Dephosphorylation of Orc2 by protein phosphatase 1 promotes the binding of the origin recognition complex to chromatin

https://doi.org/10.1016/j.bbrc.2014.04.109Get rights and content

Highlights

  • Protein phosphatase 1 physically interacts with Orc2.

  • The 119-KSVSF-123 motif of Orc2 is required for the dephosphorylation of Orc2 by PP1.

  • The dephosphorylation of Orc2 by PP1 is required for the binding of ORC to chromatin.

Abstract

Phosphorylation of Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2–5) from human chromatin and replication origins. Dephosphorylation of the phosphorylated Orc2 by protein phosphatase 1 (PP1) is accompanied by the binding of the dissociated subunits to chromatin. Here we show that PP1 physically interacts with Orc2. The binding of PP1 to Orc2 and the dephosphorylation of Orc2 by PP1 occurred in a cell cycle-dependent manner through an interaction with 119-KSVSF-123, which is the consensus motif for the binding of PP1, of Orc2. The dephosphorylation of Orc2 by PP1 is required for the binding of Orc2 to chromatin. These results support that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin and replication origins for the subsequent round of the cell cycle.

Introduction

Protein functions are often controlled by phosphorylation and dephosphorylation. The phosphorylation of the proteins that are involved in the formation of the pre-replicative and pre-initiation complexes for the cell cycle-dependent initiation of chromosome replication [1], [2], [3], [4] suggests that protein phosphatases may be necessary to reverse these phosphorylation effects. Both protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) are serine/threonine phosphatases that belong to the family of phosphoprotein phosphatases [5], [6], [7]. The association of the PP1 catalytic subunit with diverse regulatory subunits and/or interacting proteins confers distinct substrate specificities that control various physiological processes, including mitotic exit. The PP1 catalytic subunit is conserved among eukaryotic cells. Over 100 regulatory subunits or interacting proteins of PP1 have been identified. The PP1 catalytic subunit binds proteins possessing an RVXF motif, [R/K][X]0–1[V/I]X[F/W] [8], [9]. The valine (V) and phenylalanine/tryptophan (F/W) residues of this binding motif are crucial for anchoring the interacting proteins. Several regulatory subunits, such as Aurora-A, Aurora-B and Nek2, are also substrates for dephosphorylation and contain an RVXF motif [6], [10], [11], [12]. Mammalian PP1 comprises three isoforms: PP1α, PP1β/δ (which is known as PP1β throughout the remainder of this manuscript) and PP1γ. PP1α localizes in the nuclear matrix and at the centrosome, whereas PP1β is found on the chromatin [13]. PP1γ exists in the nucleoli and mitotic spindle of the cell [13], [14].

The origin recognition complex (ORC), which is composed of six different subunits, binds to the replication origins during the late M to early G1 phase of cell cycle for subsequent processes of chromosomal replication initiation [1], [2]. The bound ORC becomes dissociated from replication origin from the S phase [15]. The Orc2 subunit of ORC contains consensus sequences for phosphorylation by CDK and its mutation of the CDK phosphorylation site results in re-replication [16] and delayed cell cycle progression in yeast [17]. In human cells, Orc2 is phosphorylated by cyclin A/CDK2 at Thr116 and Thr226 during S phase, which results in dissociation of Orc2–5 subunits from the origins [15]. Dephosphorylation of Orc2 accompanies the association of ORC with chromatin [18]. The overexpression and depletion of PP1 isoforms along with specific inhibitor studies demonstrated that PP1 dephosphorylates Orc2. In this study, we show that PP1 interacts with Orc2 through the 119-KSVSF-123 motif. This interaction is essential for the dephosphorylation of Orc2 and binding to chromatin for the next round of cell cycle.

Section snippets

Cell culture and site-directed mutagenesis

U2OS, HeLa and HEK293T were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and antibiotics (penicilline/streptomycin). Inducible HeLa or U2OS Tet-On cell lines expressing the Orc2 protein were constructed and manipulated in accordance with previously described procedures [15]. To generate PP1-binding motif mutants of human ORC2, we used the following primers: For V121A/F123A, forward 5′-CACCACAAAAAAGTGCTTCAGCCA GTTTGAAGAATGATCCTGAG-3′ and reverse

The 119-KSVSF-123 motif of Orc2 is required for the binding of PP1 to Orc2

Orc2 is dephosphorylated by PP1 isoforms α, β and γ [18]. To examine the physical interaction of Orc2 with PP1 isoforms, a yeast two-hybrid assay was performed (Fig. 1(A)). The interaction between each of the PP1 isoforms and Orc2 enabled growth on the selective medium, whereas no growth on this medium was observed if PP1 isoforms were absent, and PP2A did not interact sufficiently with Orc2 to enable growth. However, no other subunit of the ORC exhibited an interaction with the PP1 isoforms (

Acknowledgments

We thank Dr. Anindya Dutta for providing us with the anti-Orc3 and anti-Orc5 antibodies. This work was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF-2013R1A1A2012109). J.S.B was supported by the BK21 Research Fellowship from the Ministry of Education, Science and Technology, Republic of Korea.

References (23)

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