THE ROLE AND REGULATION OF PROTEIN TYROSINE PHOSPHATASES IN CELL GROWTH AND PROLIFERATION

Abstract

The role and regulation of protein tyrosine phosphatases in cell growth and proliferation was studied in growing and EGF-treated quiescent Swiss 3T3 fibroblasts. Exponentially growing fibroblasts have a relatively low level of membrane phosphatase activity, which rises only as the rate of cell proliferation decreases and is maximal when cell growth is contact inhibited. At saturation density, the membrane tyrosine phosphatase specific activity is on average 8-told higher than that of cells at lower cell densities. This Increase in membrane tyrosine phosphatase activity is associated with density-dependent growth arrest and not with cellular quiescence in general, as no increase in phosphatase specific activity is detected when noncontact- inhibited cells are induced to arrest their growth through serum deprivation. The observed alterations in specific activity are attributable to a tyrosine phosphatase of M, 37,000 that was partially purified and characterized from solubilized membrane fractions of Swiss 3T3 cells. When quiescent Swiss 3T3 fibroblasts were stimulated with EGF, there was a rapid decrease in cytosolic tyrosine phosphatase specific activity. On successive chromatographies, this cytosolic tyrosine phosphatase activity was found to be a composite of two low molecular mass tyrosine phosphatases of 35 kDa (HS 1) and 50 kDa (HS2}. Treatment of A431 cells with EGF also resulted in an inhibition of cytosolic tyrosine phosphatase /specific activity. Two cytosolic tyrosine phosphatases that share similar molecular masses and biochemical properties (HS1 and HS2) appear to be the major target of regulation by EGF. The results suggest that early phosphorylation-dephosphorylation events of the signal transduction pathway mediated by EGF may be common to Swiss 3T3 and A431 cells. However, mitogenesis may involve downstream regulatory elements that are unique to each cell system. HS 1 and HS2 display different kinetics of regulation by EGF. Maximum inhibition of HS1 tyrosine phosphatase activity was observed after 10 minutes, and HS2 tyrosine phosphatase activity 30 minutes after treatment of the cells with the ligand. This suggests that HS1 may act upstream of HS2 in the EGF signal transduction pathway. Both HS1 and HS2 tyrosine phosphatases may function to maintain a low level of tyrosine phosphorylation in the cell under non-proliferating conditions. Inactivation of HS1 and HS2 following EGF stimulation may be necessary for the enhanced tyrosine phosphorylation of specific target proteins, thereby allowing proper transduction of the EGF signal. The results demonstrate that tyrosine phosphatases may have diverse roles in the regulation of cell growth. While the membrane-associated tyrosine phosphatase may have a potential role as a negative regulator in densitydependent growth, HS1 and HS2 tyrosine phosphatase activities may participate in maintenance of basal phosphorylation levels in nonproliferating cells.