- Research article
- Open Access
The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin
© Brauksiepe et al; licensee BioMed Central Ltd. 2008
- Received: 26 March 2008
- Accepted: 23 September 2008
- Published: 23 September 2008
Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed.
The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association.
We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.
- Intermediate Filament
- Kinase Assay
- Intermediate Filament Protein
- African Swine Fever Virus
- Head Domain
STK33/Stk33 is a serine/threonine kinase discovered in the course of sequencing the human chromosome 11 region 11p15 and mouse chromosome 7 . The Stk33 gene in the mouse (and also STK33 in human) is expressed differentially in a number of specific tissues and cells like testes, lung epithelia, alveolar macrophages, and horizontal cells in the retina. In mouse embryos Stk33 expression is found in the developing heart, brain and spinal cord . Based on sequence comparison with other kinases the STK33/Stk33 protein was classified as a member of the Ca2+/calmodulin-dependent kinase family (CAMK) [1, 3–5].
The CAMK group is a family of multifunctional kinases: CAMK I, CAMK II and CAMK IV. Among the most well characterized CAMKs is Ca2+/calmodulin-dependent protein kinase II. CAMK II can phosphorylate a wide range of substrates and regulates numerous cellular functions including cell division, differentiation, cardiac contraction, and synaptic plasticity . CAMK II is abundantly expressed in the brain  and a major effector for calcium-dependent signaling in neurons. The important neuronal function of CAMK II α has been demonstrated by analysing mice with certain mutated forms of CAMK II α [8–11]. In comparison with CAMK II α little is known about the CAMK II β subunit despite its prevailing appearance in the central nervous system. Alternative splicing variants of CAMK II β in brain with different kinase activity were identified . In contrast to α and β isoforms predominantly expressed in neural tissues, the δ isoforms of CAMK II prevail in the heart . The CAMK II γ isoform is mainly expressed in differentiated smooth muscle cells (dSMC). A novel variant of the γ isoform, CAMK II γ G-2, can be found in several smooth muscles, in heart and brain, but not in skeletal muscle and liver . In unstimulated dSMCs it colocalizes with vimentin. Activation with a depolarizing stimulus leads to autophosphorylation of CAMK II and phosphorylation of vimentin at CAMK II specific sites. As a consequence CAMK II bound to cytoskeletal vimentin is now translocated into the cytosol. This targeting is essential for signaling in differentiated smooth muscle cells because prevention of CAMK II targeting by antisense knockdown of CAMK II γ G-2 leads to inhibition of ERK (extracellular signal-related kinase) activation as well as to inhibition of muscle contraction . Anchoring CAMK II γ G-2 to vimentin in unstimulated cells is discussed as a prerequisite for optimal kinase activation or for spatial separation of the kinase and its substrate .
Serine 38 and serine 82 of vimentin are the major in vitr o and in vivo phosphorylation sites by CAMK II . In cells infected with the cytoplasmatic DNA virus ASFV (African Swine Fever Virus) viral DNA replication resulted in activation of CAMK II and phosphorylation of vimentin on serine 82 by CAMK II. Incubation of cells with an inhibitor of CAMK II, KN93, prevented phosphorylation of vimentin and blocked both viral DNA replication and late gene expression. This underlines that CAMK activation is required for late ASFV gene expression, but the precise role played by CAMK II in ASFV DNA replication is still unknown. In virus infected cells vimentin phosphorylated on serine 82 disassembles into aggregates which are transported along microtubules and are reorganized into a cage like structure around virus assembly site. This vimentin cage has on the one hand a cytoprotective function by preventing the diffusion of viral components into cytoplasm and on the other hand it concentrates late structural proteins at site of virus assembly .
The phosphorylation/dephosphorylation state regulates the dynamic behaviour of the intermediate filament cytoskeleton. The major vimentin phosphatase in vivo is type 1 protein phosphatase (PP1) . PP1c is in vivo associated with vimentin and dephosphorylates the CAMK II-specific phosphorylation sites of vimentin Ser38 and Ser82. Phospho-Ser82 of vimentin is dephosphorylated much slower than phospho-Ser38 by PP1c . This delayed Ser82 dephosphorylation might influence the dynamics of vimentin filament assembly/disassembly. A requirement for cell division during mitosis is the reorganization of the intermediate filament system through phosphorylation of vimentin as demonstrated by site-specific mutation of vimentin . In the case of Polo-like kinase 1 (Plk1), a kinase which also phosphorylates vimentin Ser82 , elevated Ser82 phosphorylation by Plk1 may play a role in efficient segregation of vimentin filaments during mitosis  as phospho-Ser82 on vimentin is hardly dephosphorylated by PP1 in mitosis. Phospho-Ser82 may act as a memory phosphorylation site .
