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Insulin-induced p21ras activation does not require protein kinase C, but a protein sensitive to phenylarsine oxide
R H Medema1, B M Burgering, J L Bos
1Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
Abstract:
Insulin treatment of fibroblasts overexpressing the insulin receptor causes a rapid accumulation of the GTP-bound form of p21ras. We have studied the involvement of protein kinase C (PKC) in, and the effect of phenylarsine oxide (PAO), a putative inhibitor of tyrosine phosphatase activity on, this process. Activation of p21ras was not observed when the cells were stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and pretreatment with TPA for 16 h, sufficient to down-regulate PKC activity, did not abolish p21ras activation by insulin. These results show that PKC is not involved in the insulin-induced activation of p21ras. Pretreatment of the cells with PAO for 5 min completely blocked insulin-induced p21ras activation. Addition of 2,3-dimercaptopropanol prevented this inhibition by PAO. Also, addition of PAO after insulin stimulation could reverse the activation of p21ras. Since PAO did not affect overall phosphorylation of the insulin receptor beta-chain, we conclude that a PAO-sensitive protein is involved in the induction of p21ras activation by insulin.
Insights
Insulin rapidly activates p21ras in fibroblasts. Protein kinase C (PKC) is not involved, but a tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), blocks this insulin-induced p21ras activation, indicating a PAO-sensitive protein
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Biochemistry
Background:
- Insulin receptor signaling is crucial for cellular metabolism and growth.
- p21ras activation is a key event in insulin signal transduction.
- The precise mechanisms regulating insulin-induced p21ras activation require further elucidation.
Purpose of the Study:
- To investigate the role of Protein Kinase C (PKC) in insulin-induced p21ras activation.
- To determine the effect of phenylarsine oxide (PAO), a tyrosine phosphatase inhibitor, on this signaling pathway.
Main Methods:
- Fibroblasts overexpressing the insulin receptor were treated with insulin.
- Cells were pretreated with phorbol ester (TPA) to down-regulate PKC.
- Phenylarsine oxide (PAO) was used to inhibit tyrosine phosphatase activity.
- p21ras activation was assessed by measuring GTP-bound p21ras levels.
Main Results:
- Insulin treatment led to rapid accumulation of GTP-bound p21ras.
- PKC down-regulation by TPA did not affect insulin-induced p21ras activation.
- PAO pretreatment completely blocked insulin-induced p21ras activation.
- PAO's inhibitory effect was reversible with 2,3-dimercaptopropanol.
- PAO also reversed existing p21ras activation post-insulin stimulation.
- PAO did not alter insulin receptor beta-chain phosphorylation.
Conclusions:
- Protein Kinase C (PKC) is not involved in insulin-induced p21ras activation.
- A phenylarsine oxide (PAO)-sensitive protein, likely a tyrosine phosphatase, plays a critical role in mediating insulin's effect on p21ras activation.
- This suggests a novel regulatory step in insulin signaling downstream of the insulin receptor.