Keratin Phosphorylation Research Articles

Phosphorylation of KRT8 (keratin 8) by excessive mechanical load-activated PKN (protein kinase N) impairs autophagosome initiation and contributes to disc degeneration

ABSTRACT Excessive mechanical load (overloading) is a well-documented pathogenetic factor for many mechano stress-induced pathologies, i.e. intervertebral disc degeneration (IDD). Under overloading, the balance between anabolism and catabolism within nucleus pulposus (NP) cells are badly thrown off, and NP cells undergo apoptosis. However, little is known about how the overloading is transduced to the NP cells and contributes to disc degeneration. The current study shows that conditional knockout of Krt8 (keratin 8) within NP aggravates load-induced IDD in vivo, and overexpression of Krt8 endows NP cells greater resistance to overloading-induced apoptosis and degeneration in vitro. Discovery-driven experiments shows that phosphorylation of KRT8 on Ser43 by overloading activated RHOA-PKN (protein kinase N) impedes trafficking of Golgi resident small GTPase RAB33B, suppresses the autophagosome initiation and contributes to IDD. Overexpression of Krt8 and knockdown of Pkn1 and Pkn2, at an early stage of IDD, ameliorates disc degeneration; yet only knockdown of Pkn1 and Pkn2, when treated at late stage of IDD, shows a therapeutic effect. This study validates a protective role of Krt8 during overloading-induced IDD and demonstrates that targeting overloading activation of PKNs could be a novel and effective approach to mechano stress-induced pathologies with a wider window of therapeutic opportunity. Abbreviations: AAV: adeno-associated virus; AF: anulus fibrosus; ANOVA: analysis of variance; ATG: autophagy related; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; CEP: cartilaginous endplates; CHX: cycloheximide; cKO: conditional knockout; Cor: coronal plane; CT: computed tomography; Cy: coccygeal vertebra; D: aspartic acid; DEG: differentially expressed gene; DHI: disc height index; DIBA: dot immunobinding assay; dUTP: 2’-deoxyuridine 5’-triphosphate; ECM: extracellular matrix; EDTA: ethylene diamine tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPS: group-based prediction system; GSEA: gene set enrichment analysis; GTP: guanosine triphosphate; HE: hematoxylin-eosin; HRP: horseradish peroxidase; IDD: intervertebral disc degeneration; IF: immunofluorescence staining; IL1: interleukin 1; IVD: intervertebral disc; KEGG: Kyoto encyclopedia of genes and genomes; KRT8: keratin 8; KD: knockdown; KO: knockout; L: lumbar vertebra; LBP: low back pain; LC/MS: liquid chromatograph mass spectrometer; LSI: mouse lumbar instability model; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMP3: matrix metallopeptidase 3; MRI: nuclear magnetic resonance imaging; NC: negative control; NP: nucleus pulposus; PBS: phosphate-buffered saline; PE: p-phycoerythrin; PFA: paraformaldehyde; PI: propidium iodide; PKN: protein kinase N; OE: overexpression; PTM: post translational modification; PVDF: polyvinylidene fluoride; qPCR: quantitative reverse-transcriptase polymerase chain reaction; RHOA: ras homolog family member A; RIPA: radio immunoprecipitation assay; RNA: ribonucleic acid; ROS: reactive oxygen species; RT: room temperature; TCM: rat tail compression-induced IDD model; TCS: mouse tail suturing compressive model; S: serine; Sag: sagittal plane; SD rats: Sprague-Dawley rats; shRNA: short hairpin RNA; siRNA: small interfering RNA; SOFG: safranin O-fast green; SQSTM1: sequestosome 1; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VG/ml: viral genomes per milliliter; WCL: whole cell lysate.

