Deimination, Intermediate Filaments and Associated Proteins.

Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes.

Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.

Cytoplasmic intermediate filaments (cIFs) are found in all eumetazoans, except arthropods. To investigate the compatibility of cIFs in arthropods, we expressed human vimentin (hVim), a cIF with filament-forming capacity in vertebrate cells and tissues, transgenically in Drosophila Transgenic hVim could be recovered from whole-fly lysates by using a standard procedure for intermediate filament (IF) extraction. When this procedure was used to test for the possible presence of IF-like proteins in flies, only lamins and tropomyosin were observed in IF-enriched extracts, thereby providing biochemical reinforcement to the paradigm that arthropods lack cIFs. In Drosophila, transgenic hVim was unable to form filament networks in S2 cells and mesenchymal tissues; however, cage-like vimentin structures could be observed around the nuclei in internal epithelia, which suggests that Drosophila retains selective competence for filament formation. Taken together, our results imply that although the filament network formation competence is partially lost in Drosophila, a rudimentary filament network formation ability remains in epithelial cells. As a result of the observed selective competence for cIF assembly in Drosophila, we hypothesize that internal epithelial cIFs were the last cIFs to disappear from arthropods.-Gullmets, J., Torvaldson, E., Lindqvist, J., Imanishi, S. Y., Taimen, P., Meinander, A., Eriksson, J. E. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics.

C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3ß (GSK3ß). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li(+), D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3ß, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3ß mediated the neurofilament-neurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/2) was found to downregulate GSK3ß, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilament-neurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3ß, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3ß.

Phosphatase and actin regulator 4 is associated with intermediate filaments in adult neural stem cells and their progenitor astrocytes.

Phosphatase and actin regulator 4 (Phactr4) is a newly discovered protein that inhibits protein phosphatase 1 and shows actin-binding activity. We previously found that Phactr4 is expressed in the neurogenic niche in adult mice, although its precise subcellular localization and possible function in neural stem cells (NSCs) is not yet understood. Here, we show that Phactr4 formed punctiform clusters in the cytosol of subventricular zone-derived adult NSCs and their progeny in vitro. These Phactr4 signals were not associated with F-actin fibers but were closely associated with intermediate filaments such as nestin and glial fibrillary acidic protein (GFAP) fibers. Direct binding of Phactr4 with nestin and GFAP filaments was demonstrated using Duolink protein interaction analyses and immunoprecipitation assays. Interestingly, when nestin fibers were de-polymerized during the mitosis or by the phosphatase inhibitor, Phactr4 appeared to be dissociated from nestin, suggesting that their protein interaction is regulated by the protein phosphorylation. These results suggest that Phactr4 forms functional associations with intermediate filament networks in adult NSCs.

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration.

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.

Microtubule-independent regulation of neurofilament interactions in vitro by neurofilament-bound ATPase activities.

Neurofilaments (NFs), the major neuronal intermediate filaments, form networks in vitro that mimic the axonal NF bundles. This report presents evidence for previously unknown regulation of the interactions between NFs by NF-associated ATPases. Two opposite effects on NF gelation in vitro occur at low and high ATP concentration. These findings support the hypothesis that NF bundles in situ are dynamic structures, and raise the possibility that ATP-hydrolyzing mechanoenzymes regulate their organization.

Protein kinase Cepsilon: multiple roles in the function of, and signaling mediated by, the cytoskeleton.

Recent studies have established essential roles of protein kinase Cepsilon in signaling pathways controlling various functions of microfilaments and intermediate filaments by modulating multiple cytoskeletal proteins. This review summarizes recent progress in our understanding of the roles of protein kinase Cepsilon in the functions and signaling of microfilaments and intermediate filaments, with a focus mainly on cell-matrix and cell-cell interactions, migrations and contraction, in addition to its relevance in the development of several diseases, such as malignant tumors or cardiac disease.

Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia: Heat-Shock Protein Chaperoning and Kinase Rescue.

