kunimi Kikuchi2 and Masaki Inagaki1,*

1 Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan
2 Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan
3 Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori, Miyagi 981-1293, Japan
4 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan
5 Division of Signal Transduction, Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
In astrocytes, the PGF2{alpha} or ionomycin treatment induces the phosphorylation at Ser38 and Ser82 of vimentin, a type III intermediate filament, by Ca2+/calmodulin-dependent protein kinase II (CaMKII). We found here that vimentin phospho-Ser82 was dephosphorylated much slower than phospho-Ser38. Vimentin phospho-Ser38 was dephosphorylated quickly by purified PP1 catalytic subunit (PP1c) in vitro, whereas phospho-Ser82 was insensitive to PP1c. Because PP1c directly bound to vimentin through a VxF motif (Val83-Asp84-Phe85), the PP1c active site appeared to be unable to approach phospho-Ser82, leading to the prolongation of the phosphorylation at Ser-82. In astrocytes, PP1c{alpha} was in vivo associated with vimentin filaments. The repetitive treatment by ionomycin at a short interval resulted in the sustained elevation of Ser82 phosphorylation, leading to the marked disassembly of vimentin filaments. Taken together, these results suggest that vimentin is a novel member of binding partner of PP1c in astrocytes, and vimentin-Ser82 may act as a memory phosphorylation site.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The protein phosphorylation/dephosphorylation regulates the cytoskeletal organization. The organization of intermediate filaments (IFs) is dynamically regulated by the phosphorylation state. Phosphorylation of IF proteins by various types of serine/threonine protein kinases induces the disassembly of the filament structure (Inagaki et al. 1987, 1996). Vimentin is one of the type III intermediate filament proteins distributed widely in the cytoplasm and is phosphorylated by several kinases, including Ca2+/calmoduin-dependent protein kinase (CaMKII), in vivo (Fuchs & Weber 1994).

Our previous study showed that the major vimentin phosphatase in vivo is PP1, (Inada et al. 1999), although the molecular mechanism of the interaction of PP1 with vimentin is still unclear. The localization, activity and substrate specificity of PP1 are determined by the association with various types of endogenous regulatory subunits and protein inhibitors, in vivo. The PP1c can form complexes with > 50 regulatory subunits (Cohen 2002). Most interactors of PP1 share the hydrophobic "RVxF" binding groove and the RVxF motif binds to hydrophobic pocket on the surface of the PP1c (Egloff et al. 1997; Terrak et al. 2004). Mutation of the RVxF motif is often sufficient to inhibit the high-affinity binding of an interactor to PP1, in which VxF sequence is especially important for PP1c binding (Wakula et al. 2003).

In this study, we demonstrated that PP1c displayed the site-specific dephosphorylation activity for CaMKII-phosphorylated sites of Ser38 and Ser82 of vimentin. PP1c directly bound to vimentin through a regulatory subunit-binding motif, VxF sequence, of vimentin. Since this binding site of vimentin (Val83-Asp84-Phe85) was located in close proximity to phospho-Ser82, the PP1c active site was thought to be unable to dephosphorylate phospho-Ser82 due to steric hindrance. We propose here that vimentin is a novel member of the binding partners of PP1c in astrocytes and vimentin-Ser82 may act as a memory phosphorylation site.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
CaMKII signaling induced by PGF2{alpha} or ionomycin in astrocytes

