Mast cell-restricted p70 Stat6 isoform is a product of selective proteolysis.
Signal transducer and activator of transcription (Stat)-6 is the principle Stat protein activated by interleukin (IL)-4. We defined a role for IL-4 in mast cell homeostasis through inhibiting expression of Kit and F(c)epsilonRI, and by inducing mast cell apoptosis. These effects required Stat6 expression. A molecular mechanism by which Stat6 directs these inhibitory actions in BMMC was potentially elucidated by the discovery of a carboxyl-truncated Stat6 isoform. Expression of this 70kDa isoform was unique to cultured mast cells and mast cell lines. Furthermore, this isoform lacked a carboxyl-transactivation domain, suggesting that it might behave as a dominant negative isoform. To better understand this truncated Stat6, we characterized its origins. Using Western blotting and electrophoretic mobility shift assay analysis, we assessed BMMC p70 Stat6 expression using standard and enhanced protease inhibitor cocktails. These experiments demonstrated that p70 Stat6 is derived by proteolysis during sample preparation, and has no cellular correlate. While some Stat family members are known to exist as naturally occurring truncated forms, p70 Stat6 does not appear to be such a case. Instead, the very high concentrations of proteases released during mast cell lysis result in selective proteolysis of the full-length Stat6, with p70 being the major degradation product.
Shelburne
Christopher P
CP
Department of Biology, Virginia Commonwealth University, Richmond, VA 23284, USA.
Piekorz
Roland P
RP
Bouton
L Andrew
LA
Chong
Hey Jin
HJ
Ryan
John J
JJ
D017398:Alternative Splicing; D000818:Animals; D016477:Artifacts; D001854:Bone Marrow Cells / Q000378:metabolism; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050800:STAT6 Transcription Factor; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020033:Protein Isoforms; D012333:RNA, Messenger; D050800:STAT6 Transcription Factor; C494100:Stat6 protein, mouse; D015534:Trans-Activators