A growing body of evidence from several laboratories points at nonmechanical functions of keratin intermediate filaments (IF), such as control of apoptosis, modulation of signaling, or regulation of innate immunity, among others. While these functions are generally assigned to the ability of IF to scaffold other proteins, direct mechanistic causal relationships between filamentous keratins and the observed effects of keratin knockout or mutations are still missing. We have proposed that the scaffolding of chaperones such as Hsp70/40 may be key to understand some IF nonmechanical functions if unique features or specificity of the chaperoning activity in the IF scaffold can be demonstrated. The same criteria of uniqueness could be applied to other biochemical functions of the IF scaffold. Here, we describe a subcellular fractionation technique based on established methods of keratin purification. The resulting keratin-enriched fraction contains several proteins tightly associated with the IF scaffold, including Hsp70/40 chaperones. Being nondenaturing, this fractionation method enables direct testing of chaperoning and other enzymatic activities associated with IF, as well as supplementation experiments to determine the need for soluble (cytosolic) proteins. This method also permits to analyze inhibitory activity of cytosolic proteins at independently characterized physiological concentrations. When used as complementary approaches to knockout, knockdown, or site-directed mutagenesis, these techniques are expected to shed light on molecular mechanisms involved in the effects of IF loss of function.

Properties of several protein kinases that copurify with rat spinal cord neurofilaments.

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.

Immunoelectron microscopical localization of the catalytic subunit of cAMP-dependent protein kinases in brain microtubules and neurofilaments.

The catalytic subunit of cAMP-dependent protein kinases was localized in microtubules and neurofilaments by immunogold electron microscopy. In microtubules, the label was similarly distributed as an immunolabel for the microtubule associated protein MAP 2. The neurofilaments showed no reaction with the MAP 2-antiserum. Our results support the suggestion of an in vivo role of cAMP-dependent protein kinases in the regulation of microtubules. In addition, this is the first demonstration that cAMP-dependent protein kinase is associated with neurofilaments.

Melatonin activates PKC-alpha but not PKC-epsilon in N1E-115 cells.

Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.

Association of calcium/calmodulin-dependent kinase with cytoskeletal preparations: phosphorylation of tubulin, neurofilament, and microtubule-associated proteins.

Calcium and calmodulin have been implicated in the regulation of cytoskeletal function. In this report, we demonstrate that microtubule preparations from rat brain contain a calcium/calmodulin-dependent protein kinase that phosphorylates endogenous MAP-2, tubulin, synapsin I, and neurofilament proteins. This cytoskeletal-associated kinase has been biochemically characterized and shown to be identical to Type II calcium/calmodulin-dependent protein kinase (CaM kinase II). The subunits of CaM kinase II represented major calmodulin-binding proteins in cytoskeletal preparations. A monoclonal antibody against the 52000 Da subunit of CaM kinase II specifically labeled cytoskeletal elements in cortical neurons. These results indicate that CaM kinase II is associated with the neuronal cytoskeleton and may play a role in mediating some of the effects of calcium on cytoskeletal function.

Characterization of a neurofilament-associated kinase that phosphorylates the middle molecular mass component of chicken neurofilaments.

We have examined the properties of a chicken neurofilament (NF) kinase partially purified from NF-enriched preparations. This kinase cosediments with NFs following extraction with Triton X-100 and can be separated in an active form from NFs by treatment with 0.8 M KCl. Sequential chromatography of the salt extract on DEAE-cellulose and phosphocellulose results in an approximately 500-fold increase in specific activity over endogenous NF preparations as measured by 32P-incorporation into the middle molecular mass component of NFs (NF-M). The kinase is Mg(2+)-dependent, second messenger-independent and inhibited by high concentrations of heparin. It shows selectivity for NF-M and evidence is presented that the kinase phosphorylates NF-M solely in the tail domain. The kinase can also phosphorylate the microtubule-associated proteins tau and MAP2 as well as mammalian NF-M, all of which share putative phosphorylation sequences with chicken NF-M.

