Phosphatidylinositol 4-phosphate (PI4P) is generated by phosphorylation of phosphatidylinositol (PI) on the 4-position by phosphatidylinositol 4-kinases ( 1 ). In mammalian cells ( 2, 3 ), PI4P synthesis is predominately accounted for by the type II phosphatidylinositol 4-kinase II ␣ isoform (PI4KII ␣ ). Our most recent work has shown that loss of PI4KII ␣ activity in mice leads to late-onset neurodegeneration ( 2 ). In addition, Li et al. ( 4 ) have demonstrated that PI4KII ␣ is overexpressed in a wide range of common cancers where it has a key role in promoting angiogenesis. However, despite its emerging importance in disease, little is known about endogenous factors that regulate PI4KII ␣ , and there are currently no pharmacological reagents available to modulate its activity in cells. We previously demonstrated that PI4KII ␣ activity is sensitive to membrane cholesterol levels ( 5 ). Given the signifi cant role of the enzyme in major pathologies, we sought to investigate the biochemical and biophysical mechanisms that underlie sterol-sensitive PI4P synthesis.One possible mechanism through which cholesterol could affect PI4KII ␣ is by modulating its membrane mobility ( 6-10 ). Lateral diffusion of a membrane-associated protein determines its interaction dynamics with other membrane components (reviewed in Ref. 6 ), and in the case of an enzyme such as PI4KII ␣ , this is likely to be important for the kinetics of product formation. However, it is important to point out that nothing is known Abstract Type II phosphatidylinositol 4-kinase II ␣ (PI4KII ␣ ) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular traffi cking. Recently PI4KII ␣ has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KII ␣ mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KII ␣ activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KII ␣ to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fl uorescent protein (eGFP)-tagged PI4KII ␣ demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KII ␣ was assessed by fl uorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KII ␣ . Further measurements revealed that the reduction in the mobile fraction of PI4KII ␣ correlated with a loss of trans -Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme...

In this study, we investigated the role of PI4P synthesis by the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIβ, in epidermal growth factor (EGF)-stimulated phosphoinositide signaling and cell survival. In COS-7 cells, knockdown of either isozyme by RNA interference reduced basal levels of PI4P and PI(4,5)P2, without affecting receptor activation. Only knockdown of PI4KIIα inhibited EGF-stimulated Akt phosphorylation, indicating that decreased PI(4,5)P2 synthesis observed by loss of either isoform could not account for this PI4KIIα-specific effect. Phospholipase Cγ activation was also differentially affected by knockdown of either PI4K isozyme. Overexpression of kinase-inactive PI4KIIα, which induces defective endosomal trafficking without reducing PI(4,5)P2 levels, also reduced Akt activation. Furthermore, PI4KIIα knockdown profoundly inhibited cell proliferation and induced apoptosis as evidenced by the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase. However, in MDA-MB-231 breast cancer cells, apoptosis was observed subsequent to knockdown of either PI4KIIα or PI4KIIIβ and this correlated with enhanced proapoptotic Akt phosphorylation. The differential effects of phosphatidylinositol 4-kinase knockdown in the two cell lines lead to the conclusion that phosphoinositide turnover is inhibited through PI4P substrate depletion, whereas impaired antiapoptotic Akt signaling is an indirect consequence of dysfunctional endosomal trafficking.

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