Phosphorylation of Tip60 Tyrosine 327 by Abl Kinase Inhibits HAT Activity through Association with FE65

Materials

Abl (80 units/mg) was bought from Merck Millipore (Darmstadt, Germany). Other chemicals were of the highest grade of purity from Sigma. Monoclonal or polyclonal antibodies against, Actin, Tip60, and phosphor 327 Tyr Tip60 was purchased from Santa Cruz Biotech Inc (Santa Cruz Biotech. USA) or from Cell Signaling (Boston Ma, USA). All chemicals were purchased from Sigma–Aldrich unless otherwise stated.

Cell Culture and Transfection

HEK293 cells were maintained in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) at 37 °C with 5% CO₂. Transfection was performed with Lipofectamine and Plus Reagent (Invitrogen) according to the manufacturer's instruction.

DNA Constructions

Tip60 (WT), Tip60 (Y327F, Y345F Y409F) were cloned from Human Tip60 (isoform1; NP-874369). Full-length Tip60 was cloned into pCMV-AC-GFP vector for cell culture study and into pGE5X-1 vector for recombinant protein production. Sequence integrity of all constructs was verified by sequencing.

Overexpression and Purification of Recombinant Proteins

Recombinant GST, GST-Tip60, and GST-Tip60 Y327F were expressed in in Escherichia coli BL21. Overexpression of bacterial culture in linear growing phase (0.6 OD) were induced by 0.2 mM IPTG at 18 °C overnight, and the recombinant proteins were then purified by GST agarose beads (Amersham Biosciences Co). Concentrations of the recombinants protein were quantified by SDS/PAGE with the use of BSA as standard.

Immunprecipitation of Tip60

Total cell lysate of HEK293 cell was denatured by incubation with 0.5% SDS, 50 mM sodium phosphate, pH 8.0, and 2 mM EDTA at 90 °C, to allow immunoprecipitation. The samples were supplemented to obtain a composition of 50 mM sodium phosphate, pH 7.2, 1% sodium deoxycholate, 1% Triton X-100, 0.5% SDS, 150 mM NaCl, 2 mM EDTA, 5 mM NaF, 2 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% aprotinin, and 200 µg/ml leupeptin at 4 °C. Mouse anti-Tip60 monoclonal antibody (usually at a 1:100 dilution) was added, followed 18 h later by protein A-Sepharose. Washed immunoprecipitates were analyzed by the specific antibody after separation in a 11% sodium dodecyl sulfate-polyacrylamide gel.

Cell Cycle Analysis

HEK293 cells were transfected with 0.25 μg of GFP- Tip60 (WT), Tip60 (Y327F, Y345F Y409F) vector was transfected and the rate of apoptosis measured by Annexin V-PE apoptosis detection kit I (BD Biosciences, CA, USA), according to the manufacturer’s instructions. The cells were vortexed gently and incubated for 15 min at 25 °C in the dark. 400 μl of binding buffer was added to each tube. Within 1 hr, FACS was performed on a Coulter Epics Elite equipped with a gated amplifier and upgraded with enhanced system performance in The Core Facility of Chungbuk National University [21].

Protein Tyrosine Phosphorylation Assay

The assay kit and active Abl kinase were purchased from Merck Millipore. After Abl kinase reaction in the reaction buffer (20 mM MOPS, 1 mM EDTA, 0.01% Brij-35, 5% Glycerol, 0.1% β-mercaptoethanol, 1 mg/mL BSA, 100uM ATP) with GST- Tip 60 recombinant protein (1ug/10ul) , the reactant was western blotted with an anti- phosphor Tyr antibody of Cell Signaling Technolgy Inc (Boston MA, USA).

ELISA for Histone Acetyltransferase (HAT)

Extracts were immunoprecipitated as above, except that the high salt wash was omitted. The immunoprecipitates were washed twice in HAT assay buffer (50 mM Tris, pH 8/10% glycerol/0.1 mM EDTA/1 mM DTT), and incubated in 60 μl of HAT assay buffer containing acetyl-CoA (100 μM) and biotinylated histone H4 peptide (0.5 μg) for 30 min at 30°C. An aliquot of the reaction was immobilized onto streptavidin plates and tyrosine phosphorylation was detected via HAT ELISA according to the manufacturer's instructions (biovision). In some assays, HAT activity was analyzed with Tip60 immobilized on Glutathione-sepharose beads. Its HAT activity was monitored by recording the fluorescence emission above 430 nm after excitation at 313 and 366 nm, respectively. Sample without ATP was served as the negative control[22].

