Intermediate Filament Phosphorylation Research Articles

Consequences of Keratin Phosphorylation for Cytoskeletal Organization and Epithelial Functions.

Intermediate filaments are major phosphoproteins. The complex patterns of intermediate filament phosphorylation make up a poorly understood code reflecting cytoskeletal properties and cellular function through an intense crosstalk with multiple signaling pathways. This review focuses on the epithelial keratin intermediate filaments highlighting the tight-knit relationship of keratin phosphorylation and network organization during cell division and apoptosis, and the importance of keratin phosphorylation during epithelial stress responses. The occurrence of keratin phosphorylation in genetic skin diseases and acquired diseases of simple epithelial tissues in liver, pancreas, and colon will be discussed. Finally, we will review the role of keratin phosphorylation in cancer with an emphasis on migration.

A novel function for the MAP kinase SMA-5 in intestinal tube stability.

Intermediate filaments are major cytoskeletal components whose assembly into complex networks and isotype-specific functions are still largely unknown. Caenorhabditis elegans provides an excellent model system to study intermediate filament organization and function in vivo. Its intestinal intermediate filaments localize exclusively to the endotube, a circumferential sheet just below the actin-based terminal web. A genetic screen for defects in the organization of intermediate filaments identified a mutation in the catalytic domain of the MAP kinase 7 orthologue sma-5(kc1) In sma-5(kc1) mutants, pockets of lumen penetrate the cytoplasm of the intestinal cells. These membrane hernias increase over time without affecting epithelial integrity and polarity. A more pronounced phenotype was observed in the deletion allele sma-5(n678) and in intestine-specific sma-5(RNAi) Besides reduced body length, an increased time of development, reduced brood size, and reduced life span were observed in the mutants, indicating compromised food uptake. Ultrastructural analyses revealed that the luminal pockets include the subapical cytoskeleton and coincide with local thinning and gaps in the endotube that are often enlarged in other regions. Increased intermediate filament phosphorylation was detected by two-dimensional immunoblotting, suggesting that loss of SMA-5 function leads to reduced intestinal tube stability due to altered intermediate filament network phosphorylation.

New insights into roles of intermediate filament phosphorylation and progeria pathogenesis

Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Because of their insoluble nature, cellular IFs had been considered to be stable for a long time. The discovery that a purified protein kinase phosphorylated a purified IF protein and in turn induced the disassembly of IF structure in vitro led to the novel concept of dynamic IF regulation. Since then, a variety of protein kinases have been identified to phosphorylate IF proteins such as vimentin in a spatiotemporal regulated manner. A series of studies using cultured cells have demonstrated that preventing IF phosphorylation during mitosis inhibits cytokinesis by the retention of an IF bridge-like structure (IF-bridge) connecting the two daughter cells. Knock-in mice expressing phosphodeficient vimentin variants developed binucleation/aneuploidy in lens epithelial cells, which promoted microophthalmia and lens cataract. Therefore, mitotic phosphorylation of vimentin is of great importance in the completion of cytokinesis, the impairment of which promotes chromosomal instability and premature aging. © 2014 IUBMB Life, 66(3):195-200, 2014.

Defect of Mitotic Vimentin Phosphorylation Causes Microophthalmia and Cataract via Aneuploidy and Senescence in Lens Epithelial Cells

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

Identification and Cytoprotective Function of a Novel Nestin Isoform, Nes-S, in Dorsal Root Ganglia Neurons

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.

