Evolutionarily conserved structural changes in phosphatidylinositol 5-phosphate 4-kinase (PI5P4K) isoforms are responsible for differences in enzyme activity and localization

Jonathan H Clarke,Robin F Irvine

doi:10.1042/bj20130488

Abstract

Mammals have genes coding for three PI5P4Ks (PtdIns5P 4-kinases), and these have different cellular localizations, tissue distributions and lipid kinase activities. We describe in the present paper a detailed molecular exploration of human PI5P4Ks α, β and γ, as well as their fly and worm homologues, to understand how and why these differences came to be. The intrinsic ATPase activities of the three isoforms are very similar, and we show that differences in their G-loop regions can account for much of their wide differences in lipid kinase activity. We have also undertaken an extensive in silico evolutionary study of the PI5P4K family, and show experimentally that the single PI5P4K homologues from Caenorhabditis elegans and Drosophila melanogaster are as widely different in activity as the most divergent mammalian isoforms. Finally we show that the close association of PI5P4Ks α and γ is a true heterodimerization, and not a higher oligomer association of homodimers. We reveal that structural modelling is consistent with this and with the apparently random heterodimerization that we had earlier observed between PI5P4Kα and PI5P4Kβ [Wang, Bond, Letcher, Richardson, Lilley, Irvine and Clarke (2010), Biochem. J. 430, 215–221]. Overall the molecular diversity of mammalian PI5P4Ks explains much of their properties and behaviour, but their physiological functionality remains elusive.

Highlights

phosphatidylinositol 5-phosphate 4-kinase (PI5P4K) (PtdIns5P 4-kinases, EC 2.7.1.149) phosphorylate PtdIns5P to PtdIns(4,5)P2 [1]
PI5P4Ks (PtdIns5P 4-kinases, EC 2.7.1.149) phosphorylate PtdIns5P to PtdIns(4,5)P2 [1]. This serves as another route of PtdIns(4,5)P2 synthesis, alternative to the 5-phosphorylation of PtdIns4P catalysed by the PI4P5Ks (PtdIns4P 5-kinases), the much lower cellular levels of PtdIns5P compared with PtdIns4P [2–4], plus the evidence for the major pathway of PtdIns(4,5)P2 synthesis in vivo being via the PI4P5K route [5,6], has led to a consensus that the most likely function of PI5P4Ks is to regulate the levels of their substrate PtdIns5P
It remains a possibility that a post-translational modification in eukaryotic cells has a major effect on one or more of the isoforms to make their activity levels more similar, but as precipitation of any endogenous or transfected PI5P4K from cell lines unavoidably results in co-precipitation of another isoform(s) because of their heterodimerization ([20,21] and see below), we cannot address this issue directly

Summary

Introduction

PI5P4Ks (PtdIns5P 4-kinases, EC 2.7.1.149) phosphorylate PtdIns5P to PtdIns(4,5)P2 [1]. Additional mutation of some residues where the PI5P4Kα and PI5P4Kβ isoforms are identical and different from PI5P4Kγ , and which are likely to interact with the PtdIns5P substrate ([19] and Figure 2A), further enhanced activity (Figure 2C).

Results

Conclusion