KRT8 (keratin 8) attenuates necrotic cell death by facilitating mitochondrial fission-mediated mitophagy through interaction with PLEC (plectin)

ABSTRACT Dysregulation of mitochondrial homeostasis and accumulation of damaged mitochondria cause degenerative diseases such as age-related macular degeneration (AMD). We studied the effects of the intermediate cytofilament KRT8 (keratin 8) on mitochondrial homeostasis in relation to the morphology and function of mitochondria in retinal pigment epithelial cells under oxidative stress. When the mitochondria were damaged owing to oxidative stress, the damaged mitochondria were readily disposed of via mitophagy following mitochondrial fission. During this process, KRT8 was found to physically interact with the mitochondria through PLEC (plectin) and facilitate the mitochondrial fission-mediated mitophagy. However, the association between PLEC-anchoring mitochondria and KRT8 was dwindled by KRT8 phosphorylation under oxidative stress. The efficient KRT8-facilitated mitophagy flux suppressed the accumulation of damaged mitochondria and consequently diminished necrotic cell death under oxidative stress. Thus, by facilitating mitophagy, KRT8 protects RPE cells against necrotic cell death due to oxidative stress. Abbreviations: 3-MA: 3-methyladenine; AMD: age-related macular degeneration; DDIT3: DNA damage inducible transcript 3; DNM1L: dynamin 1 like; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCN1: GCN1 activator of EIF2AK4; IF: intermediate filament; KRT8: keratin 8; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MMP: mitochondrial membrane potential; OCR: oxygen consumption rate; PLEC: plectin; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20

Host-cell dependent role of phosphorylated keratin 8 during influenza A/NWS/33 virus (H1N1) infection in mammalian cells

In this study, we investigated the involvement of keratin 8 during human influenza A/NWS/33 virus (H1N1) infection in semi-permissive rhesus monkey-kidney (LLC-MK2) and permissive human type II alveolar epithelial (A549) cells. In A549 cells, keratin 8 showed major expression and phosphorylation levels.Influenza A/NWS/33 virus was able to subvert keratin 8 structural organization at late stages of infection in both cell models, promoting keratin 8 phosphorylation in A549 cells at early phases of infection. Accordingly, partial colocalizations of the viral nucleoprotein with keratin 8 and its phosphorylated form were assessed by confocal microscopy at early stages of infection in A549 cells. The employment of chemical activators of phosphorylation resulted in structural changes as well as increased phosphorylation of keratin 8 in both cell models, favoring the influenza A/NWS/33 virus’s replicative efficiency in A549 but not in LLC-MK2 cells.In A549 and human larynx epidermoid carcinoma (HEp-2) cells inoculated with respiratory secretions from pediatric patients positive for, respectively, influenza A virus or respiratory syncytial virus, the keratin 8 phosphorylation level had increased only in the case of influenza A virus infection.The results obtained suggest that in A549 cells the influenza virus is able to induce keratin 8 phosphorylation thereby enhancing its replicative efficiency.

Differentially phosphorylated proteins in the crimped and straight wool of Chinese Tan sheep

Proteins can be post-translationally modified and this can be important in the regulation of cellular processes and function. However, little is known about whether protein phosphorylation plays a role in regulating wool fibre properties. In this study, we used a chemical labelling method combined with a high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis to compare the phosphopeptides present in the wool of three Tan sheep with highly crimped wool and three Tan sheep with straighter wool. Thirty-six phosphopeptides that had differences in relative abundance between these two types of wool were identified. These peptides were derived from 28 to 33 different proteins, including two keratins (Ks) and 7 to 12 keratin-associated proteins (KAPs), with these proteins being common structural components of the wool fibre. The crimped wool had a higher relative abundance of phosphorylated K38, K72 and KAP13-x, whereas the straighter wool had a higher relative abundance of phosphorylated KAP2-1, KAP6-1, KAP4-x, KAP10-x and KAP13-y. These results confirm the phosphorylation of wool Ks and KAPs, and suggest that differential phosphorylation of Ks and KAPs may affect wool fibre crimping in Tan sheep. SignificanceProtein phosphorylation can alter the structural conformation and interaction of a protein, and hence affect the cellular processes that the protein undertakes. In this study, we compared the suite of phosphorylated proteins in crimped and straight wool from Chinese Tan sheep and found that some keratins and keratin-associated proteins were phosphorylated. Crimped wool had more keratin phosphorylation, while straight wool had more keratin-associated protein phosphorylation, with this suggesting that wool fibre crimping may be a regulated by phosphorylation of some wool proteins. This suggests that wool traits may be under epigenetic control and that post-translation modifications need to be considered in breeding for different wool types.

Targeting desmosomal adhesion and signalling for intestinal barrier stabilization in inflammatory bowel diseases-Lessons from experimental models and patients.

Inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and Ulcerative colitis (UC) have a complex and multifactorial pathogenesis which is incompletely understood. A typical feature closely associated with clinical symptoms is impaired intestinal epithelial barrier function. Mounting evidence suggests that desmosomes, which together with tight junctions (TJ) and adherens junctions (AJ) form the intestinal epithelial barrier, play a distinct role in IBD pathogenesis. This is based on the finding that desmoglein (Dsg) 2, a cadherin-type adhesion molecule of desmosomes, is required for maintenance of intestinal barrier properties both in vitro and in vivo, presumably via Dsg2-mediated regulation of TJ. Mice deficient for intestinal Dsg2 show increased basal permeability and are highly susceptible to experimental colitis. In several cohorts of IBD patients, intestinal protein levels of Dsg2 are reduced and desmosome ultrastructure is altered suggesting that Dsg2 is involved in IBD pathogenesis. In addition to its adhesive function, Dsg2 contributes to enterocyte cohesion and intestinal barrier function. Dsg2 is also involved in enterocyte proliferation, barrier differentiation and induction of apoptosis, in part by regulation of p38MAPK and EGFR signalling. In IBD, the function of Dsg2 appears to be compromised via p38MAPK activation, which is a critical pathway for regulation of desmosomes and is associated with keratin phosphorylation in IBD patients. In this review, the current findings on the role of Dsg2 as a novel promising target to prevent loss of intestinal barrier function in IBD patients are discussed.

Leukamenin E Induces K8/18 Phosphorylation and Blocks the Assembly of Keratin Filament Networks Through ERK Activation.

Leukamenin E is a natural ent-kaurane diterpenoid isolated from Isodon racemosa (Hemsl) Hara that has been found to be a novel and potential keratin filament inhibitor, but its underlying mechanisms remain largely unknown. Here, we show that leukamenin E induces keratin filaments (KFs) depolymerization, largely independently of microfilament (MFs) and microtubules (MTs) in well-spread cells and inhibition of KFs assembly in spreading cells. These effects are accompanied by keratin phosphorylation at K8-Ser73/Ser431 and K18-Ser52 via the by extracellular signal-regulated kinases (ERK) pathway in primary liver carcinoma cells (PLC) and human umbilical vein endothelial cells (HUVECs). Moreover, leukamenin E increases soluble pK8-Ser73/Ser431, pK18-Ser52, and pan-keratin in the cytoplasmic supernatant by immunofluorescence imaging and Western blotting assay. Accordingly, leukamenin E inhibits the spreading and migration of cells. We propose that leukamenin E-induced keratin phosphorylation may interfere with the initiation of KFs assembly and block the formation of a new KFs network, leading to the inhibition of cell spreading. Leukamenin E is a potential target drug for inhibition of KFs assembly.

Phosphorylation of keratin 18 serine 52 regulates mother-daughter centriole engagement and microtubule nucleation by cell cycle-dependent accumulation at the centriole.

Serine-52 (Ser52) is the major physiologic site of keratin 18 (K18) phosphorylation. Here, we report that serine-52 phosphorylated K18 (phospho-Ser52 K18) accumulated on centrosomes in a cell cycle-dependent manner. Moreover, we found that phospho-Ser52 K18 was locatedat theproximalendof themother centriole. Transfection with the K18 Ser52 → Ala (K18 S52A) mutant prevented centriole localization of phospho-Ser52 K18 and resulted in separation of the mother-daughter centrioles. Inhibition of microtubulepolymerization led to the disappearance of aggregated phospho-Ser52 K18 on the centrosome; removal of inhibitors resulted in reaccumulation of phospho-Ser52 K18 in microtubule-organizing centers. Transfection with a K18 S52A mutant inhibited microtubule nucleation. These results reveal a cell cycle-dependent change in centrosome localization of phospho-Ser52 k18 and strongly suggest that the phosphorylation status of Ser52 K18 of mother centrioles plays a critical role in maintaining a tight engagement between mother and daughter centrioles and also contributes to microtubule nucleation.