The different sites available for serine/threonine phosphorylation in vimentin are targeted by different kinases . Since we found the striking colocalization of vimentin and Stk33 in various cell types and tissues (manuscript in preparation), we were prompted to investigate whether Stk33 might be another kinase to phosphorylate vimentin.
Stk33 is a serine/threonine kinase of so far unknown function. In the present study we used bacterially expressed recombinant mouse Stk33 with several artificial vimentin deletion mutant polypeptides also expressed as recombinant proteins in E. coli for in vitro phosphorylation assays. In addition we performed co-immunoprecipitation experiments with protein extracts obtained from the mouse Sertoli cell culture SerW3. We know from previous studies (manuscript in preparation) that Stk33 and vimentin are coexpressed and colocalized in Sertoli cells of mouse testes. So, Sertoli cells should be an ideal resource for gaining native interacting Stk33 and vimentin.
However, from our previous studies it is also clear, that Stk33 is not always expressed in cells together with vimentin. We find Stk33 in vimentin-negative cells, too.
The differential expression pattern of Stk33 in mice and men resembles those of some members of the CAMK-Group . Stk33 and CAMKII might have similar functions for example in the dynamic regulation of the intermediate filament system by phosphorylation in the course of the separation of daughter cells during mitosis. Stk33 is expressed very specifically in some organs of the developing mouse embryo . Thus, Stk33 could play also a role in organ development in addition to its function in phosphorylating vimentin. In this study, however we show that vimentin is a target for phosphorylation by Stk33 in vitro and that Stk33 and vimentin can be co-immunoprecipitated indicating a close interaction also in vivo.
Recombinant mouse Stk33 kinase was incubated with γ 32P ATP with the various substrates under optimized conditions tested previously. The reaction products were separated by SDS-PAGE and detected by direct autoradiography. The results clearly demonstrate (autoradiographs shown in Figure 2C, D, E) that: i) Stk33 (complete kinase domain) is able to perform autophosphorylation. The incubation of Stk33 without any other substrate (lane Stk33 only; Figure 2C, lane 6; Figure 2D, lanes 3, Figure 2E, lane 12) leads to a strong radiolabeled band with an apparent molecular weight corresponding to the one of Stk33 (black arrowhead); ii) The derivative of Stk33, Stk33δ in which part of the kinase domain is deleted is not able to perform autophosphorylation (Figure 2C, lanes 1–3; white arrowhead). Furthermore, Stk33δ is not able to phosphorylate any of the tested substrates (Figure 2C, lane 2 and 3) in contrast to Stk33 (Figure 2C, lane 4); iii) Stk33 clearly phosphorylates vimentin in vitro (Figure 2D, lane 5 and 7; Figure 2E, lanes 1, 3, 5, 7, 9; arrows). By using the vimentin tetramer as substrate, differentiation between autophosphorylated Stk33 and phosphorylated vimentin wildtype is clearly possible (Figure 2D, lane 5; arrow).
When Stk33 is incubated together with wildtpye vimentin and the deletion variants Δ12 to Δ50, a preferred phosphorylation of vimentin over Stk33 is observed, however, there is always a basic autophosphorylation of Stk33 recognizable (black arrowhead in Figure 2E, lanes 1, 3, 5, 7, 9). So far it is not clear, whether the autophosphorylation is a prerequisite for the kinasing activity of Stk33 or whether also unphosphorylated Stk33 is able to phosphorylate vimentin. The limited resolution of deletion variants Δ30 and Δ42 in the gel might be related to a different extent of phosphorylation in truncated vimentin Δ30 compared to Δ42 and therefore a changed electrophoretic mobility might be the consequence, but this is speculation.
Notably, the truncation mutants Δ12, Δ20 and Δ30 are phosphorylated to a higher extent than Δ42 and Δ50 (Figure 2E). Therefore we conclude that, since headless vimentin is not phosphorylated at all, only the head domain of vimentin is phosphorylated and furthermore that sites both on the first 30 amino acids and sites after amino acid 30 up to the end of the head domain are phosphorylated.
