title: Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIbeta.
abstract: Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIalpha and beta. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1-90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIalpha behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIbeta, only 50% of PI4KIIbeta is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIbeta to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIbeta undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIbeta is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIbeta is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and alpha/beta chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIalpha and beta.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75239, USA.
authors.forename: Gwanghyun
authors.initials: G
authors.lastname: Jung
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Pawel
authors.initials: P
authors.lastname: Wlodarski
authors.forename: Barbara
authors.initials: B
authors.lastname: Barylko
authors.forename: Derk D
authors.initials: DD
authors.lastname: Binns
authors.forename: Hongjun
authors.initials: H
authors.lastname: Shu
authors.forename: Helen L
authors.initials: HL
authors.lastname: Yin
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Albanesi
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D054878:Lipoylation; D008856:Microscopy, Fluorescence; D015778:Minor Histocompatibility Antigens; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D020744:rac GTP-Binding Proteins