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Serine 111 phosphorylation regulates OCT4A protein subcellular distribution and degradation.

ABSTRACT: Embryonic stem cell self-renewal properties are attributed to critical amounts of OCT4A, but little is known about its post-translational regulation. Sequence analysis revealed that OCT4A contains five putative ERK1/2 phosphorylation sites. Consistent with the hypothesis that OCT4A is a putative ERK1/2 substrate, we demonstrate that OCT4A interacts with ERK1/2 by using both in vitro GST pulldown and in vivo co-immunoprecipitation assays. MS analysis identified phosphorylation of OCT4A at Ser-111. To investigate the possibility that ERK1/2 activation can enhance OCT4A degradation, we analyzed endogenous ubiquitination in cells transfected with FLAG-OCT4A alone or with constitutively active MEK1 (MEK1(CA)), and we observed that the extent of OCT4 ubiquitination was clearly increased when MEK1(CA) was coexpressed and that this increase was more evident after MG132 treatment. These results suggest an increase in OCT4A ubiquitination downstream of MEK1 activation, and this could account for the protein loss observed after FGF2 treatment and MEK1(CA) transfection. Understanding and controlling the mechanism by which stem cells balance self-renewal would substantially advance our knowledge of stem cells.

SUBMITTER: Spelat R

PROVIDER: S-EPMC3488096 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Serine 111 phosphorylation regulates OCT4A protein subcellular distribution and degradation.

Spelat Renza R Ferro Federico F Curcio Francesco F

The Journal of biological chemistry 20120928 45

Embryonic stem cell self-renewal properties are attributed to critical amounts of OCT4A, but little is known about its post-translational regulation. Sequence analysis revealed that OCT4A contains five putative ERK1/2 phosphorylation sites. Consistent with the hypothesis that OCT4A is a putative ERK1/2 substrate, we demonstrate that OCT4A interacts with ERK1/2 by using both in vitro GST pulldown and in vivo co-immunoprecipitation assays. MS analysis identified phosphorylation of OCT4A at Ser-111 ...[more]

PMID: 23024368

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Project description:Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123-3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

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Serine 111 phosphorylation regulates OCT4A protein subcellular distribution and degradation.

Publications

Serine 111 phosphorylation regulates OCT4A protein subcellular distribution and degradation.

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