Molecular insights into the membrane-associated phosphatidylinositol 4-kinase IIα

Abstract

Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KIIα in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KIIα with the ‘integral’ membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KIIα to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KIIα’s activity. We conclude from our results that PI4KIIα’s activity is regulated indirectly through changes in the membrane environment.

Puzzle out the regulation mechanism of PI4KIIα activity

“…In addition, PI4KII contains a '-CCPCC-' motif that is palmitoylated in vivo and such palmitoylation is important for the kinase activity of PI4KII. Thus currently structural information available on PI3Ks and the well-developed specific PI3K inhibitors are not helpful for understanding the substrate binding specificity and the activity regulation of PI4KIIs.Recently, the first crystal structure in the PI4K family, the catalytic domain of human PI4KII in an ADP-bound form was solved [2], and three novel insertions of PI4KII, namely I1 (a palmitoylation insertion), I2 (an RK-rich insertion) and I3 were found in this crystal structure ( Figure 1B). These three insertions distinguish PI4KII from the structures of PI3Ks ( Figure 1C).…”

“…Recently, the first crystal structure in the PI4K family, the catalytic domain of human PI4KII in an ADP-bound form was solved [2], and three novel insertions of PI4KII, namely I1 (a palmitoylation insertion), I2 (an RK-rich insertion) and I3 were found in this crystal structure ( Figure 1B). These three insertions distinguish PI4KII from the structures of PI3Ks ( Figure 1C).…”

“…(iii) Study compounds that can regulate the activity of PI4KIIvia tuning the fluctuation of membrane. Although the current work [2] has shown that cholesterol can regulate the kinase activity of PI4KIIvia non-direct interactions, more quantitative investigations are needed to precisely define how the activity of PI4KII is regulated via the changes of the membrane physical properties. It is also valuable to develop other compounds that can increase or decrease the physical fluctuation and fluidity of membrane and study how these new compounds affect the kinase activity of PI4KII.…”

“…Current work [2] has provided insight into how the kinase activity of PI4KII is regulated at the molecular level. It also lays a foundation for designing specific PI4KII inhibitors and activators for future therapeutic applications.…”

“…Thus the ideal inhibitors of phospholipid kinases would be designed base on the phospholipid-binding pocket. While a putative PI-binding pocket of PI4KII has been discovered via molecular dynamics analysis [2], more experimental structural work (crystallography, NMR and EPR) should be performed to define precisely the interactions between PI4KII and PI, which is important to develop the specific inhibitors of blocking the binding of PI. (iii) Study compounds that can regulate the activity of PI4KIIvia tuning the fluctuation of membrane.…”