A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism.

Angel Loza-Valdes,Filip Dziaczkowski,Toufic Kassouf,Andreas Schlosser,Jonathan Trujillo-Viera,Michael Leitges,Grzegorz Sumara,Alexander E Mayer,Werner Schmitz

doi:10.26508/lsa.202000863

Abstract

Members of the protein kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver of animals fed a high-fat diet. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified more than 300 putative targets of PKD3. Furthermore, biochemical analysis revealed that PKD3 regulates cAMP-dependent PKA activity, a master regulator of the hepatic response to glucagon and fasting. PKA regulates glucose, lipid, and amino acid metabolism in the liver, by targeting key enzymes in the respective processes. Among them the PKA targets phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine. Consistently, we showed that PKD3 is activated by glucagon and promotes glucose and tyrosine levels in hepatocytes. Therefore, our data indicate that PKD3 might play a role in the hepatic response to glucagon.

Highlights

Previous research has delineated the role of PKD3 in the regulation of hepatic glucose and lipid metabolism in mice fed a high-fat diet (Mayer et al, 2019)
Phospho(Ser/Thr) Protein kinase D (PKD) substrate LxRxx[S*/T*] antibody was used for immunoprecipitation to enrich proteins that have a phosphorylated PKD motif in lysates from primary hepatocytes deficient for PKD3 expressing either EGFP control or PKD3ca (Fig 1A)
The protein with highest enrichment induced by PKD3ca was PKD3 itself, suggesting that PKD3 might be subjected to autophosphorylation

Summary

Introduction

Protein kinase D (PKD) family members integrate multiple hormonal and metabolic signals to coordinate homeostasis of the organism (Sumara et al, 2009; Rozengurt, 2011; Loffler et al, 2018; Mayer et al, 2019; Kolczynska et al, 2020; Trujillo-Viera et al, 2021). PKD3 plays a role in a wide range of cellular processes in both physiological and pathological conditions

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Conclusion