To dissect the CaMKII signaling to vimentin, we monitored the site-specific phosphorylation of vimentin. Ser38 and Ser82 of vimentin are identified as the major in vitro and in vivo phosphorylation sites by CaMKII, while Ser6, Ser28, Ser33, Ser50, Ser55, Ser71, and Ser72 are phosphorylated not by CaMKII but by other kinases (Inagaki et al. 1996) (Fig. 1A,B). Previous studies have demonstrated the existence of CaMKII in astrocytes (Yano et al. 1994). CaMKII activated by Ca2+ was shown to phosphorylate vimentin in these cells (Yano et al. 1994; Ogawara et al. 1995). Stimulation of primary cultured astrocytes with 5 µM PGF2{alpha} or 1 µM ionomycin led to a remarkable increase in the phosphorylation at Ser38 and Ser82, indicating that CaMKII was the kinase responsible for the vimentin phosphorylation after the stimulation by PGF2{alpha} or ionomycin (Fig. 1Bc,d). We next observed the time course of phosphorylation at Ser38 and Ser82 after stimulation of 5 µM PGF2{alpha}. Phosphorylation of Ser38 and Ser82 increased to a maximal level in first 5 min. Then the phosphorylation of Ser38 decreased to a basal level in about 40 or 50 min, but Ser82 phosphorylation remained at a higher level for 1 h (Fig. 1C). These results show that after stimulation of PGF2{alpha}, Ser82 phosphorylation continued for over 1 h, which was distinct from the time course of Ser38.


Figure 1
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  		Figure 1  Structure, amino acid sequence and phosphorylation sites of vimentin protein. (A) Map of the molecule showing in vitro phosphorylation sites by protein kinases. The sites are indicated by P within a circle. The head domain contains a number of phosphorylation sites. The phosphorylation sites recognized by the phosphorylation specific monoclonal antibodies are indicated. (B) Recombinant vimentin was phosphorylated by CaMKII (a, b), A-kinase, C-kinase, Cdk1, Rho-kinase, and Aurora-B kinase (a). Samples were lyzed in sample buffer, resolved by SDS-PAGE and stained with Commasie Brilliant Blue or immunoblotted by 4H4, MO6, TM28, YT33, TM38, TM50, 4A4, TM71, TM72, MO82. Western blotting analysis of vimentin phosphorylation at Ser38 and Ser82 by stimulation of PGF2{alpha} or ionomycin. Astrocytes were stimulated by 5 µM PGF2{alpha} for 10 min (c) or 1 µM ionomycin for 5 min (d). C, control astrocyte; S: stimulated astrocyte; V, non-phospho-vimentin; PV, CaMKII phosphorylated vimentin. (C) Time course of Ser38 and Ser82 of vimentin dephosphorylation by stimulation of PGF2{alpha}. Astrocytes were stimulated by 5 µM PGF2{alpha} for indicated time. Samples were lyzed in sample buffer and resolved by SDS-PAGE. Phosphorylation and total vimentin protein were detected by immunoblots by monoclonal antibodies TM38, MO82 or 4H4. Optical density measurement of phosphorylated vimentin protein was plotted. The results are expressed as means ± S.E. of three assays.

 
Dynamics of vimentin dephosphorylation of phospho-Ser38 and -Ser82 under a Ca2+-depleted condition

We then monitored the time course of dephosphorylation of phospho-Ser38 and -Ser82 under a Ca2+-depleted condition that are supposed to depress CaMKII activities (Fig. 2). For this, primary cultured astrocytes were first stimulated with 1 µM ionomycin for 5 min, and then the buffer was changed into Ca2+-free HEPES-buffered Krebs Ringer solution containing 10 mM EGTA to deplete Ca2+. After the stimulation of ionomycin, Ser38 and Ser82 were phosphorylated for 5 min. The phosphorylation at Ser38 disappeared according to the decrease of [Ca2+]i, whereas the phosphorylation at Ser82 persisted for a considerably long time after Ca2+ depletion (Fig. 2A,B). To further completely inactivate kinase activities, astrocytes were treated in a buffer containing 10 mM EGTA and staurosporine, a kinase inhibitor, after the stimulation with 1 µM ionomycin for 5 min. The time course of Ser38 and Ser82 dephosphorylation were not affected with or without addition of staurosporine (Fig. 2A). Taken together, these results suggested that phospho-Ser82 was dephosphorylated much slower than phospho-Ser38.