Phosphorylation of Tyrosine 327 on TIP60 by Abl Kinase

Based on the identification of the tyrosine phosphorylation site in TIP60 by consensus sequences analysis (Fig. 1A) and the information on the protein-protein interaction between TIP60 and Abl kinase (unpublished data, [18, 19, 22]), we examined whether TIP60 is phosphorylated by Abl kinase in HEK293 cells. To detect the tyrosine phosphor modifications of the Tip60, we used anti phosphor tyrosine rabbit antibody which recognized specifically the phosphorylated tyrosine (Fig. 1A). Human Tip60 (isoform1; NP-874369) has 546aa amino acids, and functional domains and several key tyrosine phosphorylation acids (Y327 or Y345) [5, 18]. The putative FE65 binding motif (³²⁴NeiY³²⁷) of Tip60 binds to PID1 of FE65, depending on its 327 tyrosine residue phosphorylation by Abl kinase [5, 6]. Further because it has been demonstrated that FE65 interacts with Tip60 through its PID1 domain [18, 19], we attempted to test whether the putative tyrosine phosphorylation site in human Tip60, each tyrosine residue was replaced with the phenylalanine (Y327F, Y345F). The functional domains (chromodomain, Zn finger, the conserved HAT domain, and NR box) in Tip60 are indicated with the amino acid number (Fig. 1A). In order to know whether Tip60 is a tyrosine phosphorylated protein or not, we performed the experiment with the site directed mutant (Y327 or Y345). The panel is the representative data for immunoblotting with an anti-Phosphor Tyr specific antibody and Tip60 antibody (Fig. 1B). After transfected HEK293 cell with each EGFP DNA constructs which are indicated above, each cell lysate was immunoprecipitated with an anti-GFP antibody. Each immunoprecipitant was immunoblotted with an anti-Phosphor specific antibody or Tip60 antibody. Because only EGFP-Tip60 Y327F was not recognized by anti-Phosphor specific antibody, the 327 tyrosine residue is the phosphorylation site by Abl kinase (above panel in Fig. 1B). The expression of EGFP Tip60 protein in each construct was monitored with the western blot (bottom panel in Fig. 1B). The negative control (NC) indicates the untransfected HEK293 cell lysate. To monitor the point mutation effect on Tip60 binding FE65, EGFP Tip60 Y409F mutant was constructed (Fig. 1A). The mutant Tip60 (Y409F) did not ablate the interaction with FE65 (above panel in Fig. 1B).

In order to further demonstrate our observation, GST fusion protein with Tip60 containing WT or Y327F was prepared from E. coli, and subjected to a tyrosine kinase assay in vitro (as shown in Fig. 1C). The reactant was immunoblotted with an anti-Phosphor specific antibody. The reactant of Tip60 WT was recognized by an anti-Phosphor specific antibody, while Tip60 Y327F was not (above panel in Fig, 1C). The protein amount of GST fusion protein with Tip60 containing WT or Y327F was monitored with the Coomassie Blue staining (bottom panel in Fig. 1C). It has been reported that Abl kinase forms a protein complex with Tip60 in the cell. We also observed the protein-protein interaction between Tip60 and Abl kinase (data not shown). Thus, this result also suggests that Tip60 is a tyrosine phosphorylation protein which is modified by Abl kinase.

Phosphorylation on Tyrosine 327 Enhances Interaction between Tip60 and FE65 but Not HAT Activity

Because a number of physiological functions of Tip60 are associated with several proteins, including FE65, we suspected that the tyrosine phosphorylation of Tip60 could effect on its Fe65 binding activity in cells exposed to various cell death stimuli [5, 6, 13, 18, 22]. Further, because it has been reported that the tyrosine residue of Tip60 binds to PID1 of FE65 and inhibits its HAT activity, we investigated which position tyrosine phosphorylation could affect its Tip60 inhibition activity. To compare the tyrosine phosphorylation effect on Tip60, we used Tip60 Y327F mutant in parallel with both WT and Y345F. As shown Fig. (2A), theinteraction between Tip60 Y327F mutant and FE65 was ablated, compared with that of either WT or Y345F. The result is contradicted with other’s report, suggesting that Tip60 Y345 phosphorylation was responsible to interact with PID1 of FE65. Thus, our results suggested that the interaction between Tip60 and FE65 is dependent on the Y327 residue, but not Y345 residue where can be phosphorylated by Abl kinase.

