Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase.
InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)-IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.
Brehm
Maria A
MA
NIEHS/NIH, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
Schenk
Tobias M H
TM
Zhou
Xuefei
X
Fanick
Werner
W
Lin
Hongying
H
Windhorst
Sabine
S
Nalaskowski
Marcus M
MM
Kobras
Mario
M
Shears
Stephen B
SB
Mayr
Georg W
GW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology
D017931:DNA Primers; D012333:RNA, Messenger; D017853:Phosphotransferases (Alcohol Group Acceptor); C502806:inositol 1,3,4,5,6-pentakisphosphate 2-kinase
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