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Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the
1Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA 52242, USA.
Abstract:
Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.
Insights
Insulin rapidly inactivates Rap1 GTPase, leading to Raf1 kinase activation. This process involves Ras pathway activation and CrkII-C3G complex dissociation, crucial for insulin signaling.
Area of Science:
- Molecular Cell Biology
- Signal Transduction
- Endocrinology
Background:
- Insulin receptor signaling is critical for glucose homeostasis and cellular metabolism.
- The Ras and Rap1 GTPases are key regulators of downstream signaling pathways.
- Understanding the interplay between these GTPases is essential for elucidating insulin action.
Purpose of the Study:
- To investigate the role of Rap1 and its interaction with Raf1 in insulin signaling.
- To elucidate the mechanism by which insulin regulates Rap1 activity.
- To determine the relationship between Rap1 inactivation, Ras activation, and downstream signaling.
Main Methods:
- Utilized Chinese hamster ovary cells expressing the human insulin receptor.
- Assessed GTP binding to Rap1 and protein-protein interactions (Rap1-Raf1, Raf1-Ras, CrkII-C3G) via biochemical assays.
- Employed in vitro assays with GST-C3G fusion proteins and site-directed mutagenesis (Tyr221Phe) to confirm mechanisms.
Main Results:
- Insulin stimulation caused a time-dependent decrease in Rap1-GTP, indicating Rap1 inactivation.
- Rap1 inactivation correlated with decreased Rap1-Raf1 binding and increased Raf1-Ras association.
- Insulin induced Ras-independent dissociation of the CrkII-C3G complex, reversible by dephosphorylation and mutation of Tyr221.
Conclusions:
- Insulin treatment de-represses Rap1's inhibitory function on Raf1 kinase.
- Insulin signaling involves concomitant Ras activation and stimulation of the Raf1/MEK/ERK cascade.
- The CrkII-C3G complex dissociation is a key Ras-independent event in insulin-stimulated pathways.