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Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells.

ABSTRACT: Epithelial cell keratins make up the type I (K9-K20) and type II (K1-K8) intermediate filament proteins. In glandular epithelia, K8 becomes phosphorylated on S73 ((71)LLpSPL) in human cultured cells and tissues during stress, apoptosis, and mitosis. Of all known proteins, the context of the K8 S73 motif (LLS/TPL) is unique to type II keratins and is conserved in epidermal K5/K6, esophageal K4, and type II hair keratins, except that serine is replaced by threonine. Because knowledge regarding epidermal and esophageal keratin regulation is limited, we tested whether K4-K6 are phosphorylated on the LLTPL motif. K5 and K6 become phosphorylated in vitro on threonine by the stress-activated kinase p38. Site-specific anti-phosphokeratin antibodies to LLpTPL were generated, which demonstrated negligible basal K4-K6 phosphorylation. In contrast, treatment of primary keratinocytes and other cultured cells, and ex vivo skin and esophagus cultures, with serine/threonine phosphatase inhibitors causes a dramatic increase in K4-K6 LLpTPL phosphorylation. This phosphorylation is accompanied by keratin solubilization, filament reorganization, and collapse. K5/K6 LLTPL phosphorylation occurs in vivo during mitosis and apoptosis induced by UV light or anisomycin, and in human psoriatic skin and squamous cell carcinoma. In conclusion, type II keratins of proliferating epithelia undergo phosphorylation at a unique and conserved motif as part of physiological mitotic and stress-related signals.

SUBMITTER: Toivola DM

PROVIDER: S-EPMC117609 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells.

Toivola Diana M DM Zhou Qin Q English Luc S LS Omary M Bishr MB

Molecular biology of the cell 20020601 6

Epithelial cell keratins make up the type I (K9-K20) and type II (K1-K8) intermediate filament proteins. In glandular epithelia, K8 becomes phosphorylated on S73 ((71)LLpSPL) in human cultured cells and tissues during stress, apoptosis, and mitosis. Of all known proteins, the context of the K8 S73 motif (LLS/TPL) is unique to type II keratins and is conserved in epidermal K5/K6, esophageal K4, and type II hair keratins, except that serine is replaced by threonine. Because knowledge regarding epi ...[more]

PMID: 12058054

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Project description:BackgroundThe appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.Methodology/principal findingsWe have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.Conclusions/significanceThis study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis.

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Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells.

Publications

Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells.

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