PP2, a Src kinase inhibitor, was obtained from Calbiochem (San Diego, CA). [γ-32P]ATP was obtained from New England Nuclear (Boston, MA). The antibodies used and their sources were as follows: The monoclonal anti-phosphotyrosine antibody (PY99), the monoclonal anti-Src antibody (B12), the goat anti-rabbit and the goat anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal anti-Src pY418 antibody and anti-Src pY529 were from Biosource International (Camarillo, CA). The monoclonal anti-His antibody was from Invitrogen (Carlsbad, CA). Purified recombinant Src and the assay kit for Src kinase activity, anti-phosphotyrosine antibody, and protein G Agarose were obtained from Upstate Biotechnology (Lake Placid, NY). Plasmids pGFP2-C, pRluc-N, and DeepBlueC were purchased from Biosignal Packard (Montreal, Canada). Plasmids pEYFP-C1 and pECFP-N1 were purchased from Clontech (Palo Alto, CA), and pGEX-4T-1 and pTrc-His were from Invitrogen. All secondary antibodies were conjugated to horseradish peroxidase; therefore, the immunoreactive bands were developed using chemiluminescence (Pierce, Rockford, IL). Glutathione beads were from Amersham Bioscience (Uppsala, Sweden). The Optitran nitrocellulose membranes were obtained from Schleicher & Schuell (Keene, NH).
The preparation of chicken c-Src lacking the SH4 domain and GST-Src mutants were done as described (Ma et al., 2000
). GST-NT (amino acid residue 6-90), GST-CD2 (amino acid residue 152-288), and GST-CD3 (amino acid residue 350-785) expression vectors were constructed based on the sequence of pig kidney Na+
-ATPase α1 subunit (see ). GST-H+
-CD3 and GST-SERCA-CD3 were constructed based on the rat H+
-ATPase cDNA and rat cardiac SERCA 2a cDNA, respectively. His-tagged Src constructs were generated by excising the corresponding Src cDNA from the GST-Src vectors and then inserting them into pTrc-His A vector. Src-ECFP and Src-Rluc for fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) assays were constructed by cloning the full-length c-Src in frame into pECFP-N1 or pRluc vector. The rat Na+
-ATPase α1 cDNA was excised from the expression vector provided by Dr. Pressley (Texas Tech University) and inserted in frame into pEYFP-C1, and the canine Na+
-ATPase α1 cDNA was cloned into pGFP2
vectors. All constructs were verified by DNA sequencing.
Figure 3. Identification of the Src domains involved in the interaction with the Na+/K+-ATPase. (A) Schematic presentation of structures of Src. (B) Coomassie blue staining of GST-Src, GST-SH2, GST-SH3, GST-SH3SH2, and GST-kinase. (C) Binding of GST-Src, GST-SH3SH2, (more ...)
Cell Preparation, Culture, and Transient Transfection
Pig kidney proximal LLC-PK1, human embryo kidney 293T cells, and mouse fibroblast SYF and SYF + Src cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 μg/ml). LLC-PK1 cells and 293T cells were serum-starved for 24 h, whereas SYF and SYF + Src cells were cultured in the medium containing 0.5% FBS for 24 h and used for the experiments. Cells were transfected with various plasmids using Lipofectamine 2000 (Wang et al., 2004
). Experiments were performed 24 h after transfection unless indicated otherwise.
Preparation of Src, Na+/K+-ATPase, GST-fused Proteins, and His-tagged Proteins
Src, without the first 85 amino acid residues, was purified from sf-9 cells as described (Ma et al., 2000
) and used in the initial binding assays to ensure that Src binds to the Na+
-ATPase, but not the lipid composition in the purified Na+
-ATPase preparation. In subsequent experiments (e.g., phosphorylation and activity assays), purified recombinant full-length Src from Upstate Biotechnology was used. Na+
-ATPase was purified from pig kidney outer medulla using the Jorgensen method as we previously described (Xie et al., 1996
) and the preparations with specific activities between 1200 and 1400 μmol Pi/mg/h were used in this work. Under our experimental conditions either 100 μM vanadate or 10 μM ouabain caused a complete inhibition of the ATPase activity of the purified pig kidney Na+
-ATPase. GST fusion proteins or His-tagged proteins were expressed in Escherichia coli BL21
and purified on glutathione beads or nickel column.
Immunoprecipitation and GST Pulldown
Cells were lysed in RIPA buffer containing 1% Nonidet P40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 50 mM Tris-HCl (pH 7.4). Cell lysates were cleared by centrifugation at 16,000 × g
for 15 min, and the supernatants (1 mg) were either immunoprecipitated with anti-α1 antibody or incubated with different GST-fusion proteins. The complexes were then pulled down by either protein G agarose or glutathione beads as we previously described (Ma et al., 2000
; Haas et al., 2002
) and analyzed by Western blot.
Src Kinase Activity
The Src kinase activity was assayed using a commercial kit as we previously described (Haas et al., 2000
). To determine how Na+
-ATPase affects Src kinase activity, the purified Src (4.5 U) was incubated with 5 μg of the purified Na+
-ATPase in the Src assay buffer for 30 min at room temperature. Afterward, both control Src or the Na+
-ATPase-bound Src were exposed to 10 μM ouabain and Src kinase activity was determined. In other experiments, the Src pY418 was measured by anti-pY418 antibody to indicate Src activation (Ma et al., 2000
). To do so, the purified Src (4.5 U) was incubated with different amount of the purified Na+
-ATPase or GST-Na+
-ATPase constructs in phosphate-buffered saline (PBS) for 30 min at 37°C. Afterward, 2 mM ATP/Mg2+
was added. The reaction continued for 5 min at 37°C and was stopped by addition of SDS sample buffer.
