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The B56?3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1.

ABSTRACT: The B56?-containing protein phosphatase 2A (PP2A-B56?) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56?3. B56?3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56?3 decreased p27KIP1 nuclear localization. B56?3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56?3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56?3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56?3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56?3 are required for interaction between p27 and B56?3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56? levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56? levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56?3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56?3 holoenzyme plays its tumor suppressor role.

SUBMITTER: Lai TY

PROVIDER: S-EPMC4826225 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1.

Lai Tai-Yu TY Yen Chia-Jui CJ Tsai Hung-Wen HW Yang Yu-San YS Hong Wei-Fu WF Chiang Chi-Wu CW

Oncotarget 20160101 4

The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly ...[more]

PMID: 26684356

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Project description:BACKGROUND:Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential ?-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56?. METHODS AND RESULTS:Phosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56? in adult rat ventricular myocytes (ARVM) exposed to the ?-adrenergic receptor (?AR) agonist isoprenaline (ISO). Phosphorylation of B56? occurred at S573, primarily through stimulation of the ?1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56?, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56?-WT or GFP-B56?-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56?-WT but not GFP-B56?-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56?-WT or GFP-B56?-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation. CONCLUSIONS:In cardiomyocytes, ?AR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56? at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56?-PP2A substrates, which remain to be identified.

Dataset Information

The B56?3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1.

Publications

The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1.

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