Specific interaction of KIF11 with ZBP1 regulates the transport of β-actin mRNA and cell motility - PubMed (original) (raw)
. 2015 Mar 1;128(5):1001-10.
doi: 10.1242/jcs.161679. Epub 2015 Jan 14.
Affiliations
- PMID: 25588836
- PMCID: PMC4342582
- DOI: 10.1242/jcs.161679
Specific interaction of KIF11 with ZBP1 regulates the transport of β-actin mRNA and cell motility
Tingting Song et al. J Cell Sci. 2015.
Abstract
ZBP1-modulated localization of β-actin mRNA enables a cell to establish polarity and structural asymmetry. Although the mechanism of β-actin mRNA localization has been well established, the underlying mechanism of how a specific molecular motor contributes to the transport of the ZBP1 (also known as IGF2BP1) complex in non-neuronal cells remains elusive. In this study, we report the isolation and identification of KIF11, a microtubule motor, which physically interacts with ZBP1 and is a component of β-actin messenger ribonucleoprotein particles (mRNPs). We show that KIF11 colocalizes with the β-actin mRNA, and the ability of KIF11 to transport β-actin mRNA is dependent on ZBP1. We characterize the corresponding regions of ZBP1 and KIF11 that mediate the interaction of the two proteins in vitro and in vivo. Disruption of the in vivo interaction of KIF11 with ZBP1 delocalizes β-actin mRNA and affects cell migration. Our study reveals a molecular mechanism by which a particular microtubule motor mediates the transport of an mRNP through direct interaction with an mRNA-binding protein.
Keywords: Cell motility; Cell polarity; KIF11; ZBP1; mRNA transport.
© 2015. Published by The Company of Biologists Ltd.
Figures
Fig. 1.
KIF11 binds to ZBP1 and is a component of the β-actin mRNA complex. (A) Co-immunoprecipitation was performed to identify the proteins directly associated with ZBP1 in extracts of MDA231/GFP-FLAG-ZBP1 (lane 2) and MDA231/GFP (lane 3) cells. Precipitates were resolved on 4–12% SDS-PAGE and stained with Coomassie Blue. Two protein bands with molecular mass of ∼90 kDa and 110 kDa, were identified in the precipitates of the MDA231/FLAG-GFP-ZBP1 cell extract (lane 4, asterisks), but not in the MDA231/GFP cell extract (lane 5). Western blots showed that the faster migrating band was FLAG-GFP–ZBP1 (not shown). (B) The upper migrating protein band was excised and sent for peptide sequence analysis. Red sequences indicate sequenced peptides that completely match with the sequences of human KIF11. (C) β-actin mRNP was purified from cytoplasmic extracts of MEF cells expressing the β-actin-3′UTR-MBS24, using amylose–MBP resin or amylose–MBP-MCP resin. Total RNAs were isolated from starting extracts (Ext), flow-through of amylose–MBP-MCP resin (FT) and amylose–MBP resin [FT(c)], and the precipitates of amylose–MBP-MCP (Prec.) and amylose–MBP resin control [Prec.(c)]. RT-PCR was performed to detect the presence of β-actin mRNA in the RNA samples. Lane L, DNA ladder. (D) Western blots to analyze the presence of KIF11 using rabbit anti-KIF11 antibody and ZBP1 using mouse anti-ZBP1 antibodies in the precipitates. KIF11 and ZBP1 were co-precipitated with β-actin mRNA using amylose–MBP-MCP resin (Prec.), but were not precipitated in the control sample using amylose–MBP resin control [Prec.(c)]. Ext, total extracts. (E) Primary 14-day-old mouse embryo fibroblasts (MEF) of an MBS mouse were cultured on fibronectin-coated coverslips, fixed and processed for immunofluorescence using antibodies against KIF11 (green) and followed by FISH experiments using Cy3-labeled oligonucleotides for β-actin mRNA (red). Blue color indicates the nuclear position. KIF11 was mostly cytoplasmic and colocalized with β-actin mRNA. Scale bar: 10 µm.
Fig. 2.