1α,25-Dihydroxyvitamin D3 mechanism of action: Modulation of L-type calcium channels leading to calcium uptake and intermediate filament phosphorylation in cerebral cortex of young rats

The involvement of calcium-mediated signaling pathways in the mechanism of action of 1α,25-dihydroxyvitamin D3 (1,25D) is currently demonstrated. In this study we found that 1,25D induces nongenomic effects mediated by membrane vitamin D receptor (VDRm) by modulating intermediate filament (IF) phosphorylation and calcium uptake through L-type voltage-dependent calcium channels (L-VDCC) in cerebral cortex of 10day-old rats. Results showed that the mechanism of action of 1,25D involves intra- and extracellular calcium levels, as well as the modulation of chloride and potassium channels. The effects of L-VDCCs on membrane voltage occur over a broad potential range and could involve depolarizing or hyperpolarizing coupling modes, supporting a cross-talk among Ca2+ uptake and potassium and chloride channels. Also, the Na+/K+-ATPase inactivation by ouabain mimicked the 1,25D action on 45Ca2+ uptake. The Na+/K+-ATPase inhibition observed herein might lead to intracellular Na+ accumulation with subsequent L-VDCC opening and consequently increased 45Ca2+ (calcium, isotope of mass 45) uptake. Moreover, the 1,25D effect is dependent on the activation of the following protein kinases: cAMP‐dependent protein kinase (PKA), Ca2+/calmodulin‐dependent protein kinase (PKCaMII), phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase p38 (p38MAPK). The modulation of calcium entry into neural cells by the 1,25D we are highlighting, might take a role in the regulation of a plethora of intracellular processes. Considering that vitamin D deficiency can lead to brain illness, 1,25D may be a possible candidate to be used, at least as an adjuvant, in the pharmacological therapy of neuropathological conditions.

A tension-induced mechanotransduction pathway promotes epithelial morphogenesis

Mechanotransduction refers to the transformation of physical forces into chemical signals. It generally involves stretch-sensitive channels or conformational change of cytoskeleton-associated proteins. Mechanotransduction is crucial for the physiology of several organs and for cell migration. The extent to which mechanical inputs contribute to development, and how they do this, remains poorly defined. Here we show that a mechanotransduction pathway operates between the body-wall muscles of Caenorhabditis elegans and the epidermis. This pathway involves, in addition to a Rac GTPase, three signalling proteins found at the hemidesmosome: p21-activated kinase (PAK-1), the adaptor GIT-1 and its partner PIX-1. The phosphorylation of intermediate filaments is one output of this pathway. Tension exerted by adjacent muscles or externally exerted mechanical pressure maintains GIT-1 at hemidesmosomes and stimulates PAK-1 activity through PIX-1 and Rac. This pathway promotes the maturation of a hemidesmosome into a junction that can resist mechanical stress and contributes to coordinating the morphogenesis of epidermal and muscle tissues. Our findings suggest that the C. elegans hemidesmosome is not only an attachment structure, but also a mechanosensor that responds to tension by triggering signalling processes. We suggest that similar pathways could promote epithelial morphogenesis or wound healing in other organisms in which epithelial cells adhere to tension-generating contractile cells.

Unconventional actin conformations localize on intermediate filaments in mitosis

Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel “lower dimer” actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

Nongenomic actions of thyroxine modulate intermediate filament phosphorylation in cerebral cortex of rats

The developmental effects of thyroid hormones (TH) in mammalian brain are mainly mediated by nuclear receptors regulating gene expression. However, there are increasing evidences of nongenomic mechanisms of these hormones associated with kinase- and calcium-activated signaling pathways. In this context, the aim of the present work was to investigate the signaling pathways involved in the mechanism of action of TH on cytoskeletal phosphorylation in cerebral cortex of 15-day-old male rats. Results showed that l-thyroxine (L-T 4) increased the intermediate filament (IF) phosphorylation independently of protein synthesis, without altering the total immunocontent of these proteins. Otherwise, neither 3,5,3′-triiodo- l-thyronine (L-T 3) nor neurotransmitters (GABA, ATP, l-glutamate or epinephrine) acted on the IF-associated phosphorylation level. We also demonstrated that the mechanisms underlying the L-T 4 effect on the cytoskeleton involve membrane initiated actions through G i protein–coupled receptor. This evidence was reinforced by the inhibition of cyclic adenosine 5′-monophosphate (cAMP) levels. Moreover, we showed the participation of phospholipase C, protein kinase C, mitogen-activated protein kinase, calcium/calmodulin-dependent protein kinase II, intra- and extracellular Ca 2+ mediating the effects of L-T 4 on the cytoskeleton. Stimulation of 45Ca 2+ uptake by L-T 4 was also demonstrated. These findings demonstrate that L-T 4 has important physiological roles modulating the cytoskeleton of neural cells during development.

