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 PTPM EG and an inactive mutant form in which the active site cysteine is mu tated to serine (PTPMEGCS). We found that both endogenous and recombin ant enzyme were primarily located in the membrane and cytoskeletal fra ctions of COS-7 cells. Endogenous PTPMEG accounts far only 1/3000th of the total tyrosine phosphatase activity in COS-7 cells and transfecte d cells expressed 2- to 7-fold higher levels of the enzyme. These leve ls of overexpression did not result in detectable changes in either to tal tyrosine phosphatase activity or the state of protein tyrosine pho sphorylation as determined by immunoblotting of cell homogenates with anti-phosphotyrosine antibodies. Despite the low levels of activity fo r PTPMEG, we found that overexpressing cells grew slower and reached c onfluence at a lower density than vector transfected cells. Surprising ly, PTPMEGCS-transfected cells also reach confluence at a lower densit y than vector-transfected cells, although they grow to higher density than PTPMEG-transfected cells. Both constructs inhibited the ability o f COS-7 cells to form colonies in soft agar, with the native PTPMEG ha ving a greater effect (30-fold) than PTPMEGCS (10-fold). These results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit cell pr oliferation, reduce the saturation density, and block the ability of t hese cells to grow without adhering to a solid matrix.