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Genetic evidence for Pak1 autoinhibition and its release by Cdc42
Abstract:
Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.
Insights
Pak1 protein kinase is regulated by an internal interaction that keeps it inactive. Cdc42 binding disrupts this interaction, activating Pak1 for cellular processes like differentiation.
Area of Science:
- Molecular biology
- Cell signaling
- Yeast genetics
Background:
- Pak1 protein kinase in Schizosaccharomyces pombe is a member of the p21-GTPase-activated protein kinase (PAK) family.
- PAK proteins are involved in crucial cellular processes such as sexual differentiation and morphogenesis.
- The regulatory domain of PAKs is believed to autoinhibit the kinase catalytic domain, as its removal increases kinase activity.
Purpose of the Study:
- To investigate the intramolecular interaction between the regulatory and catalytic domains of Pak1.
- To elucidate the mechanism by which Cdc42 activates Pak1.
Main Methods:
- Yeast two-hybrid system to detect protein interactions.
- Mutant analysis to assess protein function.
- Genetic analysis in Schizosaccharomyces pombe.
Main Results:
- The Pak1 regulatory domain directly interacts with its kinase catalytic domain.
- This interaction occurs at the same conserved region that binds the activating GTPase, Cdc42.
- Evidence suggests this intramolecular interaction maintains Pak1 in an inactive, closed conformation.
- Cdc42 binding was shown to induce an open, active conformation of Pak1.
Conclusions:
- Pak1 undergoes autoinhibition through an intramolecular interaction between its regulatory and catalytic domains.
- Cdc42 binding disrupts these autoinhibitory interactions, leading to Pak1 activation.
- This mechanism explains how Cdc42 regulates Pak1 activity in cellular signaling pathways.