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Biochem Biophys Res Commun
2018 Jun 22;5012:387-393. doi: 10.1016/j.bbrc.2018.04.212.
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Novel phosphorelay-dependent control of ZFP36L1 protein during the cell cycle.
Kondo M
,
Noguchi A
,
Matsuura Y
,
Shimada M
,
Yokota N
,
Kawahara H
.
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The ZFP36 family is a prototypical member of a highly conserved group of proteins with CCCH-type RNA-binding domains, whose functional role and regulatory mechanism in mitotic cells remain obscure. In this study, we provide the first evidence that ZFP36L1 phosphorylation is modulated in a cell cycle-dependent manner. The C-terminal region of ZFP36L1 is critical for its cell cycle-dependent phosphorylation of this protein. We also suggest that the phosphorelay-dependent regulation of ZFP36L1 influences mitotic spindle organization. Thus, our data demonstrate a new class of regulatory mechanism for CCCH-type zinc-finger proteins in cell cycle control.
Fig. 1. ZFP36L1 is phosphorylated in a cell cycle-dependent manner. (A) Cell extracts of Xenopus fertilized eggs were blotted with anti-ZFP36L1 antiserum. Endogenous ZFP36L1 protein was detected as multiple bands (arrowheads) with time-dependent fluctuations during the cell cleavage cycle. (B) Schematic diagram of the microinjection experiments performed in this study. (C) After injecting 1 ng mRNA, embryos were harvested at either the cleaving stage (I) or interphase stage (II), and were probed with an anti-Flag antibody. Asterisk indicates a non-specific band. (D) Flag-tagged ZFP36L1 was treated with (+) or without (-) CIAP. White arrows indicate CIAP-sensitive bands. (E) Cell cycle-dependent fluctuation of ZFP36L1 protein in dividing Xenopus embryos. Eggcleavage points are indicated by arrows. (F, H) Schematic diagrams of the truncated ZFP36L1 proteins used in this study. CCCH-zinc-finger domains, ZF1 and ZF2, are indicated by black boxes. The C-terminal site (C30-ZFP36L1) required for phosphorylation is boxed in gray. Numbers denotes the corresponding amino acid positions. (G, I) mRNAs encoding Flag-tagged ZFP36L1 and its truncated derivatives were microinjected into two-cell stage Xenopus embryos. Embryos were harvested and used for dephosphorylation assays with (+) and without (-) CIAP. CIAP-sensitive bands are indicated by arrowheads.
Fig. 2. Ser cluster in the C-terminus of ZFP36L1 are essential for its phosphorylation in vivo. (A) The C30 amino acid sequences of Xenopus ZFP36L1 and its mutant derivatives used in this assay are shown. Substituted residues are indicated as magenta. (B) Flag-tagged ZFP36L1 (wild-type: WT) and its mutated derivatives were expressed in Xenopus embryos and their susceptibility to phosphorylation was monitored. (C) C-terminus of Xenopus ZFP36L1 and the corresponding regions of human homologs are shown. Identical residues to Xenopus ZFP36L1 are colored pink. XL: Xenopus laevis, HS: Homo sapiens. (D) In vitro synthesized Flag-ZFP36L1 protein was phosphorylated gradually during incubation in Xenopus meiotic CSF extracts (left panel). Incubation periods with CSF extracts at 20 °C are shown. These mobility shifts were completely reversed by CIAP treatment (right panel). (E) Phosphorylation of in vitro synthesized Flag-ZFP36L1 and a series of its mutant proteins in Xenopus CSF extracts. (F) The GST-fused recombinant protein with the C-terminal 34 amino acids of ZFP36L1 (GST-C34) and its mutant derivatives (GST-SA1-2 and GST-S325A) were tested as substrates for in vitro phosphorylation. (G) In vitro phosphorylation of ZFP36L1 at Ser325 by p42 MAP kinase. Purified GST and its fusion proteins were mixed with a series of kinases in the presence of γ-[32]P-ATP and incubated at 25 °C for 1 h. As a control, an equal amount of bacterially-produced GST was used. SDS-PAGE gels were stained with Coomassie brilliant blue (CBB). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Ser318 residue of human ZFP36L1 determines its cell cycle-dependency. (A) Cell cycle stage-dependent change of human ZFP36L1 protein. HeLa cells were transfected with an expression plasmid encoding Flag-tagged human ZFP36L1 and synchronized to the respective cell cycle stage [1]. Amounts of ZFP36L1 protein in each cell cycle stage were detected using an anti-Flag antibody. Actin was used as a loading control. (B,C) Mutation analysis was used to identify the region required for the cell cycle-dependency of ZFP36L1 protein. WT ZFP36L1 and its mutated derivatives were expressed in HeLa cells. Actin was used as a loading control. (D) ZFP36L1 proteins (WT and S318A) were expressed in HeLa cells and then chased with 20 μg/mL CHX for the indicated periods. The graph shows the quantification of anti-Flag immuno-signals normalized to the actin signal. The data represent mean ± S.D. calculated from 3 independent experiments.
Fig. 4. Forced expression of ZFP36L1 causes disorganization of the mitotic spindle. (A) Sections of ZFP36L1-expressing (a, b) and control embryos (c, d) that were stained with an anti-α-tubulin antibody (a, c) or Hoechst (b, d). Scale bar, 5 μm. (B) Merged images of misshaped mitotic spindles (magenta) and misaligned chromosomes (blue) in ZFP36L1-expressing Xenopus embryonic cells. Vertical line indicates the equatorial plane of the mitotic apparatus. Arrowheads indicate misaligned chromosomes. Scale bar, 0.75 μm. (C) Schematic diagrams of the ZFP36L1 mutant proteins used in this study. Mutated regions (or residues) are indicated by black vertical lines. Numbers denote the corresponding amino acid positions. (D) The forced expression of WT ZFP36L1 protein blocked the division of the corresponding blastomeres, while the expression of the C136R mutant and phosphorylation-defective mutants of ZFP36L1 (SA1, SA2, S325A) reduced these mitotic effects. Data are shown as means ± SD, and total numbers of embryos examined are indicated. Representative results of three independent experiments are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