Figure 2
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  		Figure 2  Time course of vimentin dephosphorylation at Ser38 and Ser82 in Ca2+ depletion condition. (A) Astrocytes were stimulated by 1 µM ionomycin for 5 min. After stimulation, buffer was changed into HEPES buffered Krebs-Ringer Solution containing 10 mM EGTA with staurosporine ({square},{blacksquare}) or without staurosporine ({circ},•). Phosphorylation and total vimentin protein were detected by immunoblots with monoclonal antibodies TM38 ({square},{circ}), MO82 ({blacksquare},•) or 4H4. Optical density measurement of phosphorylated vimentin protein by densitometer and concentration of [Ca2+]i (• with a broken line) was plotted. The results are expressed as mean ± S.E. of three assays. (B) Astrocytes treated as described in (A) were immunostained with TM38, MO82 or 4H4. Bar, 10 µm.

 
Phosho-Ser82 on vimentin can hardly be dephosphorylated by PP1

In order to identify a phosphatase(s) involved in the in vivo dephosphorylation of CaMKII-phosphorylated vimentin, we utilized protein phosphatase inhibitors, Calyculin A and Okadaic acid (Fig. 3A). Okadaic acid has a 50–100-fold weaker effect than Calyculin A on PP1. Okadaic acid and Calyculin A are reported to inhibit PP2A with a similar potency. After the stimulation with ionomycin, buffer was changed into Ca2+-free HEPES-buffered Krebs Ringer solution containing 10 mM EGTA with Okadaic acid, Calyculin A or DMSO only. The dephosphorylation of phospho-Ser38 of vimentin was inhibited by Calyculin A but not by Okadaic acid, indicating that Ser38 dephosphorylation is mainly dependent on PP1 (Fig. 3A). Interestingly, the inhibition of PP1 or PP2A, in vivo, did not markedly affect the phosphorylation level of Ser82. Considering that the major phosphatase of vimentin is PP1 (Inada et al. 1999), these data suggest the possibility that PP1 may be unable to dephosphorylate phospho-Ser82 through an unidentified mechanism. To further elucidate the difference between the dephosphorylation of phospho-Ser38 and that of phospho-82 by PP1, we next examined the in vitro dephosphorylation of CaMKII-phosphorylated vimentin, by using purified catalytic subunit of PP1 (PP1c) (Fig. 3B). After phosphorylation of recombinant vimentin by CaMKII, purified PP1c was applied to phosphorylated vimentin. PP1c dephosphorylated vimentin at Ser38 much more efficiently than at Ser82, in vitro, which is consistent with the in vivo results obtained by use of the phosphatase inhibitors (Fig. 3A,B).


Figure 3
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  		Figure 3  Characterization of dephosphorylation of CaMKII-phosphorylated vimentin by protein phosphatases. (A) Astrocytes were stimulated with 1 µM ionomycin for 5 min. After stimulation, cells were treated with HEPES buffered Krebs-Ringer Solution containing 10 mM EGTA with phosphatase inhibitor, Calyculin A or Okadaic Acid. Phosphorylation and total vimentin protein were detected by immunoblots by monoclonal antibodies TM38, MO82 or 4H4. (B) Recombinant vimentin phosphorylated by CaMKII was dephosphorylated by PP1c. At the indicated time, aliquots were removed, lyzed in sample buffer and resolved by SDS-PAGE and detected by immunoblots by monoclonal antibodies TM38, MO82 or 4H4. Optical density measurement of phosphorylated vimentin protein by densitometer was also plotted. (C) Far Western analyses of the binding of PP1c{alpha} to vimentin. Recombinant full length vimentin was separated by SDS-PAGE and transferred on to PVDF membrane. The membranes were incubated with PP1c{alpha} at an indicated concentration. Binding of PP1c{alpha} was detected by anti-PP1c{alpha} antibody and quantified. (D) Far Western analyses of the binding of PP1c to vimentin mutants. The full length (upper column) or head domain (lower column) of wild-type and V83A/F85A mutant of vimentin were resolved by SDS-PAGE. After transfer on to PVDF membrane, the membranes were incubated by 5 µg/mL PP1c{alpha}, and detected by anti-PP1c{alpha} antibody. Quantification of binding of PP1 was plotted in right panel. (E) Amino acid sequence of the synthetic wild-type and mutant vimentin peptides used in this study. Wild-type and mutant peptides were phosphorylated by CaMKII or Plk1 and incubated with PP1c containing 1 mM Mn2+ for 30 min. Phosphorylation of peptides were quantified by radio reactivity of 32P-incorporartion of phosphopeptides.