Casein kinase II is associated with neurofibrillary tangles but is not an intrinsic component of paired helical filaments.

Neurofibrillary tangles (NFT) are pathological cytoskeletal structures composed of paired helical filaments (PHF), and are found in neurons of patients afflicted with many neurodegenerative disorders, including Alzheimer's disease (AD). We previously found that an antiserum against casein kinase II (CK-II) stained NFT intensely in the brain tissue of AD patients. In the current study, we found that the anti-CK-II antiserum stains NFT and neuronal inclusions in many other neurodegenerative diseases as well, including Guam-Parkinson dementia complex, chromosome 18 deletion syndrome, progressive supranuclear palsy, Kufs' disease, and Pick's disease. This antiserum reacted, in crude brain homogenates, with both a doublet of Mr 43,000 and a Mr 27,000 Da protein which could correspond to the alpha, alpha', and beta chains of CK-II. The staining of these bands was adsorbed by preincubating anti-CK-II antiserum with purified CK-II. Preincubation of brain sections with purified CK-II strongly intensified the immunostaining of NFT with anti-CK-II, suggesting that NFT may bind CK-II. In the AD brain homogenates, the particulate CK-II levels are increased whereas the cytosolic levels are decreased without a change in total CK-II levels, consistent with the idea that CK-II binds to the particulate PHF, a major constituent of NFT. In accord with these findings, purified PHF bound CK-II, but purified PHF did not contain CK-II as its component. These results suggest that CK-II might be an extraneously deposited component of NFT. Thus, the altered CK-II compartmentalization might have significant consequences in the pathogenesis of AD.

Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase.

Glycogen synthase kinase-3 (GSK-3) reduced the mobility of human tau on SDS-PAGE, prevented binding of the monoclonal antibody (mAb), Tau.1, and induced binding of the mAb 8D8. Recombinant tau phosphorylated by GSK-3 aligned on SDS-PAGE with the abnormally phosphorylated tau (PHF-tau) associated with the paired helical filaments in Alzheimer's disease brain. Phosphorylated serine396 (numbering of the largest human brain tau isoform) was identified as a binding site on tau for mAb 8D8. The localisation of GSK-3 within granular structures in pyramidal cells indicates that GSK-3 alpha and GSK-3 beta may have a role in the production of PHF-tau in Alzheimer's disease.

Bovine neurofilament-enriched preparations contain kinase activity similar to casein kinase I--neurofilament phosphorylation by casein kinase I (CKI).

Neurofilament (NF)-enriched preparations from bovine spinal cord contain regulator-independent kinase activities that phosphorylate NF subunits as well as alpha-casein. CKI-7 (N-2-amino ethyl, 5-chloroisoquinoline, 8-sulfonamide), a specific inhibitor of casein kinase I (CKI), inhibits the phosphorylation of NF subunits in the neurofilament preparation. This inhibition occurs at a concentration range identical to concentrations where CKI-7 inhibits rabbit reticulocyte CKI phosphorylation of alpha-casein. Heparin, a specific inhibitor of casein kinase II (CKII), produced only 20% inhibition of 32P incorporation into NF subunits, and only at concentrations 5 to 10-fold higher than those required to inhibit CKII from reticulocytes. CKI from rabbit reticulocytes phosphorylated all three NF subunits (NF-H, NF-M and NF-L). Comparison of the tryptic phosphopeptide maps of NF-M, phosphorylated by the NF-associated kinase and CKI, indicates that the casein kinase I phosphorylates many of the peptides phosphorylated by the NF-associated kinase and this phosphorylation occurs at the carboxy terminal tail domain of the NF-M subunit. These studies suggest that the major independent kinase activity associated with NFs is CKI.

Occurrence and distribution of different neurochemical markers in the human dental pulp.

