Endoplasmic Reticulum (ER) Stress Enhances Tip60 (A Histone Acetyltransferase) Binding to the Concanavalin A

Abstract

Herein, we report that the concanavalin A binding of Tip60 (a target of the human immunodeficiency virus type 1-encoded transactivator Tat interacting protein 60 KD; a histone acetyltransferase; HAT) is enhanced as the result of endoplasmic reticulum (ER) stress. The cell expression of Tip60 combined with site-directed mutagenesis analysis was used to identify the glutamine 324 residue as the lecithin binding (Concanavalin A; Con A) site. The Tip60 N324A mutant strain, which seems to be the Con A binding-deficient, was attenuated the protein-protein interactions with FE65 and its protein stability, but its ability of G0-G1 cell cycle arrest was not interrupted. Interestingly, both HAT activity and the nuclear localization of Tip60 N324A mutant were enhanced than those of Tip60 WT. Thus, our results indicate that the Con A binding deficient of Tip60 seems to be one of the most pivotal posttranslational modifications (such as N-glycosylation) for its functional regulation signal, which is generated in response to ER stress.

Keywords: Cell cycle.; ER stress; HAT; N-glycosylation; Tip60.

Fig. (1) The association Tip60 with concanavalin A in Vivo and the putative Site of Tip60 N-glycosylation

A) After treatment HEK293 cell lysate with concanavalin A (Con A, Jack bean lectin), the extracts were then analyzed by Western blotting using an anti -Tip60 antibody (A left). To control the N-glycosylation of Tip60, we also added Con A to HEK293 cell lysate was pretreated with PNGase F, before Con A treatment (A right). B) The reactivity of Tip60 to Con A was reduced with the tunicamycin (2.5mg/ml) treatment for different times, as indicated above (B upper lane). To control the amount of Tip60, the each cell lysate was immuno blotted with its specific antibody (B bottom lane). * indicated the putative glucosylated Tip60. C) With the http://www.cbs.dtu.dk/services/SignalP/output.html program, the putative N-glucosylation sites in Tip60 was predicted, as indicated in Fig. 1C above. The domains (chromodomain for the histone binding, Zn finger, the conserved HAT domain, NR for the nuclear receptor binding) are also indicated [6, 7]. D) After transfected with GFP Tip60 WT, N206A, N324A, N342A, the cell lysates was precipitated either Con A (D upper lane) or GFP Ab (D bottom lane). To detect Tip60 amount, immunoblot was performed with its specific Ab.

Fig. (2) The glutamine 324 residue of Tip60 is required for the interaction with FE65 and its HAT Activity

A) After immunoprecipitating the transfected GFP Tip60 WT, N324A, N342A with monoclonal GFP Ab, the precipitant was immuno blotted with FE65 Ab, B) Acetylated-lysine Ab, or C) Tip60 Ab. D) In parallel, HAT activity of purified Tip60 WT, N324A, or N342A expressed in HEK 293 cell were compared with ELISA method in arbitrary units. For the detail, see the material and method. * P < 0.05 significance versus to each other.

Fig. (3) The reduction of Tip 60 G0-G1 cell cycle arrest by the mutation of ConA binding site mutation.

A) After transfection of HEK293 cells with each Tip60 construct as indicated below with Etoposide (5ug/ml), the G0-G1 cell cycle was sorted with the cell sorter. as a percent unit to the total cell numbers. The value represents the mean of five time repeats. B) Each Tip60 amount (indicated above) was monitored with its specific antibody.

Fig. (4) Protein stability of Tip60 (wt), or N324A mutant.

A) GFP-Tip60 or Tip60 N324A mutant was transfected into HEK293 cells and the cells were treated with cyclohexamide. The Tip60 proteins were chased for the indicated time periods. Western blotting with a monoclonal Tip60 antibody of SDS-PAGE subjected GFP-Tip60 proteins is shown. To monitor the protein amount, an equal amount of cell lysate was subjected to western blotting with an actin antibody. Results shown are one of five repeated experiments. B) Quantification of the pulse-chase experiment is shown by image analysis with the Fuji Image Quant software.

Fig. (5) The confocal microscopy of Tip65 WT, N324A, or N342A with FE65.

The confocal microscopic images transfected with GFP-Tip60 WT, N324A N342A (all green color) to the endogenous FE65 (all red color) were compared each other in the direct immunofluorescence microscopy. Results shown are one of five repeated experiments.

Fig. (6) Brefeldin which induces ER stress increases the Con A binding of Tip60.

A) After treatment of brefeldin to HEK293 cell in the indicated time and concentration, Tip60 glycosylation, the glycosylation of Tip60 which was detected by Con A bead (upper lane). For the control, each sample was immunoblotted with Tip60Ab (bottom lane). Result shown is one of five repeated experiments. B) The relative quantification of Tip60 glycosylation increase with brefeldin treatment is shown by image analysis with the Fuji Image Quant software. The relative arbitrary unit to the maximum value on the specific time in each brefeldin treatment concentration (indicated above) was shown.