In Vitro Binding Assay
The purified Na+/K+-ATPase was solubilized in 1% Triton X-100 PBS and centrifuged at 100,000 × g for 30 min. The supernatant was collected for the binding assay. GST-fusion proteins (5 μg) were conjugated on glutathione beads and incubated with the solubilized Na+/K+-ATPase in 500 μl PBS in the presence of 0.5% Triton X-100 at room temperature for 30 min. The beads were washed with the same buffer for four times. The bound Na+/K+-ATPase was resolved on 10% SDS-PAGE and detected by Western blot. Reciprocal binding assay using GST-Na+/K+-ATPase constructs (5 μg) and purified Src lacking of first 85 amino acids (200 ng) or His-tagged Src constructs (100 ng) was done similarly. To test if native Na+/K+-ATPase binds Src, the above experiments were repeated in the absence of Triton X-100. To make the Na+/K+-ATPase/Src complex, 2-5 μg of the purified Na+/K+-ATPase was incubated with 4.5 U of Src (~10 ng) in PBS in the absence of Triton X-100 at room temperature for 30 min. The complex was either used for the experiments directly as indicated or collected by centrifugation at 100,000 × g for 30 min. Control experiments showed that the Na+/K+-ATPase-bound, but not the free, Src could be copelleted by the centrifugation.
FRET Analysis by Acceptor Photobleaching
Using pECFP-N1 and pEYFP-C1 vectors described above, the enhanced cyan fluorescent protein (ECFP) was fused to the C-terminus of Src, and the enhanced yellow fluorescent protein (EYFP) was fused to the N-terminus of rat Na+/K+-ATPase α 1 subunit. Src-ECFP and EYFP-rat α1 plasmids were then cotransfected into LLC-PK1 cells. Cells transfected with either ECFP/EYFP or ECFP/EYFP-rat α1 were used as a control. After 24 h, cells growing on glass coverslip were fixed with ice-cold methanol for 15 min at -20°C and washed twice with PBS solution. The coverslip was then used for FRET measurement with Leica DMIRE2 confocal microscope (Wetzlar, Germany). The laser lines of 456 nm and 515 nm were used to illuminate fluorescence, and the emission intensities were recorded at 465-509 nm for Src-ECFP and 530-570 nm for EYFP-rat α1. The cell that expresses both Src-ECFP and EYFP-rat α1 was chosen to perform the FRET analysis. A membrane region of interest (ROI 1) was selected and photobleached by applying 100% intensity of 515-nm laser. The emission intensities of Src-ECFP and EYFP-rat α1 before and after the photobleaching process in the selected ROI 1 region were used to calculate the FRET efficiency. The FRET efficiency was also calculated at a nonphotobleached region (ROI 2) and used as a control.
FRET Analysis in Live Cells
LLC-PK1 cells were cotransfected with Src-ECFP and EYFP-rat α1 and grown on a glass coverslip for 24 h. The coverslip was then mounted in a metal chamber and analyzed with a Leica DMIRE2 confocal microscope. The laser lines of 456 nm and 515 nm were used to illuminate fluorescence, and the emission intensities were recorded at 465-509 nm for Src-ECFP and 530-570 nm for EYFP-rat α1. The cell that expresses both Src-ECFP and EYFP-rat α1 was chosen and illuminated by only 456-nm laser. The cells express only Src-ECFP or EYFP-rat α1 were used for correction and determination of the laser intensity as well as the gain and offset settings. The emission intensities for both Src-ECFP and EYFP-rat α1 in selected membrane region was recorded at 465-509 nm (FECFP) and 530-570 nm (FEYFP), respectively. The FRET efficiency was reflected by the ratio of FEYFP/FECFP. After 50 s of recording, the same cell was exposed to ouabain and the recording was continued for indicated time.
BRET assay was done as described by Lowry et al.
). Briefly, 24 h after transfection with GFP-Na+
-ATPase and Src-Rluc or other constructs as indicated, cells were seeded in triplicate in a 96-well microplate. After treatment with indicated concentration of ouabain, cells were exposed to equal volume of BRET analysis buffer containing 10 μM DeepBlue C, the substrate of Rluc. The emission at 410 nm (for Rluc) and 515 nm (for GFP) was immediately acquired using a Fluoroskan Ascent FL (Labsystems, Franklin, MA) with microplate luminometric detection. The BRET ratio was calculated as follows: (Emission at 515 nm - Background at 515 nm)/(Emission at 410 nm - Background at 410 nm), where Background signal was assessed in each experiment by measuring the signal of a sample of nontransfected cells.
LLC-PK1 cells were cultured for 24 h on glass coverslips, briefly washed twice with PBS, and then fixed with ice-cold methanol for 15 min. The cells were washed again with PBS and blocked using SignalEnhancer (Molecular Probes). Rabbit polyclonal anti-Src antibody and monoclonal anti-Na+/K+-ATPase antibody were mixed in 3% BSA and incubated with the coverslip overnight at 4°C. After three washes with PBS, Alexa fluor 546-conjugated anti-mouse antibody and Alexa fluor 488-conjugated anti-rabbit antibody were added and incubated for 1 h at room temperature. The coverslip was washed again with PBS for three times. The Na+/K+-ATPase was visualized by excitation at 546 nm and emission at 566-620 nm. Src was visualized by excitation at 488 nm and emission at 505-535 nm. To avoid the crosstalk between the two fluorescence dyes, we used sequential method featured by Leica confocal microscope to measure colocalization of the two proteins, in which, the two laser lines 488 nm and 546 nm were applied to the cells alternatively. Colocalization analysis was performed with Leica Confocal Software, version 2.5 build 1347.
Data are given as mean ± SE. Statistical analysis was performed using the Student's t test, and significance was accepted at p < 0.05.