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N-terminal phosphorylation of protein phosphatase 2A/B?2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival.

ABSTRACT: The neuron-specific B?2 regulatory subunit of protein phosphatase 2A (PP2A), a product of the spinocerebellar ataxia type 12 disease gene PPP2R2B, recruits heterotrimeric PP2A to the outer mitochondrial membrane (OMM) through its N-terminal mitochondrial targeting sequence. OMM-localized PP2A/B?2 induces mitochondrial fragmentation, thereby increasing susceptibility to neuronal insults. Here, we report that PP2A/B?2 activates the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) by dephosphorylating Ser656, a highly conserved inhibitory phosphorylation site targeted by the neuroprotective protein kinase A-A kinase anchoring protein 1 complex. We further show that translocation of PP2A/B?2 to mitochondria is regulated by phosphorylation of B?2 at three N-terminal serines. Phosphomimetic substitution of Ser20, Ser21, and Ser22 renders B?2 cytosolic, blocks Drp1 dephosphorylation and mitochondrial fragmentation, and abolishes the ability of B?2 overexpression to induce apoptosis in cultured hippocampal neurons. Alanine substitution of Ser20-Ser22 to prevent phosphorylation has the opposite effect, promoting association of B?2 with mitochondria, Drp1 dephosphorylation, mitochondrial fission, and neuronal death. OMM translocation of B?2 can be attenuated by mutation of residues in close proximity to the catalytic site, but only if Ser20-Ser22 are available for phosphorylation, suggesting that PP2A/B?2 autodephosphorylation is necessary for OMM association, probably by uncovering the net positive charge of the mitochondrial targeting sequence. These results reveal another layer of complexity in the regulation of the mitochondrial fission-fusion equilibrium and its physiological and pathophysiological consequences in the nervous system.

SUBMITTER: Merrill RA

PROVIDER: S-EPMC3549015 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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N-terminal phosphorylation of protein phosphatase 2A/Bβ2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival.

Merrill Ronald A RA Slupe Andrew M AM Strack Stefan S

The FEBS journal 20120608 2

The neuron-specific Bβ2 regulatory subunit of protein phosphatase 2A (PP2A), a product of the spinocerebellar ataxia type 12 disease gene PPP2R2B, recruits heterotrimeric PP2A to the outer mitochondrial membrane (OMM) through its N-terminal mitochondrial targeting sequence. OMM-localized PP2A/Bβ2 induces mitochondrial fragmentation, thereby increasing susceptibility to neuronal insults. Here, we report that PP2A/Bβ2 activates the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) by d ...[more]

PMID: 22583914

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Project description:Previous studies have demonstrated that the JC virus (JCV) late regulatory protein agnoprotein is phosphorylated by the serine/threonine-specific protein kinase-C (PKC) and mutants of this protein at the PKC phosphorylation sites exhibit defects in the viral replication cycle. We have now investigated whether agnoprotein phosphorylation is regulated by PP2A, a serine/threonine-specific protein phosphatase and whether JCV small t antigen (Sm t-Ag) is involved in this regulation. Protein-protein interaction studies demonstrated that PP2A associates with agnoprotein and dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this interaction inhibited the dephosphorylation of agnoprotein by PP2A. The interaction domains of Sm t-Ag and agnoprotein with PP2A were mapped, as were the interaction domains of Sm t-Ag with agnoprotein. The middle portion of Sm t-Ag (aa 82-124) was found to be critical for the interaction with both agnoprotein and PP2A and the N-terminal region of agnoprotein for interaction with Sm t-Ag. To further understand the role of Sm t-Ag in JCV regulation, a stop codon was introduced at Ser90 immediately after splice donor site of the JCV early gene and the functional consequences of this mutation were investigated. The ability of this mutant virus to replicate was substantially reduced compared to WT. Next, the functional significance of PP2A in JCV replication was examined by siRNA targeting. Downregulation of PP2A caused a significant reduction in the level of JCV replication. Moreover, the impact of Sm t-Ag on agnoprotein phosphorylation was investigated by creating a double mutant of JCV, where Sm t-Ag stop codon mutant was combined with an agnoprotein triple phosphorylation mutant (Ser7, Ser11 and Thr21 to Ala). Results showed that double mutant behaves much like the triple phosphorylation mutant of agnoprotein during viral replication cycle, which suggests that agnoprotein might be an important target of Sm t-Ag with respect to the regulation of its phosphorylation. Collectively, these results suggest that there is an interplay between agnoprotein, Sm t-Ag and PP2A with respect to the regulation of JCV life cycle and this could be important for the progression of the JCV-induced disease, PML.

Dataset Information

N-terminal phosphorylation of protein phosphatase 2A/B?2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival.

Publications

N-terminal phosphorylation of protein phosphatase 2A/Bβ2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival.

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