IK was originally identified as a cytokine that inhibits interferon gamma (IFN-γ)-induced expression of HLA class II antigen during immune responses in K562 erythroleukemic cell line [1]. The protein got its name IK based on these findings. Human IK contains 557 amino acids and migrates at about 80 kDa on SDS-PAGE [2]. It is also named RED owing to the presence of a repetitive arginine (R), aspartic (E), and glutamic acid (D) sequence [2]. It has also been reported that IK is one of the spliceosome factors [3, 4]. Screening of an siRNA library containing 23,835 human genes reveals that depletion of IK induces mitotic arrest, primarily characterized by having a high mitotic index [5]. A recent study shows that IK is required for the localization of MAD1, a spindle checkpoint protein, to the kinetochores and involved in the regulation of the spindle assembly checkpoint [6].
In the present study, we have confirmed that depletion of IK causes mitotic arrest. Our further investigation reveals that the subcellular localization of IK is dynamic during the cell cycle. We also show that the expression of IK is cell cycle-regulated. Affinity pull-down and mass spectrometry analyses reveal that IK interacts with DHX15, a putative ATP-dependent RNA helicase which is implicated in pre-mRNA splicing. Our current study suggests that IK can be explored as a new biomarker for cell proliferation and checkpoint control.
Materials and methods
Cell culture
HeLa cell line was originally obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and antibiotics (100 μg/ml of penicillin and 50 μg/ml of streptomycin sulfate, Invitrogen) at 37°C under 5% CO2.
Antibodies and plasmids
Antibodies for IK was purchased from Bethyl Laboratory Inc (Montgomery, TX). GFP antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Coilin antibody was purchased from Abcam (Cambridge, MA). GFP-IK and His6-IK were subcloned as described in a previous study [7].
RNA interference
IK small interfering RNAs (IK siRNA) was purchased from Dharmacon, which corresponds to following sequences: NNCAUAUGAGCGGAAUGAGUU. HeLa cells were seeded at 50% confluence in an antibiotic-free culture medium and transfected with siRNAs at a final concentration of 10 nM for 24, 48, or 72 h using the LipoJet™ In Vitro Transfection Kit (Ver. II, Signagen Laboratories, Rockville, MD). Negative controls were cells transfected with 10 nM siRNAs targeting firely (Photinus pyralis) luciferase. The sequence of the control siRNA is 5’UUCCTACGCTGAGTACTTCGA3’ (GL-2, Dharmacon, Waltham, MA).
Western blot
SDS-PAGE was carried out using the mini gel system from Bio-Rad (Hercules, CA). Proteins were transferred to PVDF membranes. After blocking with TBST containing 5% nonfat dry milk for 1 h, the membrane was incubated overnight with a primary antibody, followed by incubation with a horse reddish peroxidase-conjugated secondary antibody for 1 h at room temperature. After thorough washing with TBST buffer, signals on the membranes were developed with an enhanced chemiluminescent system (Pierce, Rockford, IL).
Immunoprecipitation and pull-down assay
Immunoprecipitation and pull-down assay were performed as described previously [6, 7]. For pull down assay, HEK293K cells transfected with His6-IK were lysed in a lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, 1% NP40, pH 7.4). Ni2+ -IDA-agarose resins (Clonetech, Mountain View, CA) were then added to the cell lysates and incubated with gentle agitation at 4°C overnight. The resin was then washed three times with the washing buffer [50 mM sodium phosphate, 300 mM sodium chloride, 40 mM imidazole (pH 7.4)]. After last wash, His6-tagged products were eluted in the elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 300 mM imidazole, pH 7.4). The elutes were diluted in a lyses buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton, 2 mM sodium pyrophosphate, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 500 μM PMSF, 2 μM pepstatin A, 10 units/ml aprotinin, 20 mM NEM] and cleared by centrifugation. The anti-IK antibody or control IgG (1 μg) as well as protein G-agarose (40 μl) resins (50/50, Millipore, Billerica, MA) were then added to cell lysates and incubated at 4°C overnight. The resins were then extensively washed with the lysis buffer. Proteins bound to the resin were eluted with SDS sample buffer and then subjected to SDS-PAGE followed by Western blot with appropriate antibodies.
Fluorescence microscopy
Fluorescence microscopy was performed as described in our previous studies [8]. Briefly, HeLa cells seeded on chamber slides were transfected with various expression constructs for 48 h. At the end of transfection, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After permeabilization by incubation with 0.5% Triton X100 in PBS for 20 min, cells were incubated with 2% bovine serum albumin in PBS for 1 h followed by incubating overnight with the antibody to GFP. Cells were stained with Alexa Fluor 488. Cellular DNA was finally stained with 4’, 6-diamidino-2-phenylindole (DAPI, Molecular Probe, Eugene, OR). Fluorescence signals were detected on a Leica TCS SP5 confocal microscope or on a Leica AF6000 fluorescence microscope.
Flow cytometry
Flow cytometry was performed as described in our early studies [9]. Briefly, cells were initially fixed in 75% ethanol, then suspended in a solution of PBS containing 100 μg/ml of RNase A (Sigma, St Louis, MO) and 10 μg/ml of propidium iodide (Molecular probes, Eugene, OR) and kept at room temperature for 1 h. Cellular fluorescence was then measured using Beckman Coulter Epics XL-MCLTM Flow Cytometer (Fullerton, CA). DNA content was deconvoluted using Muticycle software (Phoenix Flow System, San Diego, CA) to estimate percent of cells in different phases of the cell cycle.
Statistical analysis
Student’s t test was used to evaluate the difference between two groups. The significant level was set at 0.05.