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. 2010 Jan 13:4:1-8.
doi: 10.2174/1874091X01004010001.

Armadillo Repeat Containing 8alpha Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

Koji Tomaru  1 , Atsuhisa Ueda, Takeyuki Suzuki, Nobuaki Kobayashi, Jun Yang, Masaki Yamamoto, Mitsuhiro Takeno, Takeshi Kaneko, Yoshiaki Ishigatsubo
Affiliations

Armadillo Repeat Containing 8alpha Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

Koji Tomaru et al. Open Biochem J. .

Abstract

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) alpha. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8alpha co-localized with HRS. ARMc8alpha promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8alpha.

Keywords: ARMc8α; FBPase; HRS; UIM.; monoubiquitination.

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Figures

Fig. (1). HRS associates with ARMc8α <i>in vivo</i>, and exogenously expressed ARMc8α co-localizes with exogenously expressed T7-tagged HRS in HEK293 cells.
Fig. (1). HRS associates with ARMc8α in vivo, and exogenously expressed ARMc8α co-localizes with exogenously expressed T7-tagged HRS in HEK293 cells.
A. AH109 yeast strains were transformed with bait constructs (pGBKT7-ARMc8α, pGBKT7-ARMc8β, pGBKT7-ARMc8α (amino acids 268–648) or pGBKT7-Mock) and prey constructs (pACT2-HRS or pACT2-Mock), and the transformants were inoculated onto a plate containing SD medium lacking leucine and tryptophan to select positive clones transformed with both constructs (bait and prey) (left panel, - Leu-Trp). Colonies positive for growth were restreaked onto a plate of SD medium lacking adenine, histidine, leucine, and tryptophan to select positive clones expressing the reporter genes ADE and HIS3 (right panel, -Ade-His-Leu-Trp). B. CHO cells were transfected with pFLAG-ARMc8α or pFLAG-Mock, and the cell extracts were immunoprecipitated with an anti-FLAG antibody. The lysates and immunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting with an anti-FLAG antibody or an anti-HRS antibody. IB, immunoblot. IP, immunoprecipitation. C. HEK293 cells were co-transfected with pEGFP-ARMc8α and pT7-HRS. Cells were immunostained with a T7•tag antibody and analyzed by confocal microscopy, as described in Material and Methods. GFP-tagged ARMc8α was distributed within the cytoplasm (panel a), as was T7-tagged HRS (panel b). Panel c is a merged image of the two exogenous proteins. Arrowheads indicate typical co-localization. Scale bar represents 10 µm.
Fig. (2). ARMc8α binds to HRS <i>via</i> the proline-rich domain.
Fig. (2). ARMc8α binds to HRS via the proline-rich domain.
A. upper panel shows the HRS domain organization. Wild type and deletion HRS constructs are shown schematically in the lower panel. Abbreviations are as follows. VHS: Vps27-HRS-STAM; FYVE: Fab1-YOTB-Vac1-EA1; UIM: ubiquitin interacting motif; PRD: prolinerich domain; CC: coiled coil; PSAP: proline, serine, alanine, and proline motif; Pro/Gln: proline-and glutamine-rich domain. B, C. pFLAGARMc8α was co-transfected with each HRS deletion construct in CHO cells. Exogenous ARMc8α was immunoprecipitated with an anti-FLAG antibody. Co-immunoprecipitation of each deleted HRS was analyzed by immunoblotting with an anti-T7•tag antibody (B) and anti-Omni probe (C). As a negative control, cells were co-transfected with pFLAG-Mock and pT7-HRS.
Fig. (3). Ubiquitinated protein binding to HRS does not affect the association between HRS and ARMc8α, and exogenously expressed ARMc8α increases the interaction of ubiquitinated proteins with exogenously expressed HRS via the UIM.
Fig. (3). Ubiquitinated protein binding to HRS does not affect the association between HRS and ARMc8α, and exogenously expressed ARMc8α increases the interaction of ubiquitinated proteins with exogenously expressed HRS via the UIM.
A. In CHO cells, pFLAG-ARMc8α was co-transfected with pT7-HRS, pT7-HRS (S270E), or pT7-HRSΔUIIM, as indicated (lanes 2 to 4). Exogenous ARMc8α was immunoprecipitated with anti-FLAG antibody. Co-immunoprecipitations of each deleted HRS and ubiquitin were analyzed by immunoblotting with the antibodies indicated. As a negative control, cells were co-transfected with pFLAG-Mock and pT7-HRS (lane 1). Arrow indicates the location of the lane beginning. B. In CHO cells, pFLAG-ARMc8α or pFLAG-Mock was co-transfected with pT7-HRS or pT7-HRSΔUIM, as indicated (lanes 1 to 3). The transfected cells were lysed and the exogenous HRS and each deleted HRS were immunoprecipitated with T7•tag antibody. This was followed by immunoblotting with the antibodies shown (lanes 4 to 6). IB, immunoblot. IP, immunoprecipitation.
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