Disruption of Fyn SH3 Domain Interaction with a Proline-Rich Motif in Liver Kinase B1 Results in Activation of AMP-Activated Protein Kinase

Eijiro Yamada,Claire C Bastie,Harpal Singh Randeva

doi:10.1371/journal.pone.0089604

Eijiro Yamada, Claire C Bastie + Show 1 more

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https://doi.org/10.1371/journal.pone.0089604

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Abstract

Fyn-deficient mice display increased AMP-activated Protein Kinase (AMPK) activity as a result of Fyn-dependent regulation of Liver Kinase B1 (LKB1) in skeletal muscle. Mutation of Fyn-specific tyrosine sites in LKB1 results in LKB1 export into the cytoplasm and increased AMPK activation site phosphorylation. This study characterizes the structural elements responsible for the physical interaction between Fyn and LKB1. Effects of point mutations in the Fyn SH2/SH3 domains and in the LKB1 proline-rich motif on 1) Fyn and LKB1 binding, 2) LKB1 subcellular localization and 3) AMPK phosphorylation were investigated in C2C12 muscle cells. Additionally, novel LKB1 proline-rich motif mimicking cell permeable peptides were generated to disrupt Fyn/LKB1 binding and investigate the consequences on AMPK activity in both C2C12 cells and mouse skeletal muscle. Mutation of either Fyn SH3 domain or the proline-rich motif of LKB1 resulted in the disruption of Fyn/LKB1 binding, re-localization of 70% of LKB1 signal in the cytoplasm and a 2-fold increase in AMPK phosphorylation. In vivo disruption of the Fyn/LKB1 interaction using LKB1 proline-rich motif mimicking cell permeable peptides recapitulated Fyn pharmacological inhibition. We have pinpointed the structural elements within Fyn and LKB1 that are responsible for their binding, demonstrating the functionality of this interaction in regulating AMPK activity.

Highlights

In the adult, glucose, fatty acid and protein metabolism must be precisely maintained to insure energy homeostasis and dysregulation of these processes results in states of weight loss or weight gain
calmodulin-dependent protein Kinase Kinase (CaMKK) have been shown to activate AMPK in the skeletal muscle under mild tetanic contraction [12], CaMKK expression is very low outside the central nervous system [13] and it is considered that Liver Kinase B1 (LKB1) is the main enzyme that regulates AMPK activity in peripheral tissues
To determine whether LKB1 directly binds to Fyn SH3 domain, we generated single point mutations in the SH3 (W119A) domain and, as a control, the SH2 (R176K) domain of Fyn kinase (Fig. S1A)

Summary

Introduction

Glucose, fatty acid and protein metabolism must be precisely maintained to insure energy homeostasis and dysregulation of these processes results in states of weight loss or weight gain. Based upon analyses of conventional Fyn knockout mice, it was observed that Fyn tyrosine kinase regulates energy expenditure and fatty acid oxidation via the increased activation of AMPK in skeletal muscle and adipose tissue [16]. This occurred through a direct Fynmediated tyrosine phosphorylation of LKB1 on Y261 and Y365. We identified the structural elements within Fyn and LKB1 that are responsible for their molecular interaction Interruption of this binding using a LKB1 prolinerich domain mimetic peptide recapitulated the pharmacological effects of Fyn kinase inhibition, thereby demonstrating the specificity and functional role of this interaction in mediating AMPK activation. Considering that AMPK dysregulation is observed in several metabolic disorders, this mechanistic analysis opens up a novel possibility of therapy for the treatment of diseases of the metabolic syndrome

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