Membraneous localization of Jpk is not essential to exert cytotoxicity in F9 teratocarcinoma cells.
A novel gene, Jopock (Jpk), which was isolated as a trans-acting factor associating with the PSRE of murine Hoxa-7, has been shown to be toxic to both prokaryotic and eukaryotic cells when overexpressed. Here we demonstrate that the overexpression of enhanced green fluorescent protein (EGFP)-tagged Jpk in F9 cells results in the induction of apoptosis, as indicated by phosphatidylserine exposure, DNA fragmentation, and the alteration of mitochondria transmembrane potential. Fluorescence microscopy showed that EGFP-fused Jpk was mainly localized in the endoplasmic reticulum (ER) and a small amount was found in the mitochondria. Deletion mutants with a transmembrane (TM) domain showed a distribution similar to that of EGFP-Jpk, whereas constructs with a deletion of the TM domain localized in the whole cells. Deletion mapping experiments showed that Jpk with an N-terminal part deleted stimulated apoptosis to almost the same extent as that of the wild-type Jpk, indicating that the localization of Jpk in the ER and the TM domain does not appear to be essential for inducing cytotoxicity. Overall, these results suggest that Jpk, particularly the C-terminal part of Jpk and/or 3'UTR, triggers apoptosis through a perturbation of mitochondrial membrane permeabilization.
Kong
Kyoung-Ah
KA
Department of Anatomy, Embryology Lab., Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
Kim
Hye Sun
HS
Park
Hyoung Woo
HW
Kim
Myoung Hee
MH
D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D005809:Genes, Regulator / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D014162:Transfection; D014407:Tumor Cells, Cultured
D018398:Homeodomain Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins