Rat osteoblasts and ROS 17/2.8 cells contain a similar protein tyrosine phosphatase

Abstract

Tyrosine phosphorylation plays a central role in intracellular signaling by many hormones and growth factors. Termination of the signal is thought to involve dephosphorylation of target proteins by phosphotyrosine phosphatases (PTPase). Soluble protein PTPases from neonatal rat osteoblasts (ROBs) and rat osteosarcoma (ROS 17/2.8) cells were chromato-graphically distinguished and characterized using 32P-labelled glutamate/tyrosine co-polymer as substrate. Two activities from both cell types were chromatographically separable. The dominant PTPase activity in the presence of 60–125 mM salt (E1), was eluted from phosphocellulose by 180–280 mM NaCl, bound weakly to a strong anion exchange column (QAE-trisacryl), had an apparent K m for [ 32P]glutamate/tyrosine copolymer of 52 μg/ml, was enhanced (5–10-fold, ROS; 1.5–3-fold, ROB) by assay in 125 mM NaCl, had no significant alkaline, acid, or serine phosphatase activity and had an M r of 53 000. A second activity (E2) was not retained by phosphocellulose but eluted from QAE-trisacryl in a single peak at 90–130 mM NaCl. It had an apparent K m for [ 32P]glutamate/tyrosine copolymer of 30 μg/ml (ROS) and its activity was not enhanced by NaCl in the assay. Activity El from both cells was 50% inhibited by 0.05 μM Na 3VO 4, 20 μM ZnCl 2, or 5–10 μM CoCl 2, but not by 1 mM NaF; activity E2 had a similar inhibition profile, but was more sensitive to ZnCl 2 (IC 50, 5 μM). Co 2+ is a relatively non-toxic metal which may be a useful tool for investigating the role of phosphotyrosine in osteoblast proliferation and function. The similarity between the El activity from ROS cells and ROBs suggests that ROS cells may be useful in studying PTPase regulation by hormones, but molecular approaches will be required to establish the identity of PTPases in ROBs and ROS cells.