COS cells expression cloning of tyrosine-phosphorylated proteins by immunocytochemistry.
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 10(6) 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.
Bianchi
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Center for the Prevention of Cardiovascular Disease, Harvard School of Public Health, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA. cbianchi@caregroup.harvard.edu
Sellke
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D000818:Animals; D000918:Antibody Specificity; D019556:COS Cells; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015151:Immunoblotting; D007150:Immunohistochemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017422:Sequence Analysis, DNA; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D019061:src-Family Kinases
D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases