Enzyme regulation. IRBIT is a novel regulator of ribonucleotide reductase in higher eukaryotes

Abstract

Ribonucleotide reductase (RNR) supplies the balanced pools of deoxynucleotide triphosphates (dNTPs) necessary for DNA replication and maintenance of genomic integrity. RNR is subject to allosteric regulatory mechanisms in all eukaryotes, as well as to control by small protein inhibitors Sml1p and Spd1p in budding and fission yeast, respectively. Here, we show that the metazoan protein IRBIT forms a deoxyadenosine triphosphate (dATP)-dependent complex with RNR, which stabilizes dATP in the activity site of RNR and thus inhibits the enzyme. Formation of the RNR-IRBIT complex is regulated through phosphorylation of IRBIT, and ablation of IRBIT expression in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression. We demonstrate a mechanism for RNR regulation in higher eukaryotes that acts by enhancing allosteric RNR inhibition by dATP.

Fig. 1.. IRBIT interacts with RNR in a dATP-dependent manner.

(A) IRBIT-RNR interaction. IRBIT was immunoprecipitated from asynchronous HeLa cell lysates; bound proteins were resolved on SDS-PAGE and stained with Coomassie brilliant blue (CBB). IRBIT and R1 proteins are indicated by arrows. (B) Minimal domain required for dATP-dependent IRBIT-RNR interaction. GST-IRBIT⁶⁴⁻⁸⁷ was mixed with R1/R2B and 3 mM dATP. Complexes were retrieved by glutathione-Sepharose and resolved by SDS-PAGE and stained with CBB. (C) Alignment of IRBIT domain from different metazoans. (D) R1-IRBIT interaction is evolutionarily conserved. GST-IRBIT (hIRBIT) or GST-CG9977 (dIRBIT) were mixed with either human 6His-R1 (hR1) or Drosophila 6His-R1 (dR1) along with 3 mM dATP and analyzed as in (B). (E) hIRBIT binds Xenopus R1. GST-hIRBIT was mixed with cytostatic factor–arrested Xenopus laevis egg extracts. IRBIT-bound proteins were purified and probed with antibodies against R1 and Symplekin (4).

Fig. 2.. IRBIT binds RNR when dATP is in the A-site and requires phosphorylation for its full binding activity.

(A) dATP occupancy of RNR’s A-site is required for IRBIT binding. GST-IRBIT was mixed with either R1/R2B or R1^D57N/R2B (input, left) and 10 mM dATP and retrieved by glutathione-Sepharose (right). (B) IRBIT-RNR interaction is sensitive to ATP competition but insensitive to S-site occupancy. GST-IRBIT was mixed with R1/R2B and 3 mM dATP followed by addition of 0.3 mM ATP, 1 mM ATP, 3 mM ATP, 1 mM dTTP, or 1 mM dGTP. (C) Endogenous phosphorylation sites that were mapped on IRBIT (flags) and corresponding recombinant phosphomimetic or nonphosphorylatable mutants. (D) IRBIT phosphorylation prevents dissociation of the IRBIT-R1-dATP complex by ATP. GST-IRBIT^4STD (STD) or GST-IRBIT^4STA (STA) was mixed with R1/R2B and 3 mM dATP followed by addition of 1 mM ATP. In all panels, complexes were analyzed as in Fig. 1B.

Fig. 3.. IRBIT stabilizes and inhibits RNR-dATP complex.

(A) IRBIT does not create additional dATP binding sites on RNR. RNR was mixed with increasing doses of [³²P]dATP in the absence (circles) or presence (squares) of hIRBIT and in the presence of dTTP (1 mM) at +4°C. (B) IRBIT inhibits dATP release from RNR. Values are means T SEM. (C) Rate constants of R1-dATP interaction. B_max (maximum number of binding sites) was derived from (A), k_off was obtained from (B), and k_on was calculated from fig. S2D. K_d, dissociation constant. (D) IRBIT enhances dATP inhibition of RNR. RNR was premixed with or without dATP and with or without 6His-IRBIT and tested for uridine diphosphate (UDP) reductase activity.

Fig. 4.. IRBIT controls the endogenous dNTP pool and is required for normal cell cycle progression.

(A) dNTP levels in HeLa and in HeLa^DIRBIT cells in mitotic (M) or G₁ phases; a/s for asynchronous population. (B) Stability of R1 and IRBIT do not depend on each other. Total lysates of HeLa, HeLa^DIRBIT, and HeLa^DR1 cells were analyzed by Western blots using antibodies against IRBIT, R1, and R2B. (C) IRBIT-RNR interaction during the cell cycle. IRBIT was immunoprecipitated from corresponding lysates and analyzed for the presence of R1. (D) Cell cycle progression of HeLa cells with or without IRBIT. M-to-M transition indicates timing between anaphase and prometaphase of the next cell cycle of an individual daughter cell (n = 20). Values are means T SEM. Statistical differences were evaluated by Student’s t test (**P < 0.01).