Tissue-specific kinase expression and activity regulate flux through the pyruvate dehydrogenase complex

Alla Klyuyeva,Alina Tuganova,Natalia Kedishvili,Kirill M Popov

doi:10.1074/jbc.ra118.006433

Abstract

The pyruvate dehydrogenase complex (PDC) is a multienzyme assembly that converts pyruvate to acetyl-CoA. As pyruvate and acetyl-CoA play central roles in cellular metabolism, understanding PDC regulation is pivotal to understanding the larger metabolic network. The activity of mammalian PDC is regulated through reversible phosphorylation governed by at least four isozymes of pyruvate dehydrogenase kinase (PDK). Deciphering which kinase regulates PDC in organisms at specific times or places has been challenging. In this study, we analyzed mouse strains carrying targeted mutations of individual isozymes to explore their role in regulating PDC activity. Analysis of protein content of PDK isozymes in major metabolic tissues revealed that PDK1 and PDK2 were ubiquitously expressed, whereas PDK3 and PDK4 displayed a rather limited tissue distribution. Measurement of kinase activity showed that PDK1 is the principal isozyme regulating hepatic PDC. PDK2 was largely responsible for inactivation of PDC in tissues of muscle origin and brown adipose tissue (BAT). PDK3 was the principal kinase regulating pyruvate dehydrogenase activity in kidney and brain. In a well-fed state, the tissue levels of PDK4 protein were fairly low. In most tissues tested, PDK4 ablation had little effect on the overall rates of inactivation of PDC in kinase reaction. Taken together, these data strongly suggest that the activity of PDC is regulated by different isozymes in different tissues. Furthermore, it appears that the overall flux through PDC in a given tissue largely reflects the properties of the PDK isozyme that is principally responsible for the regulation of PDC activity in that tissue.

Highlights

The pyruvate dehydrogenase complex (PDC) is a multienzyme assembly that converts pyruvate to acetyl-CoA
This paper reports on the expression patterns of the protein products of all four genes encoding pyruvate dehydrogenase kinase (PDK) isozymes, as well as on their contributions to the overall rate of kinase reaction in the major metabolic tissues obtained from mice maintained in a well-fed state
Analysis of the corresponding mouse strains carried out in this study revealed that, unlike PDK1 and PDK2, PDK3 displays a rather limited tissue distribution

Summary

Results

Conclusions about the contributions of different PDK isozymes to the regulation of PDC activity and metabolism, in general, were often based on the analysis of the abundance of their mRNAs by Northern blotting or RT-PCR [28, 29]. With appropriate negative controls, we were able to reliably identify PDK4 protein in whole-tissue extracts Systematic application of this approach revealed that in mouse tissues, PDK isozymes display markedly different expression patterns (Fig. 1 and Fig. S1). Some of these patterns differed significantly from the expression patterns identified previously based on the analysis of corresponding mRNAs [28].

34 Ϯ 1 39 Ϯ 1

Discussion

Experimental procedures