Multiple oxidative stress-response members of the Adapt78 family.
Adapt78 is an oxidative and calcium stress-response gene. Its protein product is a potent natural inhibitor of the intracellular calcium signaling protein calcineurin. Much of what is known about Adapt78 protein is based on cell-transfection studies. Toward understanding natural endogenous Adapt78, we used an antibody raised against cellular Adapt78 and recently determined that endogenous Adapt78 protein, like its mRNA, is oxidative and calcium stress responsive. Here we report the identification of a second endogenous form of this protein family of 41 kDa. Subcellular fractionation of human HeLa cells revealed that in contrast to results of previous transfection studies, most endogenous Adapt78, characterized as 29 and 41 kDa electrophoretic doublets, resides in the cellular cytosol. The 41 kDa form of Adapt78 was abundant and found to exhibit many characteristics in common with the previously reported oxidative stress-responsive 29 kDa form, including hypo- and hyperphosphorylation variants, rapid loss of the hypophosphorylated form following oxidative stress, response to various kinase and phosphatase inhibitors, and localization. However, it also exhibited some unique characteristics, most notably the lack of calcium inducibility. Finally, the 29 kDa form exhibited a much shorter half-life and strong stabilization following oxidant exposure compared with the 41 kDa Adapt78 form. These data reveal the presence of a novel oxidative stress-responsive 41 kDa Adapt78 species, lend further insight into the Adapt78 family of proteins and their distribution, and challenge previous conclusions obtained using transfection protocols.
Michtalik
Henry J
HJ
Center for Immunology and Microbial Disease, The Albany Medical College, Albany, NY 12208, USA.
Narayan
Ananth V
AV
Bhatt
Nishant
N
Lin
H Y
HY
Mulligan
Michael T
MT
Zhang
Shen Li
SL
Crawford
Dana R
DR
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D005810:Multigene Family; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D006133:Growth Substances; D016877:Oxidants; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003513:Cycloheximide; D010744:Phosphoric Monoester Hydrolases; D010100:Oxygen; D002118:Calcium