Dynamic regulation of Oct1 during mitosis by phosphorylation and ubiquitination
Jinsuk Kang1, Ben Goodman, Yixian Zheng
1Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.
Background:
Transcription factor Oct1 regulates multiple cellular processes. It is known to be phosphorylated during the cell cycle and by stress, however the upstream kinases and downstream consequences are not well understood. One of these modified forms, phosphorylated at S335, lacks the ability to bind DNA. Other modification states besides phosphorylation have not been described.
Methodology/Principal Findings:
We show that Oct1 is phosphorylated at S335 in the Oct1 DNA binding domain during M-phase by the NIMA-related kinase Nek6. Phospho-Oct1 is also ubiquitinated. Phosphorylation excludes Oct1 from mitotic chromatin. Instead, Oct1(pS335) concentrates at centrosomes, mitotic spindle poles, kinetochores and the midbody. Oct1 siRNA knockdown diminishes the signal at these locations. Both Oct1 ablation and overexpression result in abnormal mitoses. S335 is important for the overexpression phenotype, implicating this residue in mitotic regulation. Oct1 depletion causes defects in spindle morphogenesis in Xenopus egg extracts, establishing a mitosis-specific function of Oct1. Oct1 colocalizes with lamin B1 at the spindle poles and midbody. At the midbody, both proteins are mutually required to correctly localize the other. We show that phospho-Oct1 is modified late in mitosis by non-canonical K11-linked polyubiquitin chains. Ubiquitination requires the anaphase-promoting complex, and we further show that the anaphase-promoting complex large subunit APC1 and Oct1(pS335) interact.
Conclusions/Significance:
These findings reveal mechanistic coupling between Oct1 phosphorylation and ubquitination during mitotic progression, and a role for Oct1 in mitosis.
Insights
Oct1 phosphorylation at S335 by Nek6 prevents DNA binding and directs Oct1 to cellular structures during mitosis. This phosphorylation and subsequent ubiquitination are crucial for proper cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Oct1 is a transcription factor involved in various cellular processes.
- Oct1 phosphorylation, particularly at S335, affects its DNA binding ability.
- The kinases responsible for Oct1 phosphorylation and its downstream effects during mitosis are not fully understood.
Purpose of the Study:
- To identify the kinase responsible for Oct1 phosphorylation at S335 during mitosis.
- To elucidate the downstream consequences of Oct1 phosphorylation and ubiquitination.
- To define the role of Oct1 in mitotic progression.
Main Methods:
- Western blotting and mass spectrometry to identify Oct1 phosphorylation sites and kinases.
- Immunofluorescence microscopy to track Oct1 localization during mitosis.
- siRNA-mediated knockdown and overexpression studies to assess Oct1 function.
- Xenopus egg extract experiments to study spindle morphogenesis.
- Co-immunoprecipitation to investigate protein interactions.
Main Results:
- NIMA-related kinase 6 (Nek6) phosphorylates Oct1 at S335 during M-phase.
- Phosphorylated Oct1 (Oct1(pS335)) is excluded from mitotic chromatin and localizes to centrosomes, spindle poles, kinetochores, and the midbody.
- Oct1 depletion or overexpression leads to abnormal mitoses, with S335 being critical for the overexpression phenotype.
- Oct1 plays a role in spindle morphogenesis and colocalizes with lamin B1 at the spindle poles and midbody.
- Oct1(pS335) is ubiquitinated by the anaphase-promoting complex (APC/C) via K11-linked polyubiquitin chains, involving APC1.
Conclusions:
- Oct1 phosphorylation by Nek6 and subsequent ubiquitination by the APC/C are mechanistically linked during mitosis.
- Oct1 plays a critical, mitosis-specific role in cell division.
- These findings reveal a novel regulatory mechanism for Oct1 function during mitosis.
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