As seen in Figure 2E, lane 11 it is difficult to visualize the only minute different position between autophosphorylated Stk33 and vimentin on an autoradiograph as Stk33 and vimentin have the same electrophoretic migration behaviour in a PAGE. The samples on the gel of the autoradiography 2 D were electrophoretically resolved by a longer running time than 2 E to achieve a better separation. Therefore, in lane 11, Figure 2E (shorter running time) the resolution is not good enough to resolve the two proteins of nearly identical molecular weight and electrophoretic mobility. Vimentin phosporylated by Stk33 (lane 7, Figure 2D) or by PKA (lane 9, Figure 2D) appears to migrate slightly different. The extent of phosphorylation might be different inducing a phosphorylation-dependent mobility shift on gels.
In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. As a positive control recombinant nearly full length Stk33 protein was precipitated using protein A sepharose and anti-Stk33 antibody . For all co-immunoprecipitation experiments Sertoli cell culture SerW3 was used. All protein samples (total protein extract from SerW3, samples of washing steps, precipitated proteins and recombinant proteins as positive controls for Western detection) were analyzed by SDS-PAGE after preheating in non-reducing Laemmli buffer. Therefore, the main portion of IgG molecules is still present. The appearance of a band corresponding to the IgG heavy chain is perhaps explainable because of heating during probe preparation.
To analyse whether vimentin was co-precipitated by the precipitation with anti-Stk33, a Western Blot analysis was carried out with the identical material as used for the Western blot in Figure 4B but for the detection an anti-vimentin antibody was used. Recombinant human vimentin wildtype protein was included in the analysis as a positive control (Figure 4C, lane 6). Co-immunoprecipitation from cellular extracts suggests that Stk33 and vimentin could be associated in vivo (Figure 4C, lane 5; arrow). In addition to the detection of vimentin in the precipitate, there was some vimentin in the washing buffer of the first washing step (Figure 4C, lane 2). The following washing steps did not show any vimentin in the washing buffer (Figure 4C, lane 3 and 4), and hence it is highly improbable that insufficient washing of the precipitate is responsible for the vimentin content. The results indicate clearly, that Stk33 and vimentin are in vivo associated proteins.
Testing enzymatic activity of immunoprecipitated Stk33 by a kinase assay
The aim of the present study was to investigate whether the novel serine/threonine kinase Stk33 phosporylates the intermediate filament protein vimentin. The motivation to test this was a striking colocalization of vimentin and Stk33 in various tissues and differentiated cells (manuscript in preparation). The results of the in vitro kinase assays and of the co-immunoprecipitation studies are very clear:
Stk33 is able to phosporylate vimentin in vitro and vimentin and Stk33 form a complex in vivo which can be readily co-precipitated by the use of an anti-Stk33 antibody. Stk33 binds directly to vimentin as determined by the co-sedimentation assay. Therefore, none intermediate protein mediates this association. We conclude that Stk33 plays a specific role in the dynamic behaviour of the intermediate filament cytoskeleton by phosphorylation of vimentin. It is known that phylogenetically related genes often have similar functions. Thus it is not too surprising that Stk33 -a member of the family of Ca2+/calmodulin-dependent protein kinases - is able to phosphorylate vimentin. CAMKII among other kinases is one of the major kinases responsible for the phosphorylation of the cytoskeletal protein vimentin . In that respect it is interesting that Stk33 undergoes autophosphorylation. Whether the autophosphorylation of Stk33 is a prerequisite for the phosphorylation of vimentin is not known. However, it is known, that autophosphorylation is the key event in the phosphorylation process of other members of the CAMK group [23, 24].
Intermediate filament proteins form the largest family of cytoskeletal proteins in mammalian cells. Intermediate filament proteins can be classificated into six types based on their gene structure, sequence homology and immunological and/or assembly properties . Vimentin belongs to type III of intermediate filament proteins which also comprises desmin, GFAP and peripherin.
IF proteins are composed of an amino-terminal head, a central rod and a carboxy-terminal tail [26, 27]. The rod domain is subdivided into further segments by non-α-helical regions, called linker. The head domain is essential for IF assembly and the tail for the control of lateral association. Dimerization is mediated by the rod-domain.
Most of the kinases phosphorylate sites on IF proteins located in the amino-terminal non α-helical head domain (e.g. cdc2 kinase , cAMP-dependent protein kinase (protein kinase A) , protein kinase C , CaMKII , p21-activated kinase (PAK) [30–32]). Stk33 shows similar head domain specificity: It phosphorylates different head domain deletion derivatives, but is not able to phosphorylate vimentin in which the head domain is deleted completely. Vimentin with 50 amino acids missing from the animo-terminus is still phosphorylated by Stk33 in contrast to vimentin missing 80 amino acids. Therefore we conclude that Stk33 phosphorylates one or more of the phosphorylation sites known from other kinases. The potential phosphorylation sites beyond the head domain (downstream of amino acid 81) [33, 34] and in the tail domain of vimentin [21, 33, 35] are not phosphorylated by Stk33. We can therefore be rather confident that the phosphorylation sites for Stk33 are located in the vimentin head-domain.
In spite of the differentiation- and tissue-specific expression patterns, the function of the intermediate filament proteins has long been considered to be just structural. By forming a continuous network stretched from the nuclear surface to the cell membrane and associated in tight interaction with the nuclear lamina and the nuclear cytoskeleton, it is assumed that intermediate filaments modulate and control signal transduction . The dynamic behaviour of the intermediate filament cytoskeleton is under control of kinases and phosphatases leading to structural changes of the intermediate filament cytoskeleton like reorganization, solubilization or collapse. Various types of serine/threonine protein kinases phosphorylate intermediate filament proteins in vitro leading to disassembly of the filament structure . Up to now we do not yet know whether phosphorylation of vimentin by Stk33 causes disassembly albeit this is conceivable because high levels of vimentin phosphorylation often lead to structural alterations of the filament system.
The remodelling between polymerized intermediate filaments (long filaments and short filaments called squiggles) and non-filamentous particles is regulated by kinases . Among the different structural filament forms the non-filamentous precursors (particles) are the most interesting . It has been reported that these particles can move long distances at high speed along microtubules with the help of molecular motors [38–40]. Filament precursors are delivered to special regions within the cell, where an assembly to long intermediate filaments takes place. Such flexibility enables cell movement and reorganization of the cytoplasm. Interestingly, the bi-directional movement of vimentin intermediate filaments along microtubule enables a kinase signaling over long distances within a cell. This is of special interest in neurons, where signals generated in axonal or dendritic processes have to travel long distances to the cell body (retrograde transport), especially to the nucleus, where the kinase can affect gene expression. The transport complex consisting of phosphorylated MAP kinase Erk1/2, importin β and dynein requires vimentin particles for movement along microtubule fibres in injured neurons . Normally, adult neurons express only terminally differentiated neuronal intermediate filament proteins like neurofilament proteins, but translationally silenced vimentin mRNA is activated and synthesized in lesioned nerves. As only soluble vimentin particles are capable of binding a kinase, de novo synthesized vimentin protein has to be disassembled into particles by phosphorylation or by modification through proteolysis . De novo synthesized vimentin is therefore exposed to high calcium concentrations that prevent assembly of vimentin particles to filaments due to vimentin phosphorylation by CAMKII  or due to calpain-mediated cleavage of vimentin . The creation of the kinase/vimentin complex is only possible with a phosphorylated kinase and it is promoted at high Ca2+concentrations (e.g. near site of nerve injury). On the contrary, the complex dissociates near the cell body where the Ca2+ concentrations are low . During the retrograde transport dephosphorylation of pErk is avoided as long as vimentin stays bound to the kinase. Vimentin hides phosphorylated residues in the kinase and therefore confines the access of the phosphatase to these residues . Furthermore, other interacting partners are not capable of binding to the kinase, which in turn guarantees the specificity of the transmitted signal. Interestingly, this kinase transport mechanism described for lesioned nerves is not possible in vimentin null mice . Besides several defects (e.g. in cerebellar glia ) vimentin-null mice show a defective wound repair , which might be related to the deficit of vimentin-dependent signaling as described for lesioned nerves .
The dynamic changes of the intermediate filament organization are particularly prominent during cell movement or mitosis and cell division. There is a constant state of flux between non-filamentous components, short filaments and long filaments. In some but not all cell types, vimentin filaments disassemble into aggregates and short filaments during metaphase . The organizational changes observed during mitosis are accompanied by a significant increase in the phosphorylation state. Site-specific mutation of vimentin and therefore changes in potential phosphorylation sites have been demonstrated to induce the formation of intermediate filament bridges between unseparated daughter cells . To elucidate the precise molecular function of Stk33 in vimentin phosphorylation, it is important to determine the specific phosphorylation sites on vimentin by Stk33 which is planned for the future.
