Abstract
The specific function of PP2A, a major serine/threonine phosphatase, is mediated by regulatory targeting subunits, such as members of the B55 family. Although implicated in cell division and other pathways, the specific substrates and functions of B55 targeting subunits are largely undefined. In this study we identified over 100 binding proteins of B55α and B55β in Xenopus egg extracts that are involved in metabolism, mitochondria function, molecular trafficking, cell division, cytoskeleton, DNA replication, DNA repair, and cell signaling. Among the B55α and B55β-associated proteins were numerous mitotic regulators, including many substrates of CDK1. Consistently, upregulation of B55α accelerated M-phase exit and inhibited M-phase entry. Moreover, specific substrates of CDK2, including factors of DNA replication and chromatin remodeling were identified within the interactomes of B55α and B55β, suggesting a role for these phosphatase subunits in DNA replication. In particular, we confirmed in human cells that B55α binds RPA and mediates the dephosphorylation of RPA2. The B55-RPA association is disrupted after replication stress, consistent with the induction of RPA2 phosphorylation. Thus, we report here a new mechanism that accounts for both how RPA phosphorylation is modulated by PP2A and how the phosphorylation of RPA2 is abruptly induced after replication stress.
Highlights
Protein phosphorylation, a major form of post-translational modification, plays a crucial role in regulation of protein functions
We show that B55α associates with replication protein A (RPA), and the association is reduced upon replication stress, presumably as a mechanism to allow phosphorylation of RPA2
It was established that PP2A dephosphorylates nearly half of all Ser/Thr phospho-residues, and thereby modulating numerous cellular processes
Summary
A major form of post-translational modification, plays a crucial role in regulation of protein functions. As a major group of the PP2A targeting subunits, it is expected that B55 directs PP2A to a large number of substrates. To date only a few phosphoproteins were defined as direct substrates of PP2A/B55 To fill in this large gap in knowledge, in the current study we characterize the interactomes of B55α and B55β that each contains over 100 proteins. Among these proteins were factors involved in cell division, DNA repair and replication, components of actin, microtubule, Golgi and nucleopore, and regulators of cellular signaling, metabolism and mitochondria function. Ectopic expression of B55α suppressed RPA phosphorylation, and attenuated checkpoint signaling after replication stress
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