Characterization of Type II Phosphatidylinositol 4-Kinase Isoforms Reveals Association of the Enzymes with Endosomal Vesicular Compartments

Abstract

Phosphorylation of phosphatidylinositol (PI) to PI 4-phosphate is one of the key reactions in the production of phosphoinositides, lipid regulators of several cellular functions. This reaction is catalyzed by multiple enzymes that belong either to the type II or the type III family of PI 4-kinases. Type III enzymes are structurally similar to PI 3-kinases and are sensitive to PI 3-kinase inhibitors. In contrast, the recent cloning of the first type II PI 4-kinase enzyme defined a novel enzyme family. Here we characterize a new member of this family, the type II␤ enzyme that has been identified in the NCBI data base based on its homology to the first-cloned type II␣ enzyme. The type II␤ enzyme has a primary transcript size of ϳ3.8 kb and shows wide tissue distribution.It contains an open reading frame of 1.4 kb, encoding a protein of ϳ54 kDa. Sequence comparison reveals a high degree of similarity to the type II␣ enzyme within the C-terminal catalytic domain but significantly lower homology within the N-terminal region. Expression of both enzyme yields increased PI 4-kinase activity that is associated with the microsomal membrane fractions and is significantly lower for the type II␤ than the type II␣ form. Both enzymes use PI as their primary substrate and have no detectable activity on PI monophosphates. Epitope-tagged as well as green fluorescent proteintagged forms of both enzymes localize primarily to intracellular membranes and show prominent co-localization with early endosomes and recycling endosomes but not with the Golgi. These compartments participate in the processing of both the transferrin receptor and the G protein-coupled AT 1A angiotensin receptor. Our data indicate the existence of multiple forms of type II PI 4-kinase in mammalian cells and suggest that their functions are related to the endocytic pathway.Inositol phospholipids have long been considered primarily as precursors for important messenger molecules during activation of certain G protein-coupled receptors and receptortyrosine kinases (1, 2). Phosphatidylinositol (PI) 1 4-kinase and PI 4-phosphate (PI(4)P) 5-kinase activities were believed to maintain the small PI(4,5)P 2 pools of the plasma membrane during increased phospholipase C activity in stimulated cells. In addition to this important signaling aspect of phosphoinositide metabolism, it has been increasingly recognized in the last decade that localized phosphoinositide changes are of crucial importance in the organization of signaling microdomains (3, 4). A growing number of kinases and phosphatases that act upon inositides have been identified in recent years (5, 6). This together with the recognition and characterization of several molecular motifs that interact with inositides to regulate a large number of signaling molecules has contributed to our changing perception of how inositides contribute to cellular signaling. In light of such localized functions, the importance of PI(4)P formation has to be reevaluated. It can now be safely assumed that PI(4)P serves not only as a sy...

A phosphoinositide conversion mechanism for exit from endosomes

“…Moreover, by using membrane-permeant PIP analogues oversupply of PI(4)P in PI4K2α-depleted cells could restore endosomal localization of exocyst to control levels, whereas the phenotype persisted upon PI(3)Ptreatment ( Figure 34f). Consistent with a requirement of PI(4)P, expression of kinase-inactive (D308A) (Balla, Tuymetova et al 2002) compared to wild-type PI4K2α led to a displacement of exocyst from endomembranes, presumably by lowering endosomal PI(4)P-levels due to outcompeting the active enzyme (Figure 34g). …”

“…Besides, recruitment of active, wild-type MTM1 caused depletion of PI(3)P from these endosomes (Figure 36a). Importantly, semi-quantitative analysis of total PI levels in PI4K2α-depleted cells revealed not only a reduction in PI(4)P, consistent with its PI 4-kinase activity (Balla, Tuymetova et al 2002), but also a prominent increase in PI(3)P ( Figure 36b). Further, expression of active PI4K2α elevated PI(4)P (Figure 36c).…”

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

“…PI4KII␣ has been described in a subpopulation of endosomes (Balla et al, 2002), synaptic vesicles (Guo et al, 2003), and in the Golgi complex (Guo et al, 2003;Wang et al, 2003). In the Golgi complex, PI4KII␣ was shown to control the generation of a pool of phosphatidylinositol-4-phosphate [PI(4)P] that participates in the recruitment of the clathrin adaptor AP-1 (Guo et al, 2003;Wang et al, 2003).…”

“…The kinase extensively colocalizes with AP-3 and ZnT3 and to a lower extent with AP-1. A-C to AЈ-CЈ series represent two optical planes taken every 0.75 m. Bar, 6 m. been described in the Golgi complex (Wang et al, 2003), synaptic vesicles (Guo et al, 2003), and endosomes (Balla et al, 2002), an organelle that in PC12 cell gives raise to microvesicles by AP-3-dependent mechanisms (Faundez et al, 1997;Shi et al, 1998;Blagoveshchenskaya et al, 1999;Salazar et al, 2004a,b). As a first step to determine the potential localization of PI4KII␣ to AP-3-derived vesicles, fractions from PC12 cell glycerol velocity gradients and isopycnic sucrose gradients leading to the ZnT3-enriched vesicle preparation ( Figure 1C) were probed with antibodies directed against PI4KII␣.…”

“…Interestingly, VAMP7-TI-VAMP was shown to be sorted by AP-3-dependent mechanisms . An abundant protein previously implicated in Golgi (Wang et al, 2003), endosome (Balla et al, 2002), and synaptic vesicle traffic (Guo et al, 2003) was PI4KII␣ (GI16758554; Figure 2 and Supplemental Table 1, entry 82). This enzyme was represented by a cumulative count of 38 peptides.…”