Subcellular localization and structural function of endogenous phosphorylated phosphatidylinositol 4-kinase (PI4K92).
Anti-phosphopeptide antibodies were raised against phosphatidylinositol 4-kinase (PI4K92) phosphorylation sites (Suer, S., Sickmann, A., Meyer, H. E., Herberg, F. W., and Heilmeyer, L. M. Jr. (2001) Eur. J. Biochem. 268, 2099-2106). Characterization proved three of them (anti-pSer-294, anti-pSer-496, and anti-pThr-504 antibody) to be highly specific, recognizing solely PI4K92 phosphorylated at these sites, respectively. Indirect immunofluorescence reveals that PI4K92 phosphorylated on Ser-294 localizes exclusively at the Golgi. The enzyme phosphorylated on Ser-496 and Thr-504 is detected in nuclear speckles. Phosphorylation of Ser-294 on PI4K92 increases the lipid kinase activity and thus serves better in maintaining Golgi function and morphology (compare Hausser, A., Storz, P., Martens, S., Link, G., Toker, A., and Pfizenmaier, K. (2005) Nat. Cell Biol. 7, 880-886). Microinjection of anti-pSer-496, but not of anti-pSer-294 or anti-pThr-504 antibody, into the cytoplasm or into the nucleus of HS68 cells leads to development of hotspots, probably representing aggregated PI4K92, and in later stages, cells become apoptotic and finally die. The association of phosphorylated PI4K92 with nuclear speckles is dynamic and follows the morphological alteration of speckles upon inhibition of mRNA transcription with alpha-amanitin. Overexpressed PI4K92 phosphorylated on Ser-294 is not transported to the nucleus, and that phosphorylated on Ser-496 is found in the nucleus and mislocalized at the Golgi complex. We conclude that nuclear phosphatidylinositol 4-phosphate, and consequently, synthesis of polyphosphoinositides are required for a correct nuclear function.
Szivak
Ilona
I
Institut für Physiologische Chemie, Ruhruniversität Bochum, Universitätstrasse 150, MA 2/141, 44801 Bochum, Germany.
Lamb
Ned
N
Heilmeyer
Ludwig M G
LM
D019870:1-Phosphatidylinositol 4-Kinase / Q000737:chemistry*; D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011817:Rabbits; D012694:Serine / Q000737:chemistry; D014158:Transcription, Genetic
D010748:Phosphopeptides; D012694:Serine; D019870:1-Phosphatidylinositol 4-Kinase