Inhibition of KIF11 motor function or knockdown of KIF11 expression impairs the localization of β-actin mRNA in MEF cells. (A) The association of KIF11 with β-actin mRNA is ZBP1 dependent. Cytoplasmic extracts of MEF/MBS cells [Ext (w)] and ZBP1-deleted MEF/MBS cells [Ext (ΔZBP1)] were prepared. β-actin mRNP was purified from the extracts using amylose-resin-attached MBP-MCP [Prec.(w) and Prec.(ΔZBP1)]. Total RNAs were isolated from starting extracts and the precipitates of amylose–MBP-MCP. RT-PCR was performed to detect the presence of β-actin mRNA in the RNA samples. Cont, negative control for RT-PCR. Western blotting was utilized to detect co-precipitated ZBP1(middle panel) or KIF11 (lower panel) with β-actin mRNA. (B) Primary MEF cells were cultured and serum stimulated for 30 min. FISH was performed to determine β-actin mRNA localization in MEF cells treated with monastrol. Blue color indicates the position of nuclei. Scale bars: 10 µm. (C) The localization of β-actin mRNA at the leading edge of the cell was decreased in monastrol- or colchicine-treated cells. A mean of 60 cells was counted from three independent experiments. Data show the mean±s.e.m. Statistical significance was calculated by using Student's _t_-test. (D) Western blots showing the expression levels of KIF11 protein in MDA231-ZBP1 KIF11-shRNA-treated cells. Numbers below the bands reflect the relative levels of KIF11 normalized to β-actin. The arrows indicate the detected proteins. WT, wild type. (E) Graph showing that the percentage of cells with localized β-actin mRNA was significantly reduced in MDA231-ZBP1 KIF11-shRNA-1-treated cells and MDA231-ZBP1 KIF11-shRNA-2-treated cells. The data were derived from three independent experiments. Data show the mean±s.e.m. Statistical significance was calculated by using Student's _t_-test.
Fig. 3.
The RRM12 motif of ZBP1 binds to KIF11 in vitro. (A) A schematic diagram of ZBP1 showing the organization of conserved domains. (B) Representative drawing of recombinant full-length ZBP1, ZBP1-RRM12+KH12, ZBP1-KH34, ZBP1-KH1234, ZBP1-RRM12 and ZBP1-KH12 used in this study. (C) Pulldown assays and western blotting were performed to detect co-precipitated KIF11. The results indicate that the full-length ZBP1, ZBP1-RRM12+KH12 and ZBP1-RRM12 fragments co-precipitated with KIF11 in the cell extracts.
Fig. 4.
Identification of the ZBP1-interacting domain of KIF11. (A) A schematic diagram of KIF11 showing the domain organization. The predicted regions that form coiled-coil structures are indicated. (B) A representative drawing of recombinant fragments of KIF11 used in the experiments to identify the regions responsible for binding to ZBP1. (C) MBP–KIF11 fusion proteins were attached to amylose beads and incubated with extracts of 293T cells. Co-precipitation and western blotting for ZBP1 indicates that the head domain of KIF11 does not bind to ZBP1 and the stalk domain shows a weak interaction with ZBP1, whereas the tail domain (762–1056) fragments were effectively bound to ZBP1. Ext, cell extracts; C, control co-precipitation experiment with MBP-coupled amylose beads.
Fig. 5.
