Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum

Abstract

Different phosphoinositides are synthesized in cell membranes in order to perform a variety of functions. One of the most abundant of these lipids is phosphatidylinositol (PI) 4-phosphate (PI4P), which is formed in human eukaryotes by type II and type III phosphatidylinositol 4-kinase (PI4K II and III) activities. PI4K II activity occurs in many different subcellular membranes, although no detailed analysis of the distribution of this activity has been reported. Using density gradient ultracentrifugation, we have previously found that in A431 cells the predominant PI4K activity arises from a type II alpha enzyme that is localized to a buoyant membrane fraction of unknown origin [Waugh, Lawson, Tan and Hsuan (1998) J. Biol. Chem. 273, 17115-17121]. We show here that these buoyant membranes contain an activated form of PI4K II alpha that can be separated from the bulk of the PI4K II alpha protein in A431 and COS-7 cells. Proteomic analysis revealed that the buoyant membrane fraction contains numerous endoplasmic reticulum (ER)-marker proteins, although it was separated from the bulk of the ER, ER-Golgi intermediate compartment, transitional ER, Golgi and other major subcellular membranes. Furthermore, the majority of the cytoplasmic valosin-containing protein (VCP), an AAA+ATPase implicated in various ER, transitional ER, Golgi and nuclear functions, was almost completely localized to the same buoyant membrane fraction. Co-localization of VCP and PI4K activity was confirmed by co-immunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular PI4P synthesis and VCP are localized.

Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor

“…1F). Western blotting of GFP-PtdIns4KII␣ HT1080 cell lysates with PtdIns4KII␣-specific antibodies (Waugh et al, 2003a) indicated that levels of ectopic expression were similar to those of endogenous protein and furthermore, when control and stably transfected cells were fractionated on sucrose density gradients both showed an identical distribution of endogenous protein, GFP-PtdIns4KII␣ protein and activity (data not shown). In addition, our GFP-PtdIns4KII␣ HT1080 cell line did not exhibit any disruption of the Golgi complex or mislocalisation of Golgi markers (data not shown).…”

“…The same isoform has also been found associated with synaptic vesicles (Guo et al, 2003). In addition to the Golgi complex, PtdIns4KII␣ and PtdIns4KII␤ both localise to endosomal membranes containing internalised transferrin and angiotensin (Balla et al, 2002) and EEA1 (Balla et al, 2002;Waugh et al, 2003a).…”

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P 2 and maintenance of KCNQ2/3 ion channel current

“…Adenosine targets both PI4KII types α and β isoforms, whereas wortmannin inhibits PI4KIII types α and β. Inhibition of either PI4KII or PI4KIII enzymes reduces KCNQ2/3 currents (40% with adenosine, 15% with wortmannin). PI4KIIα, originally cloned from chromaffin granules (24), traffics throughout secretory pathways and has been suggested to be present in the trans-Golgi network, endosomes, and transport vesicles (26,(65)(66)(67); PI4KIIβ is found in the cytosol, trafficking vesicles, and clathrin-coated vesicles (68)(69)(70). Taking into account the apparent localization of PI4KII isoforms along different stages of the secretory pathway, it is logical that addition of adenosine has similar effects on PM PI(4,5)P 2 as depletion of the trans-Golgi PI(4)P pool (compare Fig.…”

“…Type IIα and IIβ PI4Ks are membrane-bound proteins due to the palmitoylation of a conserved stretch of cysteines in their catalytic domains (24). Immunocytochemical analysis has revealed that they are mostly associated with trans-Golgi, endoplasmic reticulum, and endosomal membranes (25)(26)(27). These type IIα and IIβ enzymes are blocked by adenosine and calcium, but are resistant to wortmannin.…”

PI4KIIα is a novel regulator of tumor growth by its action on angiogenesis and HIF-1α regulation

“…It has been reported that PI4KIIa is important for Wnt signaling in Xenopus embryos and interacts with Dvl (Pan et al, 2008;Qin et al, 2009). Waugh et al (2003) reported that PI4KIIa colocalized with valosincontaining protein, a potential prognostic marker for cancer. Xu et al (2006) found that PI4KIIa is associated with cellugyrin-positive Glut4 vesicles.…”