Our results show that the serine/threonine kinase Stk33 phosphorylates the intermediate filament protein vimentin in vitro specifically in the vimentin head domain. Stk33 undergoes obligatory autophosphorylation, which might be a prerequisite for its kinasing activity. By co-immunoprecipitation we were able to co-isolate vimentin together with Stk33 using a polyclonal anti-Stk33 antibody. From this result we conclude that Stk33 and vimentin are interacting protein partners also in vivo. This conclusion is strongly supported by the observation that Stk33 and vimentin can be found together in many very specialized cells and tissues (manuscript in preparation). We propose that Stk33 is involved in the dynamics of intermediate filament assembly/disassembly through a specific and regulated phosphorylation of vimentin.
The kinasing activity of Stk33 was determined in an in vitro kinase assay. 0.32 μg recombinant Stk33 and 1.75 μg vimentin/vimentin deletion derivatives were incubated with 10 mM MgCl2 in 1× kinase buffer (Na-Hepes pH 7.0, 0.05% Briji). In order to use crosslinked vimentin tetramers as a substrate for Stk33 increasing concentrations of glutaraldehyde (0; 0.005; 0.01; 0.02; 0.04; 0.06%)  were used to form these complexes. As a substrate positive control casein phosphorylated by both Stk33 and Protein kinase A (PKA) catalytic subunit (Sigma) was applied to the assay. As a control for a contamination with any endogenous kinase, negative controls (assay without additionally applied Stk33 or PKA) were carried out. The reaction was initiated by adding 20 μCi γ 32P ATP. After incubation for 2 hours at 30°C, the reaction was stopped by adding SDS-sample buffer (125 mM Tris, 4% SDS, 20% Glycerol, 10 mM β-Mercaptoethanol, 2 mM EDTA, 0.04% Bromphenol blue, pH6.8). Samples were boiled for 5 min prior to loading onto polyacrylamide gels and separation by SDS-PAGE. Gels were finally autoradiographed by exposure to Kodak X-AR films.
Co-immunoprecipitation and Western Blotting
For immunoprecipitation, SerW3 cells (kindly provided by Prof. Dr. Oesch, University Hospital Mainz) were washed twice with PBS, scraped off, and solubilized in ice-cold lysis buffer containing 1% NP-40, 5 mM EDTA, 2 mM PMSF in PBS (pH 7.4) by incubation at 4°C in a shaker for 1 h. Lysed cells were centrifugated to remove particles for 20 minutes with 14 000 rpm at 4°C. Anti-Stk33  was added to the supernatant. After incubation for 2 h, protein A sepharose (Amersham Bioscience Europe GmbH, Freiburg) was added for 1 h under constant agitation. After a centrifugation step (500 rpm, 4°C, 30 seconds) the pellet was washed three times with 1 ml washing buffer (0.1% NP-40, 5 mM EDTA in PBS, pH 7.4). The final pellet was suspended in non-reducing Laemmli buffer, heated to 95°C for 3 minutes and subjected to SDS-PAGE. Western Blotting experiments were carried out as described previously  using PVDF-membrane (Roth) and Immobilion Western-HRP chemiluminescence substrate (Millipore). For the detection of immunoprecipitated proteins, a polyconal anti-vimentin antibody kindly provided by Prof. Leube, University of Mainz, Germany was applied (1:15000). Anti-guinea pig-HRP as secondary antibody was used at 1:24000 dilution in PBS-T.
For an in vitro co-sedimentation assay, recombinant Stk33 and vimentin ΔN50 protein were incubated in phosphate buffered saline for 2 h at 4°C under gentle agitation. Recombinant Stk33 was precipitated from the solution by using anti-Stk33 antibody and protein A sepharose (Amersham Bioscience Europe GmbH, Freiburg). After centrifugation the sedimented material was washed 3 times with 1 ml washing buffer (0.1% Nonidet P40, 5 mM EDTA). Aliquots of all washing samples and the final pellet were suspended in non-reducing buffer, heated and electrophoretically resolved by gel electrophoresis. To test whether Stk33 and vimentin sedimented together, Western Blotting experiments were carried out using anti-Stk33 and monoclonal anti-rabbit IgG peroxidase conjugate clone RG-96 (Sigma) and anti-mouse IgG peroxidase conjugate (Sigma) for the detection of anti-vimentin Ab-2 (Dianova).
Testing enzymatic activity of immunoprecipitated Stk33 by kinase assay
In order to test whether the immunoprecipitated Stk33 has enzymatic activity we incubated precipitated Stk33 with recombinant vimentin proteins. Co-immunoprecipitation was carried out as described before. As a discrimination between autophosphorylated Stk33 and phosphorylated vimentin is hardly possible we used recombinant vimentin with a deletion of 50 amino acids for the phosphorylation studies. The reaction was initiated by adding 20 μCi γ 32P ATP. After 1 hour at 30°C the reaction was stopped by adding non-reducing Laemmli buffer. Further steps were carried out as already described in the kinase assay protocol.
