Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK-ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells.
"Ser-12 PKA (Ando et al., 1996) Ser-23 PKA, PKC ε (Akita et al., 2007; Ando et al., 1996) Ser-33 PKA (Ando et al., 1996) Ser-36 PKA (Ando et al., 1996) Ser-42 PKA (Ando et al., 1996) Ser-50 PKA (Ando et al., 1996) Ser-73 JNK, PKC δ , MK2* (He et al., 2002; Menon et al., 2010; Ridge et al., 2005) Ser-416 PKA (Ando et al., 1996) Ser-423 PKA (Ando et al., 1996) Ser-425 PKA (Ando et al., 1996) Ser-431 ERK, JNK (Busch et al., 2012; Park et al., 2011; Park et al., 2012) Not determined AKT, AMPK, CAMK II, CK-Iα, (Kuga et al., 2013; Loschke et al., 2015; Velasco et al., 1998; Yano et al., 1991) K17 Ser-44 Not determined RSK1 US3 (Pan et al., 2011) (Murata et al., 2002) K18 Ser-33 PKC ζ (Sivaramakrishnan et al., 2009) Ser-52 MK2 (Menon et al., 2010) Not determined AMPK (Velasco et al., 1998) K19 Ser-35 Not determined (Zhou et al., 1999) Tyr-391 Src kinase (Zhou et al., 2010) K20 Ser-13 MK2, PKC* (Menon et al., 2010; Zhou et al., 2006) *No evidence for phosphorylation of residue by indicated kinase but dependent on that. "
[Show abstract][Hide abstract] ABSTRACT: Metastasis is one of hallmarks of cancer and a major cause of cancer death. Combatting metastasis is highly challenging. To overcome these difficulties, researchers have focused on physical properties of metastatic cancer cells. Metastatic cancer cells from patients are softer than benign cancer or normal cells. Changes of viscoelasticity of cancer cells are related to the keratin network. Unexpectedly, keratin network is dynamic and regulation of keratin network is important to the metastasis of cancer. Keratin is composed of heteropolymer of type I and II. Keratin connects from the plasma membrane to nucleus. Several proteins including kinases, and protein phosphatases bind to keratin intermediate filaments. Several endogenous compounds or toxic compounds induce phosphorylation and reorganization of keratin network in cancer cells, leading to increased migration. Continuous phosphorylation of keratin results in loss of keratin, which is one of the features of epithelial mesenchymal transition (EMT). Therefore, several proteins involved in phosphorylation and reorganization of keratin also have a role in EMT. It is likely that compounds controlling phosphorylation and reorganization of keratin are potential candidates for combating EMT and metastasis.
Biomolecules and Therapeutics 07/2015; 23(4):301-12. DOI:10.4062/biomolther.2015.032 · 1.73 Impact Factor
"Although a mutation may not alter the structure of a protein to affect its primary function, it may still hinder the binding of associated proteins or interfere its interaction with other cytoskeletal components, as well as affect post-translational modification by obstructing site-specific changes like phosphorylation, glycosylation, acetylation and sumoylation. This may well be the case for mutations in simple epithelia keratins –. "
[Show abstract][Hide abstract] ABSTRACT: Keratin 8 and 18 (K8/K18) mutations have been implicated in the aetiology of certain pathogenic processes of the liver and pancreas. While some K8 mutations (K8 G62C, K8 K464N) are also presumed susceptibility factors for inflammatory bowel disease (IBD), the only K18 mutation (K18 S230T) discovered so far in an IBD patient is thought to be a polymorphism. The aim of our study was to demonstrate that these mutations might also directly affect intestinal cell barrier function. Cell monolayers of genetically engineered human colonocytes expressing these mutations were tested for permeability, growth rate and resistance to heat-stress. We also calculated the change in dissociation constant (Kd, measure of affinity) each of these mutations introduces into the keratin protein, and present the first model of a keratin dimer L12 region with in silico clues to how the K18 S230T mutation may affect keratin function. Physiologically, these mutations cause up to 30% increase in paracellular permeability in vitro. Heat-stress induces little keratin clumping but instead cell monolayers peel off the surface suggesting a problem with cell junctions. K18 S230T has pronounced pathological effects in vitro marked by high Kd, low growth rate and increased permeability. The latter may be due to the altered distribution of tight junction components claudin-4 and ZO-1. This is the first time intestinal cells have been suggested also functionally impaired by K8/K18 mutations. Although an in vitro colonocyte model system does not completely mimic the epithelial lining of the intestine, nevertheless the data suggest that K8/K18 mutations may be also able to produce a phenotype in vivo.
PLoS ONE 06/2014; 9(6):e99398. DOI:10.1371/journal.pone.0099398 · 3.23 Impact Factor
"We determined, by the site-specific K8 phosphoserine antibodies anti-Ser23, anti-Ser73, and anti-Ser431, the exact K8 phosphoserine residues affected by TPA. ERK and JNK is involved in Ser431 and p38 and PKCδ is responsible for Ser73 (Park et al., 2011; Busch et al., 2012; Park et al., 2012). We found that PP2A is involved in ERK and JNK-mediated phosphorylation of K8 Ser431 (Park et al., 2012). "
[Show abstract][Hide abstract] ABSTRACT: The stiffness of cancer cells is attributable to intermediate filaments such as keratin. Perinuclear reorganization via phosphorylation of specific serine residue in keratin is implicated in the deformability of metastatic cancer cells including the human pancreatic carcinoma cell line (PANC-1). 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter and protein kinase C (PKC) activator. However, its effects on phosphorylation and reorganization of keratin 8 (K8) are not well known. Therefore, we examined the underlying mechanism and effect of TPA on K8 phosphorylation and reorganization. TPA induced phosphorylation and reorganization of K8 and transglutaminase-2 (Tgase-2) expression in a time- and dose-dependent manner in PANC-1 cells. These effects peaked after 45 min and 100 nM of TPA treatment. We next investigated, using cystamine (CTM), Tgase inhibitor, and Tgase-2 gene silencing, Tgase-2's possible involvement in TPA-induced K8 phosphorylation and reorganization. We found that Tgase-2 gene silencing inhibited K8 phosphorylation and reorganization in PANC-1 cells. Tgase-2 gene silencing, we additionally discovered, suppressed TPA-induced migration of PANC-1 cells and Tgase-2 overexpression induced migration of PANC-1 cells. Overall, these results suggested that TPA induced K8 phosphorylation and reorganization via Tgase-2 expression in PANC-1 cells.
Biomolecules and Therapeutics 02/2014; 22(2):122-8. DOI:10.4062/biomolther.2014.007 · 1.73 Impact Factor
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