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Characterization of a membrane tyrosine phosphatase in AR42J cells: regulation by somatostatin
N Tahiri-Jouti1, C Cambillau, N Viguerie
1Institut National de la Santé de la Recherche Médicale U 151, CHU Rangueil L3, Toulouse, France.
Abstract:
A phosphotyrosyl protein phosphatase (PTPase) activity has been characterized in the plasma membranes of confluent AR42J pancreatic tumor cells using 32P-labeled poly(Glu, Tyr) as substrate. Membrane PTPase activity exhibited an apparent Michaelis constant of 3 microM and an apparent maximal velocity of 0.9 nmol.min-1.mg-1. It was inhibited by orthovanadate, zinc, poly(Glu,Tyr) and was stimulated by EDTA and dithiothreitol. Gel filtration of solubilized plasma membranes gave a peak of enzyme activity at a relative molecular weight of 70,000. Plasma membrane PTPase activity was changed during AR42J cell growth. At the beginning of culture, the control PTPase activity was minimal. Over the 5 days of culture, PTPase activity increased to reach a maximum (3.5-fold over control activity) preceding confluency by 2 days. Then the high level of PTPase activity was sustained until confluency. Incubation of the cells with the stable somatostatin analogue SMS 201-995 (SMS) resulted in a rapid and transient activation of crude membrane PTPase activity. Activation reached a maximum level within 5 min of addition and return to control levels within 20 min. The effect of SMS was dose dependent with half-maximal and maximal activation occurring at 6 pM and 0.1 nM SMS respectively.
Insights
This study characterizes phosphotyrosyl protein phosphatase (PTPase) activity in AR42J pancreatic tumor cells. Somatostatin analogue SMS 201-995 rapidly activates PTPase, with activity peaking before cell confluency.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Phosphotyrosyl protein phosphatases (PTPases) play crucial roles in cellular signaling pathways.
- Dysregulation of PTPase activity is implicated in various diseases, including cancer.
- AR42J cells are a well-established model for pancreatic tumor research.
Purpose of the Study:
- To characterize the kinetic and regulatory properties of plasma membrane PTPase activity in AR42J pancreatic tumor cells.
- To investigate the modulation of PTPase activity during AR42J cell growth and confluency.
- To examine the effect of the somatostatin analogue SMS 201-995 on PTPase activity.
Main Methods:
- Enzyme kinetic assays using 32P-labeled poly(Glu, Tyr) as substrate.
- Characterization of PTPase inhibitors and activators (orthovanadate, zinc, EDTA, dithiothreitol).
- Gel filtration chromatography to determine enzyme molecular weight.
- Time-course and dose-response studies of SMS 201-995 treatment on AR42J cells.
Main Results:
- Plasma membrane PTPase activity exhibited Michaelis-Menten kinetics with specific inhibition and activation profiles.
- PTPase activity significantly increased during AR42J cell culture, peaking before confluency.
- SMS 201-995 induced rapid, transient, and dose-dependent activation of PTPase activity.
Conclusions:
- AR42J cell plasma membranes possess characterized PTPase activity modulated by cell growth.
- Somatostatin analogue SMS 201-995 acutely regulates PTPase activity in these pancreatic tumor cells.
- These findings contribute to understanding PTPase roles in pancreatic cancer cell biology and potential therapeutic targeting.