 
Previous reports suggested that the well characterized interactors of PP1c have the binding-motif, so-called "RVXF" or "KIXF" sequence. The substitution of alanines in place of either V/I or F disrupted PP1c binding (Egloff et al. 1997; Wakula et al. 2003). Very interestingly, the sequence of the head domain of vimentin contained VxF motif (Val83 and Phe85). To determine if this sequence might be a part of a PP1c-binding site, we examined the binding of PP1c{alpha} to vimentin, using Far Western analysis. We found that PP1c{alpha} bound to vimentin (Fig. 3C). The association between the recombinant full length-vimentin and PP1c{alpha} slightly diminished by the introduction of the V83A/F85A mutation (Fig. 3D, upper column). The association between the head domain of vimentin and PP1c{alpha} was strongly inhibited by the introduction of the V83A/F85A mutation (Fig. 3D, lower column). Strangely, the mutation V83A/F85A of vimentin head domain caused a band shift in SDS-PAGE (Fig. 3D, lower column), but the wild-type and mutant vimentin head did not show an apparent difference of molecular weight by MALDI-TOF/MS analysis (data not shown). These results demonstrate that PP1c{alpha} directly binds to vimentin through Val83 and Phe85 as well as through other binding sites located in the rod or tail domain of vimentin. To check if the binding of PP1c to the Val83-Phe85 motif of vimentin may affect the inability of PP1c to dephosphorylate Phospho-Ser82, we produced a set of vimentin peptides in which the various residues were changed into alanines and observed the dephosphorylation of these peptides by PP1c (Fig. 3E). Mutation of V83A or F85A resulted in the increase of dephosphorylation of phospho-Ser82 by PP1c (Fig. 3E).

PP1c was in vivo associated with vimentin in primary cultured astrocytes

Rabbit anti-PP1c{alpha} antibody specifically recognized a major band corresponding to the estimated molecular weight of 36 kDa in lysates prepared from astrocytes (Fig. 4A). Preincubation of the antibody with the immunogen peptide of PP1c{alpha} selectively inhibited the immunoreactivity, confirming the specificity of the antibody. By using the anti-PP1c{alpha} antibody, PP1c{alpha} was detected in the vimentin enriched fraction (Fig. 4B) as well as in the whole cell extracts (Fig. 4A). To examine the in vivo binding of PP1c to vimentin, vimentin was immunoprecipitated from the astrocyte cell extracts. PP1c{alpha} was clearly detected in the precipitate of vimentin, whereas a negligible amount of PP1c{alpha} was detected in the precipitate by control IgG (Fig. 4C). These results demonstrated that PP1c was associated with vimentin, in vivo. We next examined the subcellular localization of PP1c in primary cultured astrocytes (Fig. 4D,E). After the elimination of cytosolic PP1 by the treatment with 0.5% Triton X-100, we observed that a portion of PP1c{alpha} co-localized with vimentin filaments (Fig. 4D,E). These results showed that PP1c{alpha} associated with vimentin in primary cultured astrocytes.


