PMID- 10409681
OWN - NLM
STAT- MEDLINE
DCOM- 19990826
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 30
DP  - 1999 Jul 23
TI  - Characterization of NFkappaB activation by detection of green fluorescent
      protein-tagged IkappaB degradation in living cells.
PG  - 21244-50
AB  - Activation of the transcription factor NFkappaB requires rapid degradation of its
      inhibitor, IkappaBalpha. To facilitate the study of IkappaBalpha degradation, we 
      fused IkappaBalpha protein to enhanced green fluorescent protein to construct
      IkappaBalpha-enhanced green fluorescent protein (IG). We demonstrated by both
      flow cytometry and Western blot analysis that the half-life of IG in the presence
      of human tumor necrosis factor (TNF) alpha is approximately 5 min, which is
      similar to the half-life of native IkappaBalpha. The degradation coincided with
      NFkappaB translocation from the cytoplasm to the nucleus and NFkappaB-mediated
      induction of transcription. Phorbol 12-myristate 13-acetate (PMA), but not
      forskolin, also induces degradation of IG fusion protein. The half-life of IG in 
      the presence of PMA is approximately 15 min, longer than when induced with
      TNFalpha. Co-treatment with TNFalpha and PMA did not result in a synergistic
      effect on IG degradation, although they stimulate different kinases in two
      different signaling pathways. Degradation of IG was inhibited by mutations at
      serine residues 32 and 36, which are the target sites of the phosphorylation
      modification that initiates degradation of IkappaBalpha. We also demonstrated
      that basal degradation of IG in the presence of cycloheximide is inhibited by
      such mutations, suggesting that basal degradation of IkappaBalpha also requires
      phosphorylation as the signal for degradation. Finally, we showed that the rate
      of TNFalpha-induced degradation of IG remains almost constant throughout the cell
      cycle, except at the mitotic phase, in which IG degrades more slowly.
FAU - Li, X
AU  - Li X
AD  - CLONTECH Laboratories, Inc., Palo Alto, California 94303, USA. xqli@clontech.com
FAU - Fang, Y
AU  - Fang Y
FAU - Zhao, X
AU  - Zhao X
FAU - Jiang, X
AU  - Jiang X
FAU - Duong, T
AU  - Duong T
FAU - Kain, S R
AU  - Kain SR
LA  - eng
GR  - 1R43GM58288-01/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (I-kappa B Proteins)
RN  - 0 (Luminescent Proteins)
RN  - 0 (NF-kappa B)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - DNA-Binding Proteins/genetics/*metabolism
MH  - Gene Targeting
MH  - Green Fluorescent Proteins
MH  - HeLa Cells
MH  - Humans
MH  - I-kappa B Proteins
MH  - Luminescent Proteins/genetics/*metabolism
MH  - Mutation
MH  - NF-kappa B/genetics/*metabolism
MH  - *Transcriptional Activation
MH  - Transfection
EDAT- 1999/07/20 00:00
MHDA- 1999/07/20 00:01
CRDT- 1999/07/20 00:00
PHST- 1999/07/20 00:00 [pubmed]
PHST- 1999/07/20 00:01 [medline]
PHST- 1999/07/20 00:00 [entrez]
AID - 10.1074/jbc.274.30.21244 [doi]
AID - S0021-9258(19)72570-8 [pii]
PST - ppublish
SO  - J Biol Chem. 1999 Jul 23;274(30):21244-50. doi: 10.1074/jbc.274.30.21244.