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Correspondence to: Cesario Bianchi, Dept. of Surgery/Div. of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave. #DA-853, Boston, MA 02215. E-mail: cbianchi@caregroup.harvard.edu
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Summary |
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Tyrosine phosphorylation is an important post-translational modification of proteins, essential in many aspects of the cell economy, particularly in signal transduction pathways. Despite the importance of protein tyrosine phosphorylation, the approaches available for molecular cloning remain limited. We have developed a COS cell-based eukaryotic expression cloning procedure for phosphotyrosine-containing proteins by immunocytochemistry of cell monolayers. The approach takes advantage of the low basal levels of tyrosine phosphorylated, robust transient expression, availability of specific antibodies against tyrosine-phosphorylated residues, and rescue of episomal DNA after immunocytochemistry. The technique is validated by cloning the rat proto-oncogene c-fgr in its tyrosine-phosphorylated form out of a rat kidney cDNA library containing over 106 primary recombinants. This technique set the grounds for expression cloning of tyrosine-phosphorylated proteins in eukaryotic cells, and it is anticipated that further modifications and refinements will allow the identification of protein tyrosine phosphatase substrates. (J Histochem Cytochem 48:10971101, 2000)
Key Words: molecular cloning, COS, tyrosine phosphorylation, kinases, phosphatases, expression cloning, immunocytochemistry
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Introduction |
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PROTEIN TYROSINE PHOSPHORYLATION has been implicated in a myriad of physiological and pathological processes, including embryogenesis, cell differentiation, and cell transformation (for review see GT-11 libraries (
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Materials and Methods |
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cDNA Library Construction
An oligo-dT-primed cDNA library was constructed from kidneys of SpragueDawley rats' poly-A+ RNA and was ligated into the BstX I cloning sites of the pcDNAI vector (Invitrogen; Carlsbad, CA). The library was size-fractionated for inserts larger than 1.5 kb and consisted of 3 x 106 primary cDNA recombinants (Invitrogen).
Tissue Culture Techniques
COS MT3 cells that inducibly express the large T-antigen from the metallothionein promoter (
Transient Transfection
COS MT3 cells at 5080% confluence were transfected using DEAEdextran as described previously (
COS Cell Screening by Immunocytochemistry
The transfected Petri dishes were treated for 15 min with 1 mM Na3VO4 at 37C, followed by two washes in 5 ml of PBS, and were fixed in absolute ethanol (-20C) for 10 min. After rehydration in PBS (5 min each), the cells were incubated with the anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology; Lake Placid, NY) at a dilution of 1:5000. Cells were then washed three times in PBS (5 min) and incubated with a donkey anti-mouse antibody coupled to HRP at 1:200 dilution for 30 min. After an additional three washes in PBS, a colorimetric reaction was obtained with 4-chloro-1-naphthol and N,N-dimethyl-p-phenylenediamine monohydrochloride for 1015 min as described (
Visualization of Positive Cells and Plasmid Rescue
The dishes were searched at a low magnification (x40) for positive cells (dark blue) under a dissecting microscope (Leica Microsystems; Deerfield, IL). Nineteen COS cells were judged positive and subjected to plasmid rescue as follows. Dishes were allowed to dry for 20 min at RT and the putative positive cells were encircled (~0.5 cm diameter) by marking the bottom of the Petri dish with a diamond knife, and were processed to the Hirt preparation (
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Characterization of cDNA
Double-stranded DNA was prepared using a commercially available kit (Qiagen; Santa Clara, CA) and was sequenced by the chain termination reaction according to the manufacturer's instructions for the Sequenase version 1.0 kit (United States Biochemicals; Cleveland, OH). In addition, the cDNA was restriction-mapped with the enzymes Aha 2, HinP 1, Mse I, Pst I, and Stu I (New England Biolabs; Beverly, MA) for further characterization.
