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Examining BCL-2 Family Function with Large Unilamellar Vesicles
Published on: October 5, 2012
p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway
1Center Adanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane, Room 140, Piscataway, New Jersey, NJ 08854, USA.
Abstract:
A member of the small G protein family, cdc42, was isolated from a screen undertaken to identify p53-inducible genes during apoptosis in primary baby rat kidney (BRK) cells transformed with E1A and a temperature-sensitive mutant p53 using a PCR-based subtractive hybridization method. Cdc42 is a GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. In response to external stimuli, Cdc42 is known to transduce signals to regulate the organization of the actin cytoskeleton, induce DNA synthesis in quiescent fibroblasts, and promote apoptosis in neuronal and immune cells. In this study, we have demonstrated that cdc42 mRNA and protein were up-regulated in the presence of wild-type p53 in BRK cells, followed by cytoplasmic to plasma membrane translocation of Cdc42. Overexpression of Cdc42 in the presence of a dominant-negative mutant p53 induced apoptosis rapidly, indicating that Cdc42 functions downstream of p53. Furthermore, stable expression of a dominant-negative mutant of Cdc42 partially inhibited p53-mediated apoptosis. The Bcl-2 family members Bcl-xL, and the adenovirus protein E1B 19K, inhibited Cdc42-mediated apoptosis, whereas Bcl-2 did not. We provide evidence that PAK1 and JNK1 may play a role downstream of Cdc42 to transduce its apoptotic signal. Cdc42/PAK1 activates JNK1-induced phosphorylation of Bcl-2, thereby inactivating its function, and that a phosphorylation resistant mutant (Bcl-2S70,87A,T56,74A) gains the ability to inhibit Cdc42- and p53-mediated apoptosis. Thus, one mechanism by which p53 promotes apoptosis is through activation of Cdc42 and inactivation of Bcl-2.
Insights
The tumor suppressor p53 activates the small GTPase cdc42 (cell division control protein 42) to promote apoptosis. This involves cdc42 inactivating the anti-apoptotic protein Bcl-2 through phosphorylation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The tumor suppressor protein p53 plays a critical role in cellular responses to stress, including apoptosis.
- cdc42 (cell division control protein 42) is a small GTPase involved in various cellular processes, including cytoskeletal organization, DNA synthesis, and apoptosis.
- Understanding the interplay between p53 and cdc42 is crucial for elucidating apoptotic pathways.
Purpose of the Study:
- To identify genes regulated by p53 during apoptosis.
- To investigate the role of cdc42 in p53-mediated apoptosis.
- To elucidate the downstream signaling mechanisms of cdc42 in apoptosis.
Main Methods:
- PCR-based subtractive hybridization was used to identify p53-inducible genes.
- Western blotting and qRT-PCR were employed to assess cdc42 expression.
- Apoptosis assays were performed using overexpression and dominant-negative mutants of p53 and cdc42.
- Analysis of Bcl-2 family member interactions and phosphorylation status.
Main Results:
- cdc42 (cell division control protein 42) expression is upregulated by wild-type p53 during apoptosis in baby rat kidney (BRK) cells.
- Overexpression of cdc42 induces apoptosis, and dominant-negative cdc42 inhibits p53-mediated apoptosis, indicating cdc42 acts downstream of p53.
- The Bcl-2 family members Bcl-xL and adenovirus E1B 19K inhibit cdc42-mediated apoptosis, but Bcl-2 does not.
- cdc42, via PAK1 and JNK1, phosphorylates Bcl-2 at specific sites (Ser70, Ser87, Thr56, Thr74), inactivating its anti-apoptotic function.
Conclusions:
- p53 promotes apoptosis through the activation of cdc42.
- cdc42, in conjunction with PAK1 and JNK1, inactivates Bcl-2 by phosphorylation, thereby promoting apoptosis.
- This study reveals a novel signaling axis: p53 → cdc42 → PAK1/JNK1 → Bcl-2 phosphorylation → apoptosis.
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