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 IIbeta enzyme that has been identified in the NCBI data base based on its homology to the first-cloned type IIalpha enzyme. The type IIbeta enzyme has a primary transcript size of approximately 3.8 kb and shows wide tissue distribution. It contains an open reading frame of 1.4 kb, encoding a protein of approximately 54 kDa. Sequence comparison reveals a high degree of similarity to the type IIalpha 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 IIbeta than the type IIalpha form. Both enzymes use PI as their primary substrate and have no detectable activity on PI monophosphates. Epitope-tagged as well as green fluorescent protein-tagged 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY065990
Type II PI 4-kinases have been distinguished from other PI 4-kinases by their sensitivity to low concentrations of adenosine (Ki 10 –50 M) and micromolar concentrations of Ca2ϩ as well as to the anti-type II PI 4-kinase-neutralizing antibody, 4C5G [7]
Type II PI 4-kinases have been shown to be associated with virtually every membrane compartment within the cell including the plasma membrane, Golgi, secretory vesicles, and lysosomes in studies using cell or tissue fractionation [8, 9]
Summary
Vol 277, No 22, Issue of May 31, pp. 20041–20050, 2002 Printed in U.S.A. Characterization of Type II Phosphatidylinositol 4-Kinase Isoforms Reveals Association of the Enzymes with Endosomal Vesicular Compartments*. 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. In contrast to the significant progress in the field of type III PI4Ks, relatively little is known about the functions of the type II PI4Ks. Several biochemical studies demonstrate the presence of type II PI4K activity in a number of membrane compartments and organelles and indicate that the enzyme regulates PI[4]P synthesis related to several cellular processes, most notably to secretion [19]. We demonstrate that both enzymes associate with the endosomal vesicular compartment in several cell types and is involved in the regulation of endosomal membrane traffic in mammalian cells
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