Idas, a Novel Phylogenetically Conserved Geminin-related Protein, Binds to Geminin and Is Required for Cell Cycle Progression

Dafni-Eleutheria Pefani,Maria Dimaki,Magda Spella,Nickolas Karantzelis,Eirini Mitsiki,Christina Kyrousi,Ioanna-Eleni Symeonidou,Anastassis Perrakis,Stavros Taraviras,Zoi Lygerou

doi:10.1074/jbc.m110.207688

Abstract

Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development.

Highlights

Geminin, initially identified as a substrate of the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase during mitosis [2], was shown to regulate DNA replication by directly binding to and inhibiting the DNA replication licensing factor Cdt1 [3,4,5]
Geminin binding to Cdt1 during the S and G2 phases of the cell cycle ensures that licensing of a further round of DNA replication is inhibited until the end of mitosis [6, 7]
The polymerase chain reaction (PCR) product was cloned into the mammalian expression vector cDNA3.1EGFP (Invitrogen) at the NheI and KpnI restriction sites to produce a protein C-terminally fused to green fluorescent protein (GFP) under the control of the constitutive CMV promoter

Summary

EXPERIMENTAL PROCEDURES

Bioinformatics Analysis—Tblastn (NCBI) was used to search expressed sequences from the human genome using the hGeminin protein sequence as query. mRNAs deriving from 5q11.2, on chromosome 5, were identified as encoding a protein with significant similarity to Geminin. Fragments of the predicted mRNA were amplified by polymerase chain reaction (PCR) and sequenced to verify expression of this locus in HeLa cells. The PCR product was cloned into the mammalian expression vector cDNA3.1EGFP (Invitrogen) at the NheI and KpnI restriction sites to produce a protein C-terminally fused to green fluorescent protein (GFP) under the control of the constitutive CMV promoter. N-Idas was cloned into the NheI and HindIII sites of pET28a vector (Novagen) with the N-terminal His tag using PCR amplification to introduce sites. Cells were washed with PBS, 0.1% Tween and incubated for 1 h with the following secondary antibodies in blocking solution: Alexa 488-conjugated goat anti-rabbit (A11034, Molecular Probes) and Alexa 568-conjugated goat anti-mouse (A11004, Molecular Probes). The specificity of the affinity purified antibodies were tested by Western blot analysis and shown to recognize the hIdas protein in HeLa cells.

RESULTS

DISCUSSION

Taraviras and Zoi Lygerou