We thank Prof. Leube (Department of Anatomy and Cell Biology, Johannes Gutenberg-University of Mainz) for providing the anti-vimentin antibody for Western Blot experiments and Prof. Oesch (Institute of Toxicology, University Hospital Mainz) for the kind gift of the SerW3 cell culture. In the early phase the work was supported by the Federal Ministry of Education and Research by a grant to ERS
- Mujica AO, Hankeln T, Schmidt ER: A novel serine/threonine kinase gene, STK33, on human chromosome 11p15.3. Gene. 2001, 280 (1–2): 175-181. 10.1016/S0378-1119(01)00780-6.View ArticlePubMedGoogle Scholar
- Mujica AO, Brauksiepe B, Saaler-Reinhardt S, Reuss S, Schmidt ER: Differential expression pattern of the novel serine/threonine kinase, STK33, in mice and men. Febs J. 2005, 272 (19): 4884-4898. 10.1111/j.1742-4658.2005.04900.x.View ArticlePubMedGoogle Scholar
- Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S: The protein kinase complement of the human genome. Science. 2002, 298 (5600): 1912-1934. 10.1126/science.1075762.View ArticlePubMedGoogle Scholar
- Kostich M, English J, Madison V, Gheyas F, Wang L, Qiu P, Greene J, Laz TM: Human members of the eukaryotic protein kinase family. Genome Biol. 2002, 3 (9): RESEARCH0043-10.1186/gb-2002-3-9-research0043.PubMed CentralView ArticlePubMedGoogle Scholar
- Caenepeel S, Charydczak G, Sudarsanam S, Hunter T, Manning G: The mouse kinome: Discovery and comparative genomics of all mouse protein kinases. Proc Natl Acad Sci USA. 2004, 101 (32): 11707-11712. 10.1073/pnas.0306880101.PubMed CentralView ArticlePubMedGoogle Scholar
- Hudmon A, Schulman H: Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II. Biochem J. 2002, 364 (Pt 3): 593-611. 10.1042/BJ20020228.PubMed CentralView ArticlePubMedGoogle Scholar
- Babcock AM, Standing D, Bullshields K, Schwartz E, Paden CM, Poulsen DJ: In vivo inhibition of hippocampal Ca2+/calmodulin-dependent protein kinase II by RNA interference. Mol Ther. 2005, 11 (6): 899-905. 10.1016/j.ymthe.2005.02.016.View ArticlePubMedGoogle Scholar
- Silva AJ, Paylor R, Wehner JM, Tonegawa S: Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice. Science. 1992, 257 (5067): 206-211. 10.1126/science.1321493.View ArticlePubMedGoogle Scholar
- Mayford M, Bach ME, Huang YY, Wang L, Hawkins RD, Kandel ER: Control of memory formation through regulated expression of a CaMKII transgene. Science. 1996, 274 (5293): 1678-1683. 10.1126/science.274.5293.1678.View ArticlePubMedGoogle Scholar
- Cho YH, Giese KP, Tanila H, Silva AJ, Eichenbaum H: Abnormal hippocampal spatial representations in alphaCaMKIIT286A and CREBalphaDelta- mice. Science. 1998, 279 (5352): 867-869. 10.1126/science.279.5352.867.View ArticlePubMedGoogle Scholar
- Silva AJ, Stevens CF, Tonegawa S, Wang Y: Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. Science. 1992, 257 (5067): 201-206. 10.1126/science.1378648.View ArticlePubMedGoogle Scholar
- Wang P, Wu YL, Zhou TH, Sun Y, Pei G: Identification of alternative splicing variants of the beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II with different activities. FEBS Lett. 2000, 475 (2): 107-110. 10.1016/S0014-5793(00)01634-3.View ArticlePubMedGoogle Scholar
- Marganski WA, Gangopadhyay SS, Je HD, Gallant C, Morgan KG: Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells. Circ Res. 2005, 97 (6): 541-549. 10.1161/01.RES.0000182630.29093.0d.View ArticlePubMedGoogle Scholar
- Inagaki N, Tsujimura K, Tanaka J, Sekimata M, Kamei Y, Inagaki M: Visualization of protein kinase activities in single cells by antibodies against phosphorylated vimentin and GFAP. Neurochem Res. 1996, 21 (7): 795-800. 10.1007/BF02532302.View ArticlePubMedGoogle Scholar
