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Rat osteoblasts and ROS 17/2.8 cells contain a similar protein tyrosine phosphatase
L Titus1, L G Marzilli, J Rubin
1Department of Medicine, VA Medical Center, Decatur, GA 30033.
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 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.
Insights
This study characterizes two phosphotyrosine phosphatase (PTPase) activities in osteoblast cells. These enzymes are crucial for regulating cell signaling, and understanding their function is key to studying bone cell proliferation and function.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Tyrosine phosphorylation is a critical mechanism in intracellular signaling pathways regulated by hormones and growth factors.
- Phosphotyrosine phosphatases (PTPases) are essential enzymes that terminate these signals through dephosphorylation of target proteins.
- Understanding PTPase activity is vital for comprehending osteoblast proliferation and function.
Purpose of the Study:
- To chromatographically distinguish and characterize soluble protein PTPases from neonatal rat osteoblasts (ROBs) and rat osteosarcoma (ROS 17/2.8) cells.
- To investigate the properties and potential roles of different PTPase activities in osteoblast function.
- To assess the utility of ROS 17/2.8 cells as a model for studying PTPase regulation.
Main Methods:
- Soluble protein extracts from ROBs and ROS 17/2.8 cells were analyzed using phosphocellulose and QAE-trisacryl anion exchange chromatography.
- 32P-labelled glutamate/tyrosine co-polymer was used as a substrate to assay PTPase activity.
- Enzyme kinetics (apparent Km) and inhibition profiles (Na3VO4, ZnCl2, CoCl2, NaF) were determined for characterized PTPase activities.
Main Results:
- Two distinct PTPase activities (E1 and E2) were separated from both cell types.
- Activity E1, the dominant form, exhibited specific kinetic properties and was enhanced by NaCl, while E2 had different chromatographic behavior and kinetics.
- Both activities were inhibited by Na3VO4, ZnCl2, and CoCl2, with E2 being more sensitive to ZnCl2. Co2+ emerged as a potential tool for further research.
Conclusions:
- The characterization of two distinct PTPase activities provides insights into the enzymatic machinery regulating phosphotyrosine signaling in osteoblasts.
- The observed similarities between E1 activity in ROS cells and ROBs suggest that ROS cells are a valuable model for studying PTPase regulation.
- Further molecular studies are necessary to definitively identify the specific PTPases present in ROBs and ROS cells and elucidate their precise roles.