Heat Stress or Rotavirus Infection of Human Epithelial Cells Generates a Distinct Hyperphosphorylated Form of Keratin 8

Liao, Jian; Lowthert, L. A.; Omary, M. Bishr

doi:10.1006/excr.1995.1238

Cited by 47 publications

(63 citation statements)

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“…Heat stress is known to increase keratin solubility (47). HepG2 cells were subjected to heat stress to investigate if alteration in keratin 8/18 solubility would be accompanied by changes in their O-GlcNAc status.…”

Section: Alterations In Keratin Solubility Results In Simultaneous Chamentioning

confidence: 99%

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O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

Budnar¹,

Vaidya

Kalraiya

2010

Journal of Biological Chemistry

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Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.

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Section: Alterations In Keratin Solubility Results In Simultaneous Chamentioning

confidence: 99%

“…Heat stress and N-acetylglucosamine treatments are known to increase keratin solubility and cellular O-GlcNAcylation, respectively (47,48). HepG2 cells under both these conditions exhibited a simultaneous increase in K8/18 solubility and O-GlcNAcylation (Fig.…”

Section: Discussionmentioning

confidence: 97%

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

Budnar¹,

Vaidya

Kalraiya

2010

Journal of Biological Chemistry

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“…However, this seems unclear for K18 because both of these modifications exhibit similar changes under certain conditions, like mitotic arrest (48) and heat stress (49), whereas, during induced hepatotoxicity, they exhibit an inverse relationship (50). Of the two phosphorylation sites mapped on keratin 18, Ser(P) 33 causes increased solubility during mitosis and filament reorganization in response to shear stress (5,14), whereas Ser(P) 52 is known to regulate filament reorganization under stress conditions (51).…”

Section: Discussionmentioning

confidence: 99%

Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18

Kakade¹,

Budnar²,

Kalraiya³

et al. 2016

Journal of Biological Chemistry

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“…In the case of K8/18, phosphorylation regulates filament reorganization in vivo, enhances keratin solubility, plays a role in dictating the localization of K8/18 with specific cellular compartments, regulates the association with the 14-3-3 protein family, and is associated with a variety of physiologic stresses (10). With regard to cell stress, several stress modalities have been associated with hyperphosphorylation of K8/18 including heat stress and virus infection (11), and mitotic arrest (which can be considered a form of stress) of several cultured cell lines (12). Also, drug-induced hepatotoxic stress, induced in mice by feeding with a griseofulvin-supplemented diet, resulted in dramatic K8/18 hyperphosphorylation (13).…”

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confidence: 99%

“…Also, drug-induced hepatotoxic stress, induced in mice by feeding with a griseofulvin-supplemented diet, resulted in dramatic K8/18 hyperphosphorylation (13). Of note, stress-induced K8 hyperphosphorylation is associated with generation of a distinct K8 species, termed HK8 (11,12), that migrates slightly slower than K8 after one-dimensional gel analysis. To date, two in vivo K8 phosphorylation sites have been described (14).…”

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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

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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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Heat Stress or Rotavirus Infection of Human Epithelial Cells Generates a Distinct Hyperphosphorylated Form of Keratin 8

Cited by 47 publications

References 0 publications

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18*

Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18

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

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