Direct interaction of the ZBP1 RRM12 motif with the KIF11 tail domain. (A) ZBP1-RRM12 directly interacts with the KIF11 tail domain (762–1056). Left panel: lane 1, maltose beads without attached proteins were incubated with His-tagged KIF11 tail. Lanes 2 and 3, MBP-RRM12 was attached to maltose beads and incubated with the tail domain of KIF11 or with buffer containing BSA, respectively. Right panel: lane 1, Ni-beads without attached proteins were incubated with ZBP1-RRM12. Lanes 2 and 3, Ni-beads coupled to the His-tagged KIF11 tail fragment were incubated with buffer containing BSA or the ZBP1-RRM12 motif, respectively. Staining after SDS-PAGE was performed with Coomassie Blue. (B) The KIF11 tail domain interacted with endogenous ZBP1. Pull-down experiments were performed with extracts of 293T cells that were infected with lentivirus expressing MBP fusion truncation mutants of KIF11 (1–300) (lane 2), KIF11 (1–363) (lane 3) or the KIF11 tail domain (762–1056) (lane 4) using amylose affinity chromatography. The precipitates were analyzed by western blotting using ZBP1 antibodies. Lane 1, total cell extract of 293T cells. (C) The KIF5A tail domain was not bound to ZBP1 in vivo. Pulldown experiments were performed with the extracts of 293T cells infected with lentivirus expressing MBP fusion truncation mutants of the KIF11 tail domain (762–1056) (lane 2) or KIF5A tail domain (806–1032) (lane 3), and analyzed by western blots using ZBP1 antibodies. Lane 1, total cell extract of 293T cells. (D) Analysis of the in vivo interaction of the RRM12 domain of ZBP1 with endogenous KIF11. 293T cells were infected with lentivirus expressing His-tagged ZBP1-RRM12 (lane 2) or ZBP1-KH12 (lane 3). Pulldown assays were performed from the extracts of infected cells using Ni-Sepharose beads. The precipitates were analyzed by western blotting using antibodies against KIF11. Lane 1, control pulldown assay with extracts of uninfected 293T cells.
Fig. 6.
Impairing the in vivo interaction of ZBP1 with KIF11 delocalizes β-actin mRNA from the cell leading edge. MEF cells were infected with an mCherry lentivirus construct and the constructs expressing the mCherry–KIF11 (702–1056) tail domain, mCherry–KIF5A (806–1032) tail domain or the mCherry–RRM12 motif of ZBP1. After fixation and permeabilization, FISH experiments were performed to detect the localization of β-actin mRNA. (A–C) Representative images showing β-actin mRNA localization in cells expressing mCherry, mCherry–KIF11-tail or mCherry–ZBP1-RRM12. Scale bars: 10 µm. (D) Bars show the percentage of infected cells counted with localized β-actin mRNA at the leading edge. A mean of 50 cells were counted blind per coverslip in three experiments each. Data show the mean±s.e.m. Statistical significance was calculated by using Student's _t_-test.
Fig. 7.
Expression of the dominant-negative KIF11 tail fragment affects carcinoma cell motility and increases cell invasive ability. (A) Western blotting was performed to detect protein expression in the stable cells expressing mCherry (not Flag-tagged) (lane 1), Flag-tagged mCherry–KIF11-tail fusion protein (lane 2), Flag-tagged mCherry–ZBP1-RRM12 fusion protein (lane 3) and Flag-tagged mCherry–KIF5A-tail fusion protein (lane 4) using anti-Flag antibodies. The arrow indicates the position of β-actin. WT, wild-type. (B) Pulldown experiments were performed with extracts of MDA231-ZBP1 cells infected with lentivirus expressing Flag-tagged mCherry–KIF11-tail (lane 2) or mCherry–KIF5A-tail (lane 3), using anti-Flag agarose beads. The precipitates were analyzed by western blotting using ZBP1 antibodies. Lane 1, total cell extract of MDA231-ZBP1 cells. (C) Motility analysis was performed by tracking stable cells expressing mCherry or the mCherry-fused KIF11 tail domain under regular growth conditions at 2-min intervals over a 4-h period. Expression of the KIF11 tail domain did not change the random velocity (P = 0.0561; two-tailed Student's _t_-test), but decreased the directionality in cell motility (cells expressing mCherry versus cells expressing mCherry–KIF11-tail, n = 31 and 32; P = 0.027; two-tailed Student's _t_-test). Data show the mean±s.e.m. (D) Overexpression of the KIF11 tail or ZBP1-RRM12 increases the invasive ability of carcinoma cells. Stable cells expressing different mCherry fusion proteins were seeded in serum-free medium into the upper chamber of 8-µm pore Matrigel-coated transwell filters. The lower chamber contained medium with 10% serum. Cells that had invaded to the underside of the filter were stained and counted 16 h later. Invasion was quantified by visual counting of the total cells on the underside of the filter. The relative numbers of invading cells from each assay are normalized to that of the parental MDA231-ZBP1 clone and are represented as a fold change relative to the MDA231-ZBP1. Data shown in the figure represent the mean±s.e.m. of data from three experiments.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources