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Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42
M K Reeder1, I G Serebriiskii, E A Golemis
1Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Abstract:
p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.
Insights
Researchers developed novel p21-activated kinase 1 (Pak1) mutants in yeast to distinguish signaling pathways. This approach separates Pak1 activation by Cdc42 from Rac, revealing distinct upstream activators for different stimuli.
Area of Science:
- Cellular signaling
- Molecular biology
- Signal transduction pathways
Background:
- p21-activated kinase 1 (Pak1) is a key effector for small GTPases Cdc42 and Rac.
- Pak1's dual activation by Cdc42 and Rac complicates understanding upstream signaling pathways.
- Delineating specific pathways is crucial for understanding cellular responses to extracellular stimuli.
Purpose of the Study:
- To develop Pak1 mutants that selectively bind and are activated by Cdc42, but not Rac1.
- To differentiate the signaling pathways by which Cdc42 and Rac1 activate Pak1.
- To investigate the role of Cdc42 and Rac1 in Pak1 activation by specific extracellular stimuli like Ras, EGF, and calf serum.
Main Methods:
- Utilized a genetic screen in yeast to generate and identify Pak1 mutants with altered GTPase binding specificities.
- Created Pak1 mutants that selectively associate with Cdc42 but not Rac1.
- Tested the activation of these mutants in rat 3Y1 cells in response to oncogenic Ha-Ras, epidermal growth factor (EGF), and calf serum.
Main Results:
- Identified Pak1 mutants with selective Cdc42 coupling, with mutations mapping to and outside the p21 binding domain.
- Demonstrated that Cdc42 selectively activates Pak1 mutants unable to bind Rac1.
- Showed that oncogenic Ha-Ras and EGF activate Pak1 through a GTPase other than Cdc42, likely Rac1.
- Revealed that calf serum can activate Pak1 independently of Rac, suggesting alternative activation pathways.
Conclusions:
- Pak1 activation pathways are stimulus-dependent and involve distinct GTPase effectors.
- Ras and EGF signaling pathways converge on Rac1 for Pak1 activation.
- Calf serum activates Pak1 through a Rac-independent mechanism, highlighting pathway divergence.