To investigate further the effect of Y327 residue phosphorylation, we monitored Tip60 activity by ELISA measurement. Surprisingly, the HAT activity of Tip60 Y327F mutant is higher than that WT or Y345F mutant (Fig. 2B). The results suggested that the interaction between Tip60 and FE65, together with the auto-acetylation (Fig. 2B), seem to be the inhibitory effect to the HAT activity of Tip60 (Fig. 2B). Thus, these observations suggested that the tyrosine 327 phosphorylation of Tip60 influences on not only its association with FE65 but also its auto acetylation and HAT activity. Further the results also suggested that the FE65's anti Tip60 activity depends on the direct physical interaction of FE65 with Tip60 (data not shown). Therefore, Tip60 tyrosine phosphorylation may induce to increase FE65's anti Tip60 role by enhancing with the direct interaction between FE65and Tip60.

Tyrosine 327 Phosphorylation of Tip60 Affects Subcellular Localization

Through its HAT activity, Tip60 targets a number of substrates for acetylation. Tip60 also targets itself for auto acetylation, which can be used as an indicator for its activity. To determine whether the tyrosine phosphrylation could affect Tip60's activity, HEK293 cells transfected with GFP-Tip60 were treated with the tyrosine phosphorylation, which indicated that the HAT activity was affected by FE65 binding (Fig. 3). We observed that the localization of Tip60 Y327F was mainly in the nuclear, compared with Tip60 WT and Y345F (which were found in both nuclear and cytoplasm). Therefore it seems to be that the depletion of the tyrosine phoshorylation on 327 residue promotes Tip60 nuclear localization (Fig. 3 middle).

Tyrosine 327 Phosphorylation of Tip60 Promotes G0-G1

To determine further the functional role of tyrosine 327 phosphorylation of Tip 60, we compared the cell cycle regularity of Tip60 Y327F mutant in parallel with both WT and Y345F. After transfected at day 3 with each DNA construct (EGFP-Tip60 WT, Y327F, or Y345F), cells were harvested at different days post-transfection as indicated. HEK293 cells transfected with the control vector or Tip60 constructs (WT, Y327F or Y345F) were harvested and subjected to FACS analysis. As shown in Fig. (4A), Tip60 Y327F construct significantly reduced G0-G1 arrest, compared with the control vector or WT or Y345F, even though each protein expression level was similar (Fig. 4B). Thus, these results suggest that tyrosine 327 phosphorylation on Tip60 by Abl kinase is required to promote G0-G1 stage.

Hypothetical Signal Transduction to Tip60 by Abl Tyrosine Kinase Phosphorylation

We describe here for the first time an interaction between FE65 and TIP60 that was enhanced by posttranslational modification of tyrosine 327 on Tip60. This interaction resulted in cell death and the inhibition of Tip60 HAT activity. Furthermore, we observed that phosphorylation on tyrosine 327 of Tip60 by Abl kinase inhibited HAT activity, with FE65 being part of a feedback regulatory mechanism. Thus, our results revealed a new regulatory mechanism controlling the apoptotic activity of Abl kinase during environmental or ER stress [18, 22]. When a stress signal from the environment reaches the cell membrane, Abl tyrosine kinase is activated through a signaling pathway. Tip60, as an Abl substrate, is phosphorylated on tyrosine 327. Activated Abl kinase phosphorylates tyrosine 376 on Tip60, resulting in increased FE65 binding. Ultimately, the cell enters G0 arrest or apoptosis. Activated Tip60 acetylates Abl kinase as a feedback mechanism (Fig. 5).

When an environmental signal reaches the cell membrane, Abl tyrosine kinase is activated through a signal pathway and Tip60 is phosphorylated on tyrosine 327. Activated Abl kinase phosphorylates Tip60 tyrosine 376, resulting in increased FE65 binding and the cell enters G0-G1 arrest or apoptosis. Activated Tip60 acetylates Abl kinase, as a feedback mechanism.