Novel BRCA2-Interacting Protein, LIMD1, Is Essential for the Centrosome Localization of BRCA2 in Esophageal Cancer Cell

Abstract

Mutation of breast cancer 2, early onset (BRCA2) has been identified as a vital risk factor for esophageal cancer (EC). To date, several proteins have been reported as BRCA2-interacting proteins and are associated with multiple biological processes. This study's aim was to identify a novel interactive protein of BRCA2 and to explore its functional roles in EC. A yeast two-hybrid screening was performed to identify a novel BRCA2-interacting protein. Glutathione-S-transferase (GST) pull-down analysis was performed to find out how the binding domain of BRCA2 interacts with LIM domains containing 1 (LIMD1). The interaction between LIMD1 and BRCA2 at the endogenous level was confirmed by using coimmunoprecipitation and immunobloting. Furthermore, two different sequences of short hairpin RNAs (shRNAs) against LIMD1 were transfected into the human EC cell line ECA109. Afterward, the effects of LIMD1 suppression on the centrosome localization of BRCA2 and cell division were analyzed using an immunofluorescence microscope. Results showed that LIMD1 was a novel BRCA2-interacting protein, and LIMD1 interacted with the conserved region of BRCA2 (amino acids 2,750-3,094) in vitro. Importantly, after interfering with the protein expression of LIMD1 in ECA109 cells, the centrosome localization of BRCA2 was significantly abolished and abnormal cell division was significantly increased. These results suggested that LIMD1 is a novel BRCA2-interacting protein and is involved in the centrosome localization of BRCA2 and suppression of LIMD1, causing abnormal cell division in EC cells.

Figure 1

LIMD1 was a novel BRCA2-interacting protein. (A) Schematic representation of the structure of BRCA2; LIMD1 was identified as an interacting protein of BRCA2. (B) The interaction between BRCA2 and LIMD1 was analyzed by GST pull-down assay; LIMD1, LIM domains containing 1; BRCA2, breast cancer 2, early onset; NES, nuclear export sequence; CLS, centrosomal localization signal; NLS, nuclear localization signal; GST, glutathione-S-transferase.

Figure 2

LIMD1 interacted with BRCA2 at the endogenous level. The interaction between BRCA2 and LIMD1 at the endogenous level was detected by coimmunoprecipitation and immunoblot analysis. LIMD1, LIM domains containing 1; BRCA2, breast cancer 2, early onset; IP, immunoprecipitation; IB, immunoblot; IgG, immunoglobulin G.

Figure 3

LIMD1 suppression abolished the centrosome localization of BRCA2 and caused abnormal cell division. Two difference sequences of shRNAs against LIMD1 or scrambled shRNA were transfected into ECA109 cells for 24 h, respectively. (A) The efficiency of transfection was detected by immunoblot analysis. (B) The cells of BRCA2 located to the centrosome were stained with bis-benzimide (Hoechst 33258) and analyzed under an immunofluorescence microscope. (C) The cells with three or more centrosomes were stained with bis-benzimide (Hoechst 33258) and analyzed under an immunofluorescence microscope. LIMD1, LIM domains containing 1; BRCA2, breast cancer 2, early onset; shRNA, short hairpin RNA. ***p < 0.001.