Aurora-B dysfunction of multinucleated giant cells in glioma detected by site-specific phosphorylated antibodies

The origin of multinucleated giant cells in glioma has not been made clear. In a previous paper the authors studied multinucleated giant tumor cells by using mitosis-specific phosphorylated antibodies to determine the phosphorylation of intermediate filaments and demonstrated that these cells stay in the early mitotic stage, undergoing neither fusion nor degeneration. In the current study the authors investigated the possible genetic causes of multinucleated giant tumor cells. Cultured mono- or multinucleated human glioma cells were immunostained with monoclonal antibodies (mAbs) 4A4, YT33, TM71, HTA28, YG72, and alphaAIM-1. The three former antibodies revealed a particular mitotic cell cycle through site-specific phosphorylation of vimentin; that is, the early phase, mid phase, and late phase, respectively. The three later antibodies demonstrated phosphorylation of H3 at Ser28, phosphorylation of vimentin at Ser72, and aurora-B, respectively, making it possible to identify aurora-B distribution and function during mitosis. In addition, paraffin-embedded tissue sections obtained in three patients with giant cell glioblastoma were also examined. Multinucleated giant tumor cells immunoreacted with the mAb 4A4 and alphaAIM-1 but not with YT33, TM71, HTA28, and YG72 in vitro and in vivo. Findings in this study indicated that multinucleated giant tumor cells remain in the early mitotic phase because of aurora-B dysfunction, effecting aberrations in cytoplasmic cleavage without affecting nuclear division.

α-Keto-β-methylvaleric acid increases the in vitro phosphorylation of intermediate filaments in cerebral cortex of young rats through the gabaergic system

In this study we investigated the effects of α-ketoisovaleric (KIV) and α-keto-β-methylvaleric acids (KMV), metabolites accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of young rats during development (9–21 days of age) We observed that KMV significantly increased the in vitro incorporation of 32P into the IF proteins studied in cortical slices of 12-day-old rats through the PKA and PKCaMII, with no alteration at the other ages. In contrast, KIV was ineffective in altering the phosphorylating system associated with IF proteins at all ages examined. A similar effect on IF phosphorylation was achieved by incubating cortical slices with γ-aminobutiric acid (GABA). Furthermore, by using specific GABA antagonists, we verified that KMV induced a stimulatory effect on IF phosphorylation of tissue slices from 12-day-old rats mediated by GABA A and GABA B receptors. In conclusion, our results indicate the involvement of the GABAergic system in the alterations of IF phosphorylation caused by KMV, one of the branched-chain keto acids accumulating in MSUD.

Morphological assessment of the development of multinucleated giant cells in glioma by using mitosis-specific phosphorylated antibodies.

The nature and origin of multinucleated giant cells in glioma have not been made clear. To investigate the phosphorylation of intermediate filaments, the authors studied multinucleated giant cells in vitro and in vivo by using mitosis-specific phosphorylated antibodies. Cultured human glioma cells were immunostained with monoclonal antibodies (mAbs) 4A4, KT13, and TM71, which recognized the phosphorylation of vimentin at Ser55, glial fibrillary acidic protein at Serl3, and vimentin at Ser71, respectively. Subsequently, the nature of multinucleated giant cells was investigated using laser scanning confocal microscopy. In addition, paraffin-embedded tissue sections obtained in three patients with giant cell glioblastoma were also investigated. Multinucleated giant cells were immunoreacted with the mAb 4A4 and not with KT13 and TM71 in vitro and in vivo. In addition, the authors obtained these results in multinucleated giant cells under natural conditions, without drug treatments. Findings in this investigation indicated that multinucleated giant cells are those remaining in mitosis between metaphase and telophase, undergoing neither fusion nor degeneration.

Effect of propionic and methylmalonic acids on the in vitro phosphorylation of intermediate filaments from cerebral cortex of rats during development.

In this study we investigated the in vivo and in vitro effects of methylmalonic (MMA) and propionic acids (PA), at concentrations usually found in methylmalonic acidemia and propionic acidemia respectively, on the phosphorylation of intermediate filament proteins in cerebral cortex of rats during development. Rats of 9, 12, and 17 days were acutely injected with the acids and sacrificed 90 min after injection. The cerebral cortex was dissected, and slices were incubated with 32P-orthophosphate. The cytoskeletal fraction was extracted and the radioactivity incorporated into intermediate filament subunits was measured. In addition, cortical slices from nontreated rats of 9, 12, 15, 17, 21, and 60 days of life were incubated with the acids in the presence of 32P-orthophosphate, the cytoskeletal fraction was extracted and the radioactivity was measured. Results demonstrated that MMA and PA significantly decreased the radioactivity incorporated into intermediate filament proteins at day 12, both in vivo and in tissue slices. In contrast, PA increased the in vitro phosphorylation of the cytoskeletal proteins in slices of 21-day-old animals. It acts through PP2A and PP2B in 12-day-old rats and through PKA and PKCaMII in 21-day-old animals. We propose that alteration of cytoskeletal protein phosphorylation caused by methylmalonic and propionic acids may be related to the neurological dysfunction characteristic of propionic and methylmalonic acidemia.

α-Ketoisocaproic acid regulates phosphorylation of intermediate filaments in postnatal rat cortical slices through ionotropic glutamatergic receptors

In this study we investigated the effects of α-ketoisocaproic acid (KIC), the main keto acid accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of rats during development. KIC decreased the in vitro incorporation of 32P into the IF proteins studied up to day 12, had no effect on day 15, and increased this phosphorylation in cortical slices of 17- and 21-day-old rats. A similar effect on IF phosphorylation was achieved along development by incubating cortical slices with glutamate. Furthermore, the altered phosphorylation caused by the presence of KIC in the incubation medium was mediated by the ionotropic receptors NMDA, AMPA and kainate up to day 12 and by NMDA and AMPA in tissue slices from 17- and 21-day-old rats. The results suggest that alterations of IF phosphorylation may be associated with the neuropathology of MSUD.

Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments.

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

A case of giant axonal neuropathy showing focal aggregation and hypophosphorylation of intermediate filaments

We report here the clinicopathological features of a typical case of giant axonal neuropathy (GAN). Scanning electron microscopy of the hair of this case revealed an extraordinarily irregular cuticle. Focal accumulation of intermediate filaments in axons, Schwann cells, muscle fibers and skin fibroblasts were also found under an electron microscopy. When examined immunocytochemically, muscle fibers exhibited local disruption of the filamentous network in the subsarcolemmal space and in the central cytoplasm accompanied by focal accumulation of desmin. The intracellular network of vimentin was also disrupted, exhibiting global accumulation in some of the cultured skin fibroblasts. Decreased interneurofilament spacing was found in enlarged axons, suggesting the presence of hypophosphorylation of neurofilaments in this patient. These findings suggest general disorganization, abnormal distribution and possible defective phosphorylation of intermediate filaments in GAN.

Dynamic organisation of intermediate filaments and associated proteins during the cell cycle.