Immunoblotting
COS cells transfected with the cloned cDNA (6-cm diameter; 6-well Petri dishes) were solubilized in 500 µl of 1 x Laemmli buffer (62.5 mM Tris-HCl, 2% SDS, 1% ß-mercaptoethanol, 10% glycerol, and 0.0025% bromophenol blue) boiled for 5 min, and an aliquot of 10 µl was subjected to 10% SDS-PAGE using a Bio-Rad Miniprotean II apparatus. Proteins were transferred to polyvinylidene fluoride membranes in a semidry transfer apparatus (Millipore; Bedford, MA). Nonspecific binding was blocked with 3% bovine serum albumin in 50 mM Tris-HCl, 100 mM NaCl, and 0.05% Tween-20, pH 8.0. The blots were then incubated with either the anti-phosphotyrosine antibody or an anti-c-fgr antibody (1:2000) (Cambridge Research Biochemicals; Wilmington, DE). After washing three times (5 min per wash) with the same buffer, the membranes were incubated with either donkey anti-mouse antibody conjugated with HRP at 1:5000 dilution for phosphotyrosine detection or with donkey anti-sheepHRP (1:4000) for c-fgr. Unbound antibodies were removed by washing as above and detection was obtained by enhanced chemiluminescence with an ECL kit (Amersham Pharmacia Biotech; Piscataway, NJ) and exposure to X-ray film.
Immunoprecipitation
COS cells were lysed in 1 ml immunoprecipitation buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% Nonidet P-40, 0.05% Tween-20, 10 µM phenylmethylsulfonyl fluoride, and 10 mM NaF, pH 8.0) for 10 min on ice. The lysate was vortexed three times (10 sec each) and was cleared by a brief centrifugation. After incubation with 50 µl of agarose-coupled anti-phosphotyrosine antibody for 16 hr at 4C, the agarose beads were sedimented by brief centrifugation and washed by resuspending and pelleting three times with 1 ml of immunoprecipitation buffer. One hundred µl of 2 x Laemmli buffer was added to the agarose pellet and boiled for 5 min. Ten µl was fractionated on a 10% SDS-PAGE and processed for immunoblotting using the anti c-fgr antibody as described above.
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Results |
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The modifications introduced into the transfection method, i.e., the use of Serum Plus-supplemented medium instead of regular bovine serum, trypsinization (
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Sequence Analysis of the Cloned cDNA
The cDNA clone had an insert of 1.9 kb. Its nucleotide sequences from over 350 bp at either the 5' and 3' flanking regions reveal that the cDNA encodes the rat c-fgr tyrosine kinase (accession number X57018.1). Restriction mapping confirmed the presence of restriction sites and fragment sizes for Aha 2, Pst I, Mse I, Hinp I, and Stu I, as predicted from the restriction mapping of the rat c-fgr sequence (accession number X57018.1).
Immunoblotting and Immunoprecipitation
Lysates of COS cells untransfected and transfected with the cloned cDNA were subjected to immunoblotting. In transfected cells only one major tyrosine-phosphorylated band of 55 kD was detected, confirming that the cloned cDNA increases tyrosine phosphorylation in COS cells (Fig 3) and that the 55-kD protein corresponds to a band of the same molecular weight detected with the anti-c-fgr antibody (Fig 3), indicating that the tyrosine kinase is autophosphorylated.
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Immunoprecipitation with phosphotyrosine antibodies and blotting confirmed that the major tyrosine-phosphorylated protein is the 55-kD c-fgr (Fig 3).
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Discussion |
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The COS cell system is probably the most popular transient eukaryote expression system allowing rapid characterization of new clones. As a cloning approach, it was first used to clone GM-CSF by a functional assay in polls of transfected COS cells (
Compared to the approaches described before for affinity purification (GT11 (
The technique described here, despite its flexibility, has the disadvantage of not having been standardized as have prokaryote expression cloning and yeast interaction trapping cloning, for both of which kits are available. However, the COS cell expression cloning has proved useful in situations where more classical cloning techniques may have failed, e.g., the Type 1 angiotensin II receptor (
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Acknowledgments |
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We wish to thank Drs Charles Homcy and Thomas Quertermous for encouragement and support.
Received for publication February 21, 2000; accepted March 29, 2000.
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