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Phosphorylation of FADD/ MORT1 at serine 194 and association with a 70-kDa cell cycle-regulated protein kinase
C Scaffidi1, J Volkland, I Blomberg
1Tumor Immunology Program, Applied Tumorvirology, German Cancer Research Center, Heidelberg, Germany.
Abstract:
The adapter molecule Fas-associated death domain protein (FADD)/mediator of receptor-induced toxicity-1 (MORT1) is essential for signal transduction of the apoptosis-inducing receptor CD95 (APO-1/Fas) as it connects the activated receptor with the effector caspase-8. FADD also plays a role in embryonic development and the cell cycle reentry of T cells. FADD is phosphorylated at serine residues. We now show that phosphorylation exclusively occurs at serine 194. The phosphorylation of FADD was found to correlate with the cell cycle. In cells arrested at the G2/M boundary with nocodazole, FADD was quantitatively phosphorylated, whereas only nonphosphorylated FADD was found in cells arrested in G1/S with hydroxyurea. In this context, we have identified a 70-kDa cell cycle-regulated kinase that specifically binds to the C-terminal half of FADD. Because CD95-mediated apoptosis is independent of the cell cycle, phosphorylation of FADD may regulate its apoptosis-independent functions.
Insights
The adapter protein FADD (Fas-associated death domain) is phosphorylated at serine 194, correlating with the cell cycle. This phosphorylation may regulate FADD
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The adapter molecule Fas-associated death domain protein (FADD) is crucial for CD95-mediated apoptosis, linking the receptor to caspase-8.
- FADD also participates in embryonic development and T cell cycle reentry.
- FADD undergoes phosphorylation at serine residues, but the specific site and cell cycle regulation were previously unclear.
Purpose of the Study:
- To identify the specific serine residue of FADD phosphorylation.
- To investigate the relationship between FADD phosphorylation and the cell cycle.
- To identify the kinase responsible for cell cycle-regulated FADD phosphorylation.
Main Methods:
- Phosphorylation site analysis of FADD.
- Cell cycle arrest experiments using nocodazole (G2/M) and hydroxyurea (G1/S).
- Identification of a novel kinase binding to FADD.
Main Results:
- FADD phosphorylation exclusively occurs at serine 194.
- FADD phosphorylation levels correlate with the cell cycle: high in G2/M and low in G1/S.
- A 70-kDa cell cycle-regulated kinase that binds to FADD's C-terminus was identified.
Conclusions:
- FADD phosphorylation at serine 194 is a cell cycle-dependent event.
- The identified kinase likely mediates this cell cycle regulation.
- Given that CD95-mediated apoptosis is cell cycle-independent, FADD phosphorylation may regulate its non-apoptotic functions.