Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III beta protects from dephosphorylation and stabilizes lipid kinase activity.
Phosphatidylinositol-4-kinase-IIIbeta (PI4KIIIbeta) is activated at the Golgi compartment by PKD-mediated phosphorylation. Subsequent mechanisms responsible for continuous PtdIns(4)P production at Golgi membranes and potential interaction partners of activated PI4KIIIbeta are unknown. Here we identify phosphoserine/-threonine binding 14-3-3 proteins as novel regulators of PI4KIIIbeta activity downstream of this phosphorylation. The PI4KIIIbeta-14-3-3 interaction, evident from GST pulldowns, co-immunoprecipitations and bimolecular fluorescence complementation, was augmented by phosphatase inhibition with okadaic acid. Binding of 14-3-3 proteins to PI4KIIIbeta involved the PKD phosphorylation site Ser294, evident from reduced 14-3-3 binding to a S294A PI4KIIIbeta mutant. Expression of dominant negative 14-3-3 proteins resulted in decreased PI4KIIIbeta Ser294 phosphorylation, whereas wildtype 14-3-3 proteins increased phospho-PI4KIIIbeta levels. This was because of protection of PI4KIIIbeta Ser294 phosphorylation from phosphatase-mediated dephosphorylation. The functional significance of the PI4KIIIbeta-14-3-3 interaction was evident from a reduction of PI4KIIIbeta activity upon dominant negative 14-3-3 protein expression. We propose that 14-3-3 proteins function as positive regulators of PI4KIIIbeta activity by protecting the lipid kinase from active site dephosphorylation, thereby ensuring a continuous supply of PtdIns(4)P at the Golgi compartment.
Hausser
Angelika
A
Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. angelika.hausser@izi.uni-stuttgart.de
Link
Gisela
G
Hoene
Miriam
M
Russo
Chiara
C
Selchow
Olaf
O
Pfizenmaier
Klaus
K
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics / Q000378:metabolism*; D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology; D012694:Serine / Q000378:metabolism
D048948:14-3-3 Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D012694:Serine; D019870:1-Phosphatidylinositol 4-Kinase; C089212:protein kinase D; D011493:Protein Kinase C