Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIbeta.
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.
Jung
Gwanghyun
G
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75239, USA.
Wang
Jing
J
Wlodarski
Pawel
P
Barylko
Barbara
B
Binns
Derk D
DD
Shu
Hongjun
H
Yin
Helen L
HL
Albanesi
Joseph P
JP
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
D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D020744:rac GTP-Binding Proteins