HIF stabilization inhibits renal epithelial cell migration and is associated with cytoskeletal alterations

Acute kidney injury (AKI) is a common and potentially lethal complication in the hospitalized patients, with hypoxic injury being as a major cause. The loss of renal tubular epithelial cells (TEC), one of the AKI hallmarks, is potentially followed by tubular regeneration process orchestrated by the remaining uninjured TECs that undergo proliferation and migration. In this study, we used human primary TEC to investigate the initiation of tubular cell migration and associated cytoskeletal alterations in response to pharmacological HIF stabilization which resembles the pathophysiology of hypoxia. Tubular cells have been shown to migrate as cohorts in a wound healing assay. Importantly, cells of distal tubular origin moved faster than those of proximal origin. HIF stabilization impaired TEC migration, which was confirmed by live single cell tracking. HIF stabilization significantly reduced tubular cell migration velocity and promoted cell spreading. In contrast to the control conditions, HIF stabilization induced actin filaments rearrangement and cell adhesion molecules including paxillin and focal adhesion kinase. Condensed bundling of keratin fibers was also observed, while the expression of different types of keratins, phosphorylation of keratin 18, and the microtubule structure were not altered. In summary, HIF stabilization reduced the ability of renal tubular cells to migrate and led to cytoskeleton reorganization. Our data suggested an important involvement of HIF stabilization during the epithelial migration underlying the mechanism of renal regeneration in response to AKI.

YdjC chitooligosaccharide deacetylase homolog induces keratin reorganization in lung cancer cells: involvement of interaction between YDJC and CDC16.

Lung cancer is a fatal disease with a high mortality rate. The perinuclear reorganization of keratin 8 (K8) is an important biochemical phenomenon reflecting changes in the physical properties of metastatic cancer. However, there is not much of information about the regulatory molecules involved in phosphorylation and perinuclear reorganization of K8.In this study, we investigated the role and molecular mechanisms of YdjC chitooligosaccha- ride deacetylase homolog (YDJC) in sphingosylphosphorylcholine (SPC)-induced phosphorylation and reorganization of K8, and migration and invasion (SPC-induced events). SPC induced expression of YDJC in a dose- and time-dependent manner. Gene silencing of YDJC suppressed SPC-induced events. YDJC overexpression induced the SPC-induced events. YDJC deacetylase dominant negative mutant (YDJCD13A) did not induce SPC-induced events. YDJC siRNA reduced ERK activation and overexpression of YDJC induced ERK activation. The siRNA of ERK1 or ERK2 suppressed YDJC-induced phosphorylation and reorganization of K8, and migration and invasion. Co-immunoprecipitation revealed that YDJC binds to CDC16. Interestingly, CDC16 siRNA induced SPC-induced events. Overexpression of CDC16 blocked SPC-induced events. KMPLOT analysis based on public microarray data revealed the poor prognosis of lung cancer patients with high expression of YDJC compared with patients with low expression of YDJC.The collective results indicate that YDJC is involved in SPC-induced events in A549 lung cancer cells by interacting with CDC16. YDJC overexpression might be involved in the progression of lung cancer. These results also suggest that suppression of YDJC or boosting of CDC16 interaction with YDJC might be a novel way to prevent progression of lung cancer.

Threonine 150 Phosphorylation of Keratin 5 Is Linked to Epidermolysis Bullosa Simplex and Regulates Filament Assembly and Cell Viability

A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII-/-) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation of Keratin-8 in skin squamous cell carcinoma derived cell line.

Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine73 (head-domain) and Serine431 (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1T14,Y15 , EIF4EBP1T46,T50 , EIF4BS422 , AKT1S1T246,S247 , CTTN1T401,S405,Y421 , and CAP1S307/309 in K8-S73A/D mutant and CTTN1T401,S405,Y421 , BUB1BS1043 , and CARHSP1S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYCS176 , ZYXS344 , and PNNS692 could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies.

Intermediate filament reorganization dynamically influences cancer cell alignment and migration

The interactions between a cancer cell and its extracellular matrix (ECM) have been the focus of an increasing amount of investigation. The role of the intermediate filament keratin in cancer has also been coming into focus of late, but more research is needed to understand how this piece fits in the puzzle of cytoskeleton-mediated invasion and metastasis. In Panc-1 invasive pancreatic cancer cells, keratin phosphorylation in conjunction with actin inhibition was found to be sufficient to reduce cell area below either treatment alone. We then analyzed intersecting keratin and actin fibers in the cytoskeleton of cyclically stretched cells and found no directional correlation. The role of keratin organization in Panc-1 cellular morphological adaptation and directed migration was then analyzed by culturing cells on cyclically stretched polydimethylsiloxane (PDMS) substrates, nanoscale grates, and rigid pillars. In general, the reorganization of the keratin cytoskeleton allows the cell to become more ‘mobile’- exhibiting faster and more directed migration and orientation in response to external stimuli. By combining keratin network perturbation with a variety of physical ECM signals, we demonstrate the interconnected nature of the architecture inside the cell and the scaffolding outside of it, and highlight the key elements facilitating cancer cell-ECM interactions.

Novel involvement of RhebL1 in sphingosylphosphorylcholine-induced keratin phosphorylation and reorganization: Binding to and activation of AKT1.

Sphingosylphosphorylcholine induces keratin phosphorylation and reorganization, and increases viscoelasticity of metastatic cancer cells such as PANC-1 cells. However, the mechanism involved in sphingosylphosphorylcholine-induced keratin phosphorylation and reorganization is largely unknown. Sphingosylphosphorylcholine dose- and time-dependently induces the expression of RhebL1. The involvement of RhebL1 in sphingosylphosphorylcholine-induced events including keratin 8 (K8) phosphorylation, reorganization, migration and invasion was examined. Gene silencing of RhebL1 suppressed the sphingosylphosphorylcholine-induced events and overexpression of RhebL1 enhanced those events even without sphingosylphosphorylcholine treatment. We examined whether the G protein function of RhebL1 induces K8 phosphorylation using constitutively active RhebL1Q64L and dominant negative RhebL1D60K. G protein activity of RhebL1 is involved in sphingosylphosphorylcholine-induced K8 phosphorylation. We found that RhebL1 binds and activates AKT1. G protein activity of RhebL1 is involved in the binding and activation of AKT1. MK2206 (AKT inhibitor) and gene silencing of AKT1 inhibited the sphingosylphosphorylcholine-induced events, whereas overexpression of activated-AKT1 induced K8 phosphorylation, reorganization, migration and invasion even without sphingosylphosphorylcholine treatment.The collective results indicate that RhebL1 is involved in sphingosylphosphorylcholine-induced events in A549 lung cancer cells via binding to AKT1 leading to activation of it. These results suggest that suppression of RhebL1 or inhibition of RhebL1′s binding to AKT1 might be a novel way that prevents changes in the physical properties of metastatic cancer cells.

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.

Leukotriene B4 induces EMT and vimentin expression in PANC-1 pancreatic cancer cells: Involvement of BLT2 via ERK2 activation