Intermediate filaments, which form the structural framework of both the cytoskeleton and the nuclear lamina in most eukaryotic cells, have been found to be highly dynamic structures. A continuous exchange of subunit proteins at the filament surface and a stabilisation of soluble subunits by chaperone-type proteins may modulate filament structure and plasticity. Recent studies on the cell cycle-dependent interaction of intermediate filaments with associated proteins, and a detailed analysis of intermediate filament phosphorylation in defined subcellular locations at various stages of mitosis, have brought new insights into the molecular mechanisms involved in the mitotic reorganisation of intermediate filaments. Some of these studies have allowed new speculations about the possible cellular functions of cytoplasmic intermediate filaments, and increased our understanding of the specific functions of the lamins and the lamina-associated membrane proteins in the post-mitotic reassembly of the nucleus.

Two-dimensional gel analysis of glandular keratin intermediate filament phosphorylation.

Two-dimensional gel analysis of glandular keratin intermediate filament phosphorylation.

Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization.

There is ample in vitro evidence that phosphorylation of intermediate filaments, including keratins, plays an important role in filament reorganization. In order to gain a better understanding of the function of intermediate filament phosphorylation, we sought to identify the major phosphorylation site of human keratin polypeptide 18 (K18) and study its role in filament assembly or reorganization. We generated a series of K18 ser-->ala mutations at potential phosphorylation sites, followed by expression in insect cells and comparison of the tryptic 32PO4-labeled patterns of the generated constructs. Using this approach, coupled with Edman degradation of the 32PO4-labeled tryptic peptides, and comparison with tryptic peptides analyzed after labeling normal human colonic tissues, we identified ser-52 as the major K18 physiologic phosphorylation site. Ser-52 in K18 is not glycosylated and matches consensus sequences for phosphorylation by CAM kinase, S6 kinase and protein kinase C, and all these kinases can phosphorylate K18 in vitro predominantly at that site. Expression of K18 ser-52-->ala mutant in mammalian cells showed minimal phosphorylation but no distinguishable difference in filament assembly when compared with wild-type K18. In contrast, the ser-52 mutation played a clear but nonexclusive role in filament reorganization, based on analysis of filament alterations in cells treated with okadaic acid or arrested at the G2/M stage of the cell cycle. Our results show that ser-52 is the major physiologic phosphorylation site of human K18 in interphase cells, and that its phosphorylation may play an in vivo role in filament reorganization.

Dual regulation of intermediate filament phosphorylation.

Intermediate filament proteins have been isolated from ME-180, cells of a human cervical carcinoma. Eight of these proteins have been identified as keratins by immunologic cross-reactivity to antibodies raised against authentic human epidermal keratins. The ME-180 keratin proteins consist of two major subunits designated MEK-1 and MEK-2 with approximate molecular weights of 58,000 and 53,000, respectively, and six minor subunits of 59, 57, 52.5, 50.5, 45, and 40 kilodaltons. When ME-180 cells were incubated for 2-24 h in the presence of [32P]orthophosphate, MEK-1 and MEK-2 as well as the 52.5- and 40-kilodalton keratins were phosphorylated at their serine residues. V8 protease digests revealed that phosphorylation of MEK-2 is restricted to one peptide representing approximately half the molecule. Regulation of MEK-1 and MEK-2 phosphorylation has been studied by prelabeling the cells for 2 h in 32P-labeled medium. This was followed by up to 2 h of continued incubation in the same medium after the addition of a variety of perturbing agents. The phosphorylation of MEK-2 increased in the presence of 10(-4) M dibutyryl cyclic AMP (twofold), 1 mM methylisobutylxanthine (2.5-fold), 10(-5) M isoproterenol (fivefold), and 10(-9) M cholera toxin (sevenfold). In contrast, MEK-1 phosphorylation was unaffected by these agents. Neither cyclic GMP, Ca++, hydrocortisone, nor epidermal growth factor had any effect on the phosphorylation of MEK-1 or MEK-2. The results indicate that the phosphorylation of these two keratins is independently controlled by cyclic AMP-dependent kinase for MEK-2 and by cyclic nucleotide-independent kinase for MEK-1. The observed differences in control suggest distinct functions for MEK-1 and MEK-2 within the cytoskeletal network.