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Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation.

ABSTRACT: The B?/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization of Cdc6 dephosphorylation. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all PP2A holoenzymes. PR70 also binds distinctively to the catalytic subunit near the active site, which is required for PR70 to enhance phosphatase activity toward Cdc6. Our studies provide a structural basis for unique regulation of B?/PR72 holoenzymes by calcium ions, and suggest the mechanisms for precise control of substrate specificity among PP2A holoenzymes.

SUBMITTER: Wlodarchak N

PROVIDER: S-EPMC3698643 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation.

Wlodarchak Nathan N Guo Feng F Satyshur Kenneth A KA Jiang Li L Jeffrey Philip D PD Sun Tingwan T Stanevich Vitali V Mumby Marc C MC Xing Yongna Y

Cell research 20130611 7

The B″/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization ...[more]

PMID: 23752926

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Project description:BackgroundBiological effects of nuclear factor-kappaB (NF kappaB) can differ tremendously depending on the cellular context. For example, NF kappaB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NF kappaB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NF kappaB, involving a lack of inhibitor of kappaB (I kappaB alpha) protein reappearance. Permanent activation of the upstream kinase IKK beta results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized I kappaB alpha.ResultsTo investigate the mechanism underlying the general PP2A-mediated tuning of IKK beta phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKK beta and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKK beta phosphorylation thus allowing for I kappaB alpha resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKK beta phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of I kappaB alpha reappearance, is not a direct UVB action but requires instrumentality.ConclusionThe model developed here can be used as a reliable building block of larger NF kappa B models and offers comprehensive simplification potential for future modeling of NF kappa B signaling. It gives more insight into the newly discovered mechanisms for IKK deactivation and allows for substantiated predictions and investigation of different hypotheses. The evidence of constitutive activity of PP2Ac at the IKK complex provides new insights into the feedback regulation of NF kappa B, which is crucial for the development of new anti-cancer strategies.

Project description:Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

Dataset Information

Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation.

Publications

Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation.

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