Leukotriene B4 (LTB4) is a leukocyte chemoattractant and plays a major role controlling inflammatory responses including pancreatitis. LTB4 is known to be correlated with cancer progression. LTB4 induces keratin phosphorylation and reorganization by activating extracellular regulated kinase (ERK) in PANC-1 pancreatic cancer cell lines. However, the role of LTB4 in epithelial mesenchymal transition (EMT) and vimentin expression in pancreatic cancer cells is unknown.We examined whether LTB4 induces EMT and vimentin expression by Western blot, si-RNA, and RT-PCR. LTB4 induced morphological change, decreased E-cadherin expression and increased N-cadherin and vimentin expression. LTB4 increased migration and invasion of PANC-1 cancer cells. LTB4 dose-dependently upregulated expression of vimentin in PANC-1 cancer cells. LTB4-induced vimentin expression was suppressed by LY255283 (BLT2 antagonist). Comp A, a BLT2 agonist, further increased vimentin expression. Gene silencing of BLT2 suppressed LTB4-or Comp A-induced vimentin expression in PANC-1 cells. The MEK inhibitor, PD98059 suppressed Comp A-induced vimentin expression. Comp A or transfection of plasmid containing BLT2 cDNA (pCBLT2) activated ERK, and BLT2 gene silencing suppressed Comp A-induced ERK activation. ERK2 siRNA abrogated Comp A-induced vimentin expression and ERK2 overexpression enhanced vimentin expression. One of well-known cause of ras mutation, cigarette smoke extracts increased BLT2 expression in PANC-1 cancer cells.Taken together, these results suggest that BLT2 is involved in LTB4-induced vimentin expression through ERK2 in PANC-1 cells.

Mechanics of soft epithelial keratin networks depend on modular filament assembly kinetics.

Structural adaptability is a pivotal requirement of cytoskeletal structures, enabling their reorganization to meet the cellular needs. Shear stress, for instance, results in large morphological network changes of the human soft epithelial keratin pair K8:K18, and is accompanied by an increase in keratin phosphorylation levels. Yet the mechanisms responsible for the disruption of the network structure in vivo remain poorly understood. To understand the effect of the stress-related site-specific phosphorylation of the K8:K18 pair, we created phosphomimicry mutants - K8(S431E), K8(S73E), K18(S52E) - in vitro, and investigated the various steps of keratin assembly from monomer to network structure using fluorescence and electron microscopy, and using rheology characterized their network mechanical properties. We find that the addition of a charged group produces networks with depleted intra-connectivity, which translates to a mechanically weaker and more deformable network. This large variation in network structure is achieved by the formation of shorter mutant filaments, which exhibit differing assembly kinetics and a manifestly reduced capacity to form the extended structures characteristic of the wild-type system. The similarity in outcome for all the phosphomimicry mutants explored points to a more general mechanism of structural modulation of intermediate filaments via phosphorylation. Understanding the role of kinetic effects in the construction of these cytoskeletal biopolymer networks is critical to elucidating their structure-function properties, providing new insight for the design of keratin-inspired biomaterials. Structural remodeling of cytoskeletal networks accompanies many cellular processes. Interestingly, levels of phosphorylation of the human soft epithelial keratin pair K8:K18 increase during their stress-related structural remodeling. Our multi-scale study sheds light on the poorly understood mechanism with which site-specific phosphorylation induces disruption of the keratin network structure in vivo. We show how phosphorylation reduces keratin filament length, an effect that propagates through to the mesoscopic structure, resulting in the formation of connectivity-depleted and mechanically weaker networks. We determine that the intrinsically-set filament-to-filament attractions that drive bundle assembly give rise to the structural variability by enabling the formation of kinetically-arrested structures. Overall, our results shed light on how self-assembled intermediate filament structures can be tailored to exhibit different structural functionalities.

In vitro phosphorylation as tool for modification of silk and keratin fibrous materials.

An overview is given of the recent work on in vitro enzymatic phosphorylation of silk fibroin and human hair keratin. Opposing to many chemical "conventional" approaches, enzymatic phosphorylation is in fact a mild reaction and the treatment falls within "green chemistry" approach. Silk and keratin are not phosphorylated in vivo, but in vitro. This enzyme-driven modification is a major technological breakthrough. Harsh chemical chemicals are avoided, and mild conditions make enzymatic phosphorylation a real "green chemistry" approach. The current communication presents a novel approach stating that enzyme phosphorylation may be used as a tool to modify the surface charge of biocompatible materials such as keratin and silk.

