Approaches to Study Posttranslational Regulation of Intermediate Filament Proteins

Kochin, Vitaly; Pallari, Hanna‐Mari; Pant, Harish C.; Eriksson, John E.

doi:10.1016/s0091-679x(04)78014-0

Cited by 5 publications

(18 citation statements)

References 87 publications

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“…As shown in Fig. 3, this analysis suggested that K20 Ser 13 or Ser 14 is phosphorylated within the K20 tryptic peptide 10 (R)SLSSSLQAPVVSTVGMQR. Mutational analysis was then carried out by transfecting BHK-21 cells with K20 S13A or K20 S14A (together with the partner K8 to stabilize the transfected K20) (Fig.…”

Section: Identification Of Ser 13 As a K20 Phosphorylation Site That mentioning

confidence: 69%

“…After fusion and screening, one clone (termed Q6) was chosen, subcloned, and propagated. In addition, a rabbit phospho (p)-epitope-specific antibody was generated commercially (Anaspec) against the peptide CFHRSLpSSS-LQA, which recognizes the human and mouse pSer 13 -containing K20.…”

Section: Methodsmentioning

confidence: 99%

“…Mutagenesis, Transfection, and Immunofluorescence Staining-The human K20 cDNA was mutated to convert Ser 13 or Ser 14 to Ala using a QuikChange site-directed mutagenesis kit (Stratagene), and mutations were confirmed by DNA sequencing. Transfections into NIH-3T3 or BHK-21 cells were done using WT or mutant hK20 cDNA plus hK8 cDNA.…”

Section: Methodsmentioning

confidence: 99%

“…Effect of K20 Ser 13 Mutation on Keratin Filament OrganizationGiven that keratin phosphorylation can play a role in keratin filament organization (15, 23), we tested whether K20 Ser 13 3 Ala (S13A) has an effect on keratin filament assembly. Under basal conditions, co-transfection of K8 with K20 S13A into NIH-3T3 cells does not have a significant effect on keratin filament assembly as compared with K20 WT or S14A transfectants.…”

Section: Identification Of Ser 13 As a K20 Phosphorylation Site That mentioning

confidence: 99%

“…In terms of posttranslational modifications, phosphorylation is the most studied (11)(12)(13) and occurs preferentially within the head and tail domains of keratins, which are the most polymorphic domains within IF proteins. Of the more than 20 keratins, only K1, K4 -K6, K8, K18, and K19 have been studied in terms of their phosphorylation (11, 14 -17).…”

mentioning

confidence: 99%

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Keratin 20 Serine 13 Phosphorylation Is a Stress and Intestinal Goblet Cell Marker

Zhou

Cadrin

Herrmann

et al. 2006

Journal of Biological Chemistry

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Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser 13 or Ser 14 was a likely phosphorylation site, and Ser 13 was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser 13 antibody. K20 Ser 13 phosphorylation increases after protein kinase C activation, and Ser 13 -to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser 13 hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser 13 phosphorylation. Among mouse small intestinal enterocytes, K20 Ser 13 is preferentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser 13 is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser 13 phosphorylation also serves as a unique marker of small intestinal goblet cells. Keratins are intermediate filament (IF)3 cytoskeletal proteins that are preferentially expressed in epithelial cells. All IF proteins consist of a central ␣-helical "rod" domain that is flanked by non-␣-helical N-terminal "head" and C-terminal "tail" domains. Expression of unique complements of type I (keratin polypeptides 9 -20 (K9 -K20)) and type II (K1-K8) keratins, which associate at a noncovalent 1:1 ratio of type I to type II heteropolymers, distinguishes different epithelial subtypes. For example, keratinocytes preferentially express K5/K14 or K1/K10 depending on their differentiation state in the epidermis; adult hepatocytes express K8/K18 exclusively, whereas intestinal epithelial cells express K8 as the major type II keratin with varying levels of the type I keratins, K18/K19/K20, depending on the differentiation state (e.g. crypt versus villus cells) or cell type (e.g. goblet versus absorptive cell) (1-3). The functions of keratins (and other IF proteins) are becoming increasingly well understood (4 -7) as IF-null or dominant negative mouse models are developed and studied (8) and as mutations in IF proteins are linked to a growing list of human diseases (9). These functions are mechanical and nonmechanical in nature and include providing tissue and cell integrity, protecting cells from apoptosis and nonmechanical forms of injury, tissue-specific functions, cell signaling, and maintenance of subcellular organelle positioning and functions.Keratin and other IF protein funct...

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Section: Identification Of Ser 13 As a K20 Phosphorylation Site That mentioning

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Identification Of Ser 13 As a K20 Phosphorylation Site That mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Keratin 20 Serine 13 Phosphorylation Is a Stress and Intestinal Goblet Cell Marker

Zhou

Cadrin

Herrmann

et al. 2006

Journal of Biological Chemistry

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Fast track to a phosphoprotein sketch – MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity

2006

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Tryptic phosphopeptide mapping by TLC on microcrystalline cellulose has been a convenient method to get a fast and highly reproducible overview of the number of phosphopeptides present in any given (32)P-labeled phosphoprotein. This method also provides an immediate presentation of the relative phosphorylation stoichiometry between individual phosphopeptides. However, so far, traditional tryptic phosphopeptide maps have not been useful for phosphoproteomics applications, as the S/N has been very poor, due to the large number of quenching substances and contaminants present on cellulose plates. In this study, we present a rapid and easy method for phosphopeptides identification from 2-D phosphopeptide maps (2-D-PPMs). We obtain improved sensitivity (femtomole levels) upon MALDI-TOF MS analysis of phosphopeptides extracted from 2-D-PPMs. Using this approach we could confidently characterize the major phosphorylation sites of in vivo and in vitro (32)P-labeled proteins.

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Providing cellular signposts – Post‐translational modifications of intermediate filaments

et al. 2008

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Intermediate filaments are dynamically regulated by their post-translational modifications. Initially these modifications were found to regulate filament dynamics and organization. In the last few years, their roles have extended significantly to facilitating, for example, the recruitment and sequestration of signaling molecules that regulate a wide range of cellular functions. While phosphorylation has been established as the principal post-translational modification regulating intermediate filament function, other modifications with co-operative roles are emerging, adding a further dimensions to intermediate filament-mediated signaling.

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Approaches to Study Posttranslational Regulation of Intermediate Filament Proteins

Cited by 5 publications

References 87 publications

Keratin 20 Serine 13 Phosphorylation Is a Stress and Intestinal Goblet Cell Marker

Keratin 20 Serine 13 Phosphorylation Is a Stress and Intestinal Goblet Cell Marker

Fast track to a phosphoprotein sketch – MALDI-TOF characterization of TLC-based tryptic phosphopeptide maps at femtomolar detection sensitivity

Providing cellular signposts – Post‐translational modifications of intermediate filaments

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