Inhibition of protein tyrosine phosphatase activity in HER14 cells by melittin and Ca 2+ ionophore A23187

Abstract

We investigated the effect of melittin and Ca 2+ ionophore A23187 on protein tyrosine phosphatase activity in HER14 cells (NIH-3T3 cells transfected with human epidermal growth factor ‘EGF’ receptor). Cell fractions were used to measure protein tyrosine phosphatase activity in vitro using 32P-labeled poly(Glu/Tyr) (4 : 1) peptide as a substrate. Treatment of HER14 cells with melittin or with A23187, inhibited protein tyrosine phosphatase activity in the cell sonicate and homogenate, as well as in cytosolic and particulate fractions of these cells. The inhibitory effect of both drugs was prevented by preincubating cells with EGTA (ethyleneglycolbis( β-aminoethyl ether)- N, N, N′, N′-tetracetic acid). The cyclooxygenase inhibitor indomethacin enhanced the inhibitory effect of A23187, but not that of melittin. Addition of arachidonic acid to the cells partially prevented the inhibition of protein tyrosine phosphatase activity by melittin or A23187. Preexposure of cells to EGF enhanced the inhibitory effect of melittin - but not that of A23187. Addition of CaCl 2, or MgCl 2 to the cell homogenate inhibited protein tyrosine phosphatase activity. These results show that protein tyrosine phosphatase activity in HER14 cells is inhibited by melittin and Ca 2+ ionophore A23187 through a Ca 2+-dependent mechanism, and is regulated by arachidonic acid metabolism and EGF receptor activation.