The occurrence and distribution of neuronal markers in human premolar and molar pulps were studied immunohistochemically. In the apical and central parts of the pulp, evenly distributed, thick neurofilament-immunoreactive nerve bundles predominated, which in many instances accompanied blood vessels. In the coronal parts, especially in the pulp horns, such nerve bundles formed a subodontoblastic plexus, while thin neurofilament-immunoreactive fibres projected into the odontoblastic region. In the coronal parts of the pulp, thin, varicose, calcitonin gene-related peptide (CGRP)- and occasionally substance P-immunoreactive fibres were observed in the pulp-dentine zone and also in the vicinity of blood vessels. Vasoactive intestinal polypeptide (VIP) fibres were distributed in several nerve bundles, while single VIP fibres were seen projecting into the odontoblastic region as well as in the vicinity of blood vessels. Peptide histidine isoleucine amide (PHI)-immunoreactive fibres showed a similar distribution as VIP, but were less common. Furthermore, neuropeptide Y-immunoreactive fibres occurred occasionally around blood vessels in the inner parts of the pulp. Tyrosine hydroxylase-immunoreactive nerve fibres with a varicose appearance were observed in some nerve bundles, but were also frequently seen around and in blood vessels. In premolar pulps obtained from teeth with open apices a less dense neurofilament innervation was seen in the coronal pulp. However, no apparent difference in the occurrence and distribution of the other neuronal markers was found compared to mature teeth. The human dental pulp, thus, seems to have a rich occurrence of neuropeptides and tyrosine hydroxylase in thin, varicose fibres. However, the distribution of the fibres expressing immunoreactivity to these neuronal markers seems to be sparse in comparison to neurofilament-immunoreactive fibres.

PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia.

Phosphorylation of the activation domain of protein kinase C (PKC) isoforms is essential to start a conformational change that results in an active catalytic domain. This activation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This "rescue" mechanism is responsible for the maintenance of the steady-state levels of atypical PKC (aPKC [PKCι/λ and ζ]) and is blocked in inflammation. Although there is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized molecules, it is unclear what kinase performs that function during the rescue and where the rescue takes place. To identify the activating kinase during the rescue mechanism, we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool. PDK1 knockdown and two different PDK1 inhibitors-BX-912 and a specific pseudosubstrate peptide-destabilized PKCι. PDK1 coimmunoprecipitated with PKCι in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays using immunodepletion and rescue with recombinant protein. Surprisingly, we found that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and pAkt were dependent on dynamin activity. These results highlight a novel signaling function of apical endosomes in polarized cells.

Hyperphosphorylation of intermediate filament proteins is involved in microcystin-LR-induced toxicity in HL7702 cells.

Microcystin-LR (MC-LR) is commonly characterized as a hepatotoxin, which can cause disruption of keratin filaments. Keratins, however, account for only two types of intermediate filaments (IFs), and the potential involvement of other IF proteins in MC-LR-induced toxicity and the underlying mechanisms are still unclear. In this study, the human normal liver cell line HL7702 was used to investigate whether MC-LR can change the transcription, translation, and phosphorylation levels of major IF proteins and to elucidate the underlying mechanisms. The results showed that MC-LR triggered an accumulation of IFs around the nucleus and led to the formation of dense bundles. When the cells were treated with 10µM MC-LR, cell proliferation significantly decreased with an increase in apoptosis and cell cycle arrest. Moreover, the mRNA and protein levels of keratin 18, vimentin and lamin A/C were not changed; however, the phosphorylation of K8/18 and vimentin was significantly increased. Furthermore, we found MC-LR exposure caused phosphoactivation of P38, JNK and ERK1/2 in a concentration-dependent manner, and P38 and ERK1/2 were involved in MC-LR-induced hyperphosphorylation of IF proteins. Taken together, the results of this study suggest that MC-LR exerts its potential hepatotoxicity through MAPK pathway activation, which cause hyperphosphorylation of IF proteins and result in cytoskeletal architecture remodeling and cell survival/death regulation. Since IFs serve as signaling platforms and dozens of IF proteins are involved in different signaling pathways, future studies focus on different IFs may provide helpful insights into the mechanisms of MC-LR toxicity.