Rat osteoblasts and ROS 17/2.8 cells contain a similar protein tyrosine phosphatase.
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 chromatographically 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 Km for [32P]glutamate/tyrosine copolymer of 52 micrograms/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 Km for [32P]glutamate/tyrosine copolymer of 30 micrograms/ml (ROS) and its activity was not enhanced by NaCl in the assay. Activity E1 from both cells was 50% inhibited by 0.05 microM Na3VO4, 20 microM ZnCl2, or 5-10 microM CoCl2, but not by 1 mM NaF; activity E2 had a similar inhibition profile, but was more sensitive to ZnCl2 (IC50, 5 microM). Co2+ 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 E1 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.
Titus
L
L
Department of Medicine, VA Medical Center, Decatur, GA 30033.
Marzilli
L G
LG
Rubin
J
J
Nanes
M S
MS
Catherwood
B D
BD
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010006:Osteoblasts / Q000201:enzymology*; D012516:Osteosarcoma / Q000201:enzymology*; D010766:Phosphorylation; D011108:Polymers; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D005971:Glutamates; D011108:Polymers; D018698:Glutamic Acid; D014443:Tyrosine; D012965:Sodium Chloride; D017027:Protein Tyrosine Phosphatases