Figure 4
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  		Figure 4  Association of PP1c and vimentin filaments in astrocytes. (A) Detection of PP1c{alpha} in astrocytes. Lysates from primary cultured astrocytes were stained with Coomassie Brilliant Blue (CBB), detected with anti-PP1c{alpha} antibody by immnoblotting (PP1c{alpha}), or immunostained with anti-PP1c{alpha} antibody preabsorbed by PP1c{alpha} antigen peptide. Anti-PP1c{alpha} antibody specifically recognized a band of about 36 kDa in samples. (B) Copurification analyses of PP1c with vimentin. Purified cytoskeletal fraction by using lysis buffer described under Experimental procedures. Vimentin enriched fraction (V) and whole cell lysates (W) were stained with Coomassie Brilliant Blue (CBB), or detected with anti-PP1c{alpha} antibody by immunoblotting (PP1c{alpha}). (C) Co-immunoprecipitation of PP1c{alpha} and vimentin in astrocytes. Vimentin was immunoprecipitated from lysates of primary cultured astrocytes by anti-vimentin polyclonal antibody, and immunoblotted with anti-vimentin antibody (V9) or with anti-PP1c{alpha} antibody. Arrows and arrowheads show vimentin and PP1c{alpha}, respectively. (D) Primary cultured astrocytes were double-stained by anti-PP1c{alpha} antibody and anti-vimentin antibody (V9). The enlargement of the area indicated by white squares was also shown. Bar, 10 µm. (E) In primary cultured astrocytes, a part of cells showed strongly colocalization with vimentin and PP1c{alpha}. Astrocytes were double-stained by anti-PP1c{alpha} antibody and anti-vimentin antibody (V9). Bar, 10 µm.

 
Vimentin Ser82 may function as a "memory" phosphorylation site

To dissect a physiological role for the sustained phosphorylation of Ser82 in CaMKII signaling, we examined the effect of the repetitive activation of CaMKII on Ser82 phosphorylation. For this, astrocytes were stimulated with ionomycin repeatedly at intervals of 10 min (Fig. 5A). Predictably, phosphorylation of Ser38 rapidly diminished after the every removal of ionomycin. On the other hand, phosphorylation level of Ser82 gradually increased followed by the every stimulation (Fig. 5A). These results show that the repetitive activation of CaMKII at a short interval could build up the phosphorylation on Ser82, leading to the sustained elevation of Ser82 phosphorylation, whereas the same treatment only resulted in the repetition of the short-term phosphorylation of Ser38. In addition, this repeated stimulation of ionomycin caused the marked reorganization of vimentin filaments (Fig. 5B). Therefore, it is possible that the frequency of the activation of CaMKII induced by [Ca2+]i oscillation in cells may influence the level and duration of Ser82 phosphorylation and thereby may affect the equilibrium of the vimentin filament assembly and disassembly.


Figure 5
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  		Figure 5  The sustained elevation of Ser82 phosphorylation induced by the repetitive activation of CaMKII. (A) Astrocytes were stimulated by 1 µM ionomycin for 10 min at 10 min intervals. Phosphorylation was detected by immunoblots (exp. 1 and exp. 2) or immunostaining with monoclonal antibodies TM38, MO82 or 4H4. Bar, 10 µm. (B) A sample of representative vimentin staining at 0 min, 10 min or 50 min in (A) was shown. Nuclei were stained with propidium iodide (red). The enlargement of the area indicated by white squares was also shown. Bar, 10 µm. (C) A model for the PP1c binding to vimentin. PP1c binds to vimentin through Val83 and Phe85, thereby decreasing accessibility to phosphorylated Ser82. The solid yellow arrow shows strong PP1c catalytic activity against Ser38. The broken yellow arrow shows weak PP1c catalytic activity against Ser82. RBD: a regulatory subunit binding domain.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The phosphorylation state of proteins is finely regulated by protein kinases and protein phosphatases. Eukaryotic cells contain four major types of serine/threonine protein phosphatases, termed PP1, PP2A, PP2B (calcineurin) and PP2C (Cohen 1989; Wera & Hemmings 1995). In rats, PP1 catalytic subunit (PP1c) has at least four isoforms, PP1{alpha}, PP1{gamma}1, PP1{gamma}2 and PP1{delta} (Sasaki et al. 1990). These mammalian PP1c isoforms possess distinct tissue distributions and subcellular localizations through interactions of various regulatory subunits (Shima et al. 1993; da Cruze e Silva et al. 1995; Raghavan et al. 2000). Most of PP1c regulatory subunits interact with a small hydrophobic groove on the surface of PP1c through a short conserved binding motif. In this study, our data indicated that the binding of PP1c to the conserved binding motif, a VxF motif, of vimentin affected the dephosphorylation of phospho-Ser82 (Fig. 3C–E). The site of PP1c which binds to RVxF motif-containing peptides is reported to be spatially distinct from PP1c active site by analysis of a crystal structure of PP1c complexed with an RVxF containing peptide (Wakula et al. 2003). Hence, the binding of PP1c to the VxF motif located in close proximity to Ser82 may prevent PP1c active site from approaching phospho-Ser82, leading to the persistence of phosphorylation of Ser82 after the stimulation by ionomycin or PGF2{alpha} (Fig. 5C).