- Stefanovic S, Windsor M, Nagata KI, Inagaki M, Wileman T: Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II. J Virol. 2005, 79 (18): 11766-11775. 10.1128/JVI.79.18.11766-11775.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Inada H, Togashi H, Nakamura Y, Kaibuchi K, Nagata K, Inagaki M: Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments. J Biol Chem. 1999, 274 (49): 34932-34939. 10.1074/jbc.274.49.34932.View ArticlePubMedGoogle Scholar
- Oguri T, Inoko A, Shima H, Izawa I, Arimura N, Yamaguchi T, Inagaki N, Kaibuchi K, Kikuchi K, Inagaki M: Vimentin-Ser82 as a memory phosphorylation site in astrocytes. Genes Cells. 2006, 11 (5): 531-540. 10.1111/j.1365-2443.2006.00961.x.View ArticlePubMedGoogle Scholar
- Yasui Y, Goto H, Matsui S, Manser E, Lim L, Nagata K, Inagaki M: Protein kinases required for segregation of vimentin filaments in mitotic process. Oncogene. 2001, 20 (23): 2868-2876. 10.1038/sj.onc.1204407.View ArticlePubMedGoogle Scholar
- Yamaguchi T, Goto H, Yokoyama T, Sillje H, Hanisch A, Uldschmid A, Takai Y, Oguri T, Nigg EA, Inagaki M: Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis. J Cell Biol. 2005, 171 (3): 431-436. 10.1083/jcb.200504091.PubMed CentralView ArticlePubMedGoogle Scholar
- Hanks SK, Hunter T: Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. Faseb J. 1995, 9 (8): 576-596.PubMedGoogle Scholar
- Eriksson JE, He T, Trejo-Skalli AV, Harmala-Brasken AS, Hellman J, Chou YH, Goldman RD: Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments. J Cell Sci. 2004, 117 (Pt 6): 919-932. 10.1242/jcs.00906.View ArticlePubMedGoogle Scholar
- Ogawara M, Inagaki N, Tsujimura K, Takai Y, Sekimata M, Ha MH, Imajoh-Ohmi S, Hirai S, Ohno S, Sugiura H, et al.: Differential targeting of protein kinase C and CaM kinase II signalings to vimentin. J Cell Biol. 1995, 131 (4): 1055-1066. 10.1083/jcb.131.4.1055.View ArticlePubMedGoogle Scholar
- Wilmann M, Gautel M, Mayans O: Activation of calcium/calmodulin regulated kinases. Cell Mol Biol (Noisy-le-grand). 2000, 46 (5): 883-894.Google Scholar
- Colbran RJ, Smith MK, Schworer CM, Fong YL, Soderling TR: Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation. J Biol Chem. 1989, 264 (9): 4800-4804.PubMedGoogle Scholar
- Paramio JM, Jorcano JL: Beyond structure: do intermediate filaments modulate cell signalling?. Bioessays. 2002, 24 (9): 836-844. 10.1002/bies.10140.View ArticlePubMedGoogle Scholar
- Herrmann H, Aebi U: Structure, assembly, and dynamics of intermediate filaments. Subcell Biochem. 1998, 31: 319-362.PubMedGoogle Scholar
- Herrmann H, Hesse M, Reichenzeller M, Aebi U, Magin TM: Functional complexity of intermediate filament cytoskeletons: from structure to assembly to gene ablation. Int Rev Cytol. 2003, 223: 83-175. 10.1016/S0074-7696(05)23003-6.View ArticlePubMedGoogle Scholar
- Chou YH, Ngai KL, Goldman R: The regulation of intermediate filament reorganization in mitosis. p34cdc2 phosphorylates vimentin at a unique N-terminal site. J Biol Chem. 1991, 266 (12): 7325-7328.PubMedGoogle Scholar
- Inagaki M, Gonda Y, Nishizawa K, Kitamura S, Sato C, Ando S, Tanabe K, Kikuchi K, Tsuiki S, Nishi Y: Phosphorylation sites linked to glial filament disassembly in vitro locate in a non-alpha-helical head domain. J Biol Chem. 1990, 265 (8): 4722-4729.PubMedGoogle Scholar
- Li QF, Spinelli AM, Wang R, Anfinogenova Y, Singer HA, Tang DD: Critical role of vimentin phosphorylation at Ser-56 by p21-activated kinase in vimentin cytoskeleton signaling. J Biol Chem. 2006, 281 (45): 34716-34724. 10.1074/jbc.M607715200.PubMed CentralView ArticlePubMedGoogle Scholar
