The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells.
We established stable COS-7 cell lines overexpressing recombinant PTPMEG and an inactive mutant form in which the active site cysteine is mutated to serine (PTPMEGCS). We found that both endogenous and recombinant enzyme were primarily located in the membrane and cytoskeletal fractions of COS-7 cells. Endogenous PTPMEG accounts for only 1/3000th of the total tyrosine phosphatase activity in COS-7 cells and transfected cells expressed 2- to 7-fold higher levels of the enzyme. These levels of overexpression did not result in detectable changes in either total tyrosine phosphatase activity or the state of protein tyrosine phosphorylation as determined by immunoblotting of cell homogenates with anti-phosphotyrosine antibodies. Despite the low levels of activity for PTPMEG, we found that overexpressing cells grew slower and reached confluence at a lower density than vector transfected cells. Surprisingly, PTPMEGCS-transfected cells also reach confluence at a lower density than vector-transfected cells, although they grow to higher density than PTPMEG-transfected cells. Both constructs inhibited the ability of COS-7 cells to form colonies in soft agar, with the native PTPMEG having a greater effect (30-fold) than PTPMEGCS (10-fold). These results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit cell proliferation, reduce the saturation density, and block the ability of these cells to grow without adhering to a solid matrix.
Division of Hematology, Washington University School of Medicine, St. Louis, MO 63110, USA.
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Majerus
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002448:Cell Adhesion; D002455:Cell Division; D002462:Cell Membrane / Q000201:enzymology; D002999:Clone Cells; D046508:Culture Techniques / Q000379:methods; D003553:Cystine; D003599:Cytoskeleton / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012694:Serine; D014162:Transfection
D011994:Recombinant Proteins; D012694:Serine; D003553:Cystine; D017027:Protein Tyrosine Phosphatases