Recent physiological studies have suggested that the cross-talk between astrocytes and neurons is an important factor in information processing in the brain and astrocytes modulate synaptic activity by releasing transmitters (Allen & Barres 2005). Synapses in central nervous system are covered with the lamella or filopodia of astrocytes, and the neighbor astrocytes contribute to the maturation of synapses. Astrocytes release a variety of agonists, such as glutamate and ATP, in response to the oscillatory increase in [Ca2+]i and the signal from adjacent neurons (Sul et al. 2004). Astrocytes also express many types of neurotransmitter receptors and can respond to neurotransmitters by a characteristic oscillatory increase in [Ca2+]i (Yoshida et al. 2005). In primary cultured astrocytes, [Ca2+]i oscillatory process, which is mimicked by repeated stimulation with calcium ionophore, led to the reorganization of vimentin filaments. Knockout mouse studies have revealed that vimentin plays an important role in the formation of reactive astrocytes, which proliferate and migrate in brain injury and axonal regeneration (Privat 2003) and vimentin plays an important role of transcellular migration in lymphocyte (Nieminen et al. 2006). Therefore, the reorganization of vimentin filaments is predicted to increase the motility of astrocytes. In primary cultured astrocytes, [Ca2+]i oscillatory process results in the specific accumulation of phosphorylatin of vimentin-Ser82 and the marked reorganization of vimentin filaments. In central nervous system, this accumulation of phosphorylation of vimentin-Ser82 may play a key role in the function of astrocytes by increasing cell motility and perhaps affect the synaptic maturation.

We have recently found that Plk1 binds to vimentin-Ser55 phosphorylated by Cdk1 and then phosphorylates vimentin-Ser82 during mitosis (Yamaguchi et al. 2005). The elevation of Ser82 phosphorylation appears to continue until the end of mitosis even after Plk1 binding to vimentin may be diminished by the reduction of vimentin-Ser55 phosphorylation at anaphase. We consider that phospho-Ser82 on vimentin may hardly be dephosphorylated by PP1 in mitosis. This sustained Ser82 phosphorylation by Plk1 may play some roles in the efficient segregation of vimentin filaments during mitosis, although Rho-kinase and Aurora-B may have greater roles (Yamaguchi et al. 2005). The prolongation of phosphorylation of vimentin Ser82 may increase the steady state level of vimentin phosphorylation. In this condition, disassembly of vimentin filaments will easily occur when new phosphorylation by another kinase, CaMKII or Plk1, is added to vimentin. Thus, the prolongation of Ser82 phosphorylation decreases the threshold of the strength of new phosphorylation signals required for vimentin filament disassembly, and may function as a kind of "memory" for the phosphorylation status by CaMKII and Plk1.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Phosphorylation and dephosphorylation of recombinant vimentin and vimentin peptide

Production of recombinant vimentin and phosphorylation of vimentin by CaMKII were previously described (Ogawara et al. 1995). Recombinant double mutant vimentin (V83A, F85A) was prepared using QuickChange site-directed mutagenesis kits (Stratagene Inc). To dephosphorylate phosphorylated vimentin, recombinant PP1c was reacted with CaMKII-phosphorylated vimentin samples containing 1 mM MnCl2. Reaction of dephosphorylation was finished by adding SDS-sample buffer. Vimentin wild-type and mutant peptides were phosphorylated by CaMKII or Plk1. To dephosphorylate phosphorylated vimentin peptides, recombinant PP1c was reacted with CaMKII- or Plk1-phosphorylated vimentin peptides.