Epithelial membrane protein 2 regulates sphingosylphosphorylcholine-induced keratin 8 phosphorylation and reorganization: Changes of PP2A expression by interaction with alpha4 and caveolin-1 in lung cancer cells

Sphingosylphosphorylcholine (SPC) is found at increased in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. However, the detailed mechanism of SPC-induced K8 phosphorylation and reorganization is not clear.We observed that SPC dose-dependently reduced the expression of epithelial membrane protein 2 (EMP2) in lung cancer cells. Then, we examined the role of EMP2 in SPC-induced phosphorylation and reorganization of K8 in lung cancer cells. We found that SPC concentration-dependently reduced EMP2 in A549, H1299, and other lung cancer cells. This was verified at the mRNA level by RT-PCR and real-time PCR (qPCR), and intracellular variation through confocal microscopy. EMP2 gene silencing and stable lung cancer cell lines established using EMP2 lentiviral shRNA induced K8 phosphorylation and reorganization. EMP2 overexpression reduced K8 phosphorylation and reorganization. We also observed that SPC-induced loss of EMP2 induces phosphorylation of JNK and ERK via reduced expression of protein phosphatase 2A (PP2A). Loss of EMP2 induces ubiquitination of protein phosphatase 2A (PP2A). SPC induced caveolin-1 (cav-1) expression and EEA1 endosome marker protein but not cav-2. SPC treatment enhanced the binding of cav-1 and PP2A and lowered binding of PP2A and alpha4. Gene silencing of EMP2 increased and gene silencing of cav-1 reduced migration of A549 lung cancer cells.Overall, these results suggest that SPC induces EMP2 down-regulation which reduces the PP2A via ubiquitination induced by cav-1, which sequestered alpha4, leading to the activation of ERK and JNK.

Novel effects of FTY720 on perinuclear reorganization of keratin network induced by sphingosylphosphorylcholine: Involvement of protein phosphatase 2A and G-protein-coupled receptor-12

Sphingosylphosphorylcholine (SPC) evokes perinuclear reorganization of keratin 8 (K8) filaments and regulates the viscoelasticity of metastatic cancer cells leading to enhanced migration. Few studies have addressed the compounds modulating the viscoelasticity of metastatic cancer cells. We studied the effects of sphingosine (SPH), sphingosine 1-phosphate (S1P), FTY720 and FTY720-phosphate (FTY720P) on SPC-induced K8 phosphorylation and reorganization using Western blot and confocal microscopy, and also evaluated the elasticity of PANC-1 cells by atomic force microscopy.FTY720, FTY720P, SPH, and S1P concentration-dependently inhibited SPC-evoked phosphorylation and reorganization of K8, and migration of PANC-1 cells. SPC triggered reduction and narrow distribution of elastic constant K and conversely, FTY720 blocked them. A common upstream regulator of JNK and ERK, protein phosphatase 2A (PP2A) expression was reduced by SPC, but was restored by FTY720 and FTY72P. Butyryl forskolin, a PP2A activator, suppressed SPC-induced K8 phosphorylation and okadaic acid, a PP2A inhibitor, induced K8 phosphorylation. Gene silencing of PP2A also led to K8 phosphorylation, reorganization and migration. We also investigated the involvement of GPR12, a high-affinity SPC receptor, in SPC-evoked keratin phosphorylation and reorganization. GPR12 siRNA suppressed the SPC-triggered phosphorylation and reorganization of K8. GPR12 overexpression stimulated keratin phosphorylation and reorganization even without SPC. FTY720 and FTY720P suppressed the GPR12-induced phosphorylation and reorganization of K8. The collective data indicates that FTY720 and FTY720P suppress SPC-induced phosphorylation and reorganization of K8 in PANC-1 cells by restoring the expression of PP2A via GPR12. These findings might be helpful in the development of compounds that modulate the viscoelasticity of metastatic cancer cells and various SPC actions.

Enzymatic phosphorylation of hair keratin enhances fast adsorption of cationic moieties

The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by 31P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10min for cationic moiety like methylene blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance.