Empigen BB: A Useful Detergent for Solubilization and Biochemical Analysis of Keratins

Lowthert, L. A.; Ku, Nam Su; Liao, Jian; Coulombe, Pierre A.; Omary, M. Bishr

doi:10.1006/bbrc.1995.1051

Cited by 45 publications

(48 citation statements)

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“…Immunoprecipitation, Gel, and Blot Analysis-Washed cells were solubilized using 1% Nonidet P-40 or 1% Empigen BB (21) (24 h), or at 37°C in the presence of okadaic acid (OA) (1 g/ml, 2 h). An equal number of cells was solubilized with 1% Nonidet P-40 for 30 min followed by immunoprecipitation using anti-K8/18 mAb L2A1 or using mAb LJ4.…”

Section: Methodsmentioning

confidence: 99%

Stress, Apoptosis, and Mitosis Induce Phosphorylation of Human Keratin 8 at Ser-73 in Tissues and Cultured Cells

Omary¹,

Ku²,

Liao³

1997

Journal of Biological Chemistry

114

134

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Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. We previously showed that several types of cell stress such as heat and virus infection result in a distinct hyperphosphorylated form of K8 (termed HK8). To better characterize K8/18 phosphorylation, we generated monoclonal antibodies by immunizing mice with hyperphosphorylated keratins that were purified from colonic cultured human HT29 cells pretreated with okadaic acid. One antibody specifically recognized HK8, and the epitope was identified as 71 LLpSPL which corresponds to K8 phosphorylation at Ser-73. Generation of HK8 occurs in mitotic HT29 cells, basal crypt mitotic cells in normal mouse intestine, and in regenerating mouse hepatocytes after partial hepatectomy. Prominent levels of HK8 were also generated in HT29 cells that were induced to undergo apoptosis using anisomycin or etoposide. In addition, mouse hepatotoxicity that is induced by chronic feeding with griseofulvin resulted in HK8 formation in the liver. Our results demonstrate that a "reverse immunological" approach, coupled with enhancing in vivo phosphorylation using phosphatase inhibitors, can result in the identification of physiologic phosphorylation states. As such, K8 Ser-73 phosphorylation generates a distinct HK8 species under a variety of in vivo conditions including mitosis, apoptosis, and cell stress. The low steady state levels of HK8 during mitosis, in contrast to stress and apoptosis, suggest that accumulation of HK8 may represent a physiologic stress marker for simple epithelia.

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Section: Methodsmentioning

confidence: 99%

Stress, Apoptosis, and Mitosis Induce Phosphorylation of Human Keratin 8 at Ser-73 in Tissues and Cultured Cells

Omary¹,

Ku²,

Liao³

1997

Journal of Biological Chemistry

114

134

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“…The specified proteins were solubilized with Empigen BB (Calbiochem, Bad Soden, Germany) as described by Lowthert et al for K18 (32). The solubilization buffer consisted of 2% Empigen in PBS with inhibition of the proteolytic activity by addition of 600 Ag/mL of Pefabloc SC and one tablet of Complete Mini per 8 mL of solution (both from Roche Diagnostics, Mannheim, Germany).…”

Section: Western Blottingmentioning

confidence: 99%

Transfection of Keratin 18 Gene in Human Breast Cancer Cells Causes Induction of Adhesion Proteins and Dramatic Regression of Malignancy In vitro and In vivo

Bühler

Schäller²

2005

Molecular Cancer Research

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This study shows that high keratin 18 (K18) expression in tumor cells is associated with reduced invasiveness in vitro and lack of tumorigenicity in nude mice. We previously showed that high K18 expression correlated with a good prognosis and that reducing K18 expression increased the aggressiveness of established breast cancer cell lines. To confirm these observations, we transfected the human K18 gene into the human breast cancer cell line MDA-MB-231 and isolated a stable overexpressing clone. The forced K18 expression was associated with a complete loss of the previously strong vimentin expression in the parent cell line, induction of the K18 dimerization partner K8, and up-regulation of adhesion proteins. These changes were accompanied by a dramatic reduction in the aggressiveness of the K18 transfectants in vitro and in vivo. We conclude that forced reexpression of K18 causes at least partial redifferentiation of the tumor cell, followed by a corresponding regression of malignant phenotype. (Mol Cancer Res 2005;3(7):365 -71)

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“…2); in contrast, K18 (33) and K19 (15) have, for the most part, one major dynamic site. Another feature of K20 (phospho-and nonphospho-), which was unmasked using the detergent Empigen that is highly effective in solubilizing keratins (43), is that K20 is significantly less stable than the other type I keratins, K18 and K19, in its ability to form complexes with the type II keratin, K8 (Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

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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Empigen BB: A Useful Detergent for Solubilization and Biochemical Analysis of Keratins

Cited by 45 publications

References 0 publications

Stress, Apoptosis, and Mitosis Induce Phosphorylation of Human Keratin 8 at Ser-73 in Tissues and Cultured Cells

Stress, Apoptosis, and Mitosis Induce Phosphorylation of Human Keratin 8 at Ser-73 in Tissues and Cultured Cells

Transfection of Keratin 18 Gene in Human Breast Cancer Cells Causes Induction of Adhesion Proteins and Dramatic Regression of Malignancy In vitro and In vivo

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

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