Cell preparation and drug application

Primary cultured astrocytes were prepared from the cerebral cortices of newborn rats as previously described (Inagaki et al. 1997). Cells were cultured in Dulbecco's modified Eagle medium containing 10% FCS for three weeks. In some experiments, astrocytes were differentiated by incubation for 2 d with 250 µM dibutyryl cAMP in serum free medium (Trimmer et al. 1982). Ionomycin or prostaglandin F2{alpha} (PGF2{alpha}) dissolved in HEPES-buffered Krebs-Ringer solution (containing the following (in mM): NaCl, 115; KCl, 5.4; CaCl2, 2; MgCl2, 0.8; glucose, 13.8; HEPES, 20 (pH 7.4)) was applied.

[Ca2+]i measurements

The [Ca2+]i of cultured astrocytes was measured as described elsewhere (Inagaki et al. 1997). [Ca2+]i was calculated from the ratio of the fluorescence intensities obtained with excitations at 340 nm and 380 nm.

Immunocytochemistry

Cells were fixed with 3% formaldehyde in phosphate-buffered saline (PBS) for 10 min, followed by treatment with –20 °C methanol for 10 min. They were incubated with TM38, MO82 and 4H4 diluted in PBS for 2 h, followed by incubation with Alexa488-conjugated anti-mouse or anti-rat antibodies diluted 1 : 400 by PBS for 1 h. For PP1c{alpha} staining, cells were treated in 0.5% Triton X-100/PHEM (containing the following): 60 mM Pipes; 25 mM HEPES; 10 mM EGTA; 4 mM MgSO4 (pH 6.9)) for 30 s before fixation.

Far Western analysis

Far Western analysis was performed as previously reported with some modifications (Yamaguchi et al. 2005). Vimentin or vimentin head domain was transferred on to PVDF membranes and incubated with of PP1c{alpha} (Upstate). These membranes were incubated affinity purified anti-PP1c{alpha} rabbit polyclonal antibody (Shima et al. 1993) and then with HRP-conjugated anti-rabbit antibody.

Purification of vimentin enriched cytoskeletal fraction

Astrocytes were washed twice with PBS and lyzed in ice cold lysis buffer (50 mM Tris pH 7.5, 1% Triton X-100, 1 µg/mL leupetin, 1 mM PMSF, 0.6 M KCl). The lysate were centrifuged at 15 000 r.p.m. for 30 min and remove the supernatant. After addition of lysis buffer, lysate was centrifuged again. Centrifugation was repeated at least 3 times. Precipitation was confirmed by SDS-PAGE analysis.

Immunoprecipitation

Astrocytes were washed twice with PBS and lyzed in ice cold vimentin immunoprecipitation buffer (50 mM Tris pH 7.5, 1% Nonidet P-40, 1 µg/mL leupetin, 1 mM PMSF). The lysates were immunoprecipitated by Catch and Release ver 2.0 (Upstate) with anti-vimentin antibody. Anti-vimentin antibody (V9) was purchased from Sigma-Aldrich.


   Acknowledgements
 
We thank Y. Hayashi for preparing materials and technical assistance. This work was supported in part by Grants-Aid for Scientific Research and Cancer Center Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a grant-in-aid for the Third Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan, by The Naito Foundation, and by the Uehara Memorial Foundation.


   Footnotes
 
Communicated by: Noriko Osumi

* Correspondence: E-mail: minagaki{at}aichi-cc.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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