- Tang DD, Bai Y, Gunst SJ: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine. Biochem J. 2005, 388 (Pt 3): 773-783.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang L, Kasif S, Cantor CR, Broude NE: GC/AT-content spikes as genomic punctuation marks. Proc Natl Acad Sci USA. 2004, 101 (48): 16855-16860. 10.1073/pnas.0407821101.PubMed CentralView ArticlePubMedGoogle Scholar
- Ivaska J, Pallari HM, Nevo J, Eriksson JE: Novel functions of vimentin in cell adhesion, migration, and signaling. Exp Cell Res. 2007, 313 (10): 2050-2062. 10.1016/j.yexcr.2007.03.040.View ArticlePubMedGoogle Scholar
- Izawa I, Inagaki M: Regulatory mechanisms and functions of intermediate filaments: a study using site- and phosphorylation state-specific antibodies. Cancer Sci. 2006, 97 (3): 167-174. 10.1111/j.1349-7006.2006.00161.x.View ArticlePubMedGoogle Scholar
- Kochin V, Imanishi SY, Eriksson JE: Fast track to a phosphoprotein sketch – MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity. Proteomics. 2006, 6 (21): 5676-5682. 10.1002/pmic.200600457.View ArticlePubMedGoogle Scholar
- Helfand BT, Chang L, Goldman RD: Intermediate filaments are dynamic and motile elements of cellular architecture. J Cell Sci. 2004, 117 (Pt 2): 133-141. 10.1242/jcs.00936.View ArticlePubMedGoogle Scholar
- Helfand BT, Chou YH, Shumaker DK, Goldman RD: Intermediate filament proteins participate in signal transduction. Trends Cell Biol. 2005, 15 (11): 568-570. 10.1016/j.tcb.2005.09.009.View ArticlePubMedGoogle Scholar
- Prahlad V, Yoon M, Moir RD, Vale RD, Goldman RD: Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks. J Cell Biol. 1998, 143 (1): 159-170. 10.1083/jcb.143.1.159.PubMed CentralView ArticlePubMedGoogle Scholar
- Helfand BT, Mikami A, Vallee RB, Goldman RD: A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization. J Cell Biol. 2002, 157 (5): 795-806. 10.1083/jcb.200202027.PubMed CentralView ArticlePubMedGoogle Scholar
- Clarke EJ, Allan V: Intermediate filaments: vimentin moves in. Curr Biol. 2002, 12 (17): R596-598. 10.1016/S0960-9822(02)01102-8.View ArticlePubMedGoogle Scholar
- Perlson E, Hanz S, Ben-Yaakov K, Segal-Ruder Y, Seger R, Fainzilber M: Vimentin-Dependent Spatial Translocation of an Activated MAP Kinase in Injured Nerve. Neuron. 2005, 45 (5): 715-726. 10.1016/j.neuron.2005.01.023.View ArticlePubMedGoogle Scholar
- Inagaki N, Goto H, Ogawara M, Nishi Y, Ando S, Inagaki M: Spatial patterns of Ca2+ signals define intracellular distribution of a signaling by Ca2+/Calmodulin-dependent protein kinase II. J Biol Chem. 1997, 272 (40): 25195-25199. 10.1074/jbc.272.40.25195.View ArticlePubMedGoogle Scholar
- Gimenez YRM, Langa F, Menet V, Privat A: Comparative anatomy of the cerebellar cortex in mice lacking vimentin, GFAP, and both vimentin and GFAP. Glia. 2000, 31 (1): 69-83. 10.1002/(SICI)1098-1136(200007)31:1<69::AID-GLIA70>3.0.CO;2-W.View ArticleGoogle Scholar
- Eckes B, Colucci-Guyon E, Smola H, Nodder S, Babinet C, Krieg T, Martin P: Impaired wound healing in embryonic and adult mice lacking vimentin. J Cell Sci. 2000, 113 (Pt 13): 2455-2462.PubMedGoogle Scholar
- Chou YH, Khuon S, Herrmann H, Goldman RD: Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis. Mol Biol Cell. 2003, 14 (4): 1468-1478. 10.1091/mbc.E02-08-0545.PubMed CentralView ArticlePubMedGoogle Scholar
- Blom N, Gammeltoft S, Brunak S: Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol. 1999, 294 (5): 1351-1362. 10.1006/jmbi.1999.3310.View ArticlePubMedGoogle Scholar
- Nakai K, Horton P: PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci. 1999, 24 (1): 34-36. 10.1016/S0968-0004(98)01336-X.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.