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Disruption of retinoblastoma protein function by coexpression of its C pocket fragment
1Department of Biology, University of California at San Diego, La Jolla 92093-0347.
Abstract:
The growth suppression function of the retinoblastoma protein (RB) is mediated by its interaction with a variety of cellular proteins. RB contains at least two protein-binding pockets: the large A/B pocket, which interacts with E2F and the D-type cyclins, and the C pocket, which interacts with the nuclear c-Abl tyrosine kinase. The large A/B pocket and the C pocket are shown here to be functionally distinct and can be occupied simultaneously. A complex containing E2F, RB, and c-Abl is detected in vivo and can be assembled in vitro. We propose that the biological activity of RB not only depends on the inhibition of its targets but also on its ability to properly assemble specific protein complexes. Consistent with this hypothesis, a fragment of RB, SE delta, containing only the C pocket is shown to act as a dominant-negative inhibitor of RB function. SE delta does not have growth inhibitory activity of its own. When coexpressed with full-length RB, SE delta does not disrupt the RB-E2F or RB-D2 complexes nor does it affect the expression, phosphorylation, or nuclear tethering of the full-length RB. SE delta does compete with RB for binding to c-Abl and is fully capable of inhibiting the c-Abl tyrosine kinase. Thus, SE delta can inactivate RB while maintaining the inhibition of E2F and c-Abl. These results suggest that the inhibition of RB-binding proteins is not sufficient to suppress cell growth and that the assembly of RB-mediated protein complexes is also important for the promotion of cell-cycle arrest.
Insights
The retinoblastoma protein (RB) uses distinct pockets to bind E2F and c-Abl. Proper complex assembly, not just target inhibition, is crucial for RB
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Protein-protein Interactions
Background:
- The retinoblastoma protein (RB) is a key tumor suppressor regulating cell growth.
- RB interacts with various proteins, including E2F and c-Abl tyrosine kinase, via distinct binding pockets.
- The functional significance of these distinct binding pockets and their simultaneous occupation is not fully understood.
Purpose of the Study:
- To investigate the functional distinctness of RB's A/B and C pockets.
- To determine if RB's growth suppression function relies on specific protein complex assembly.
- To analyze the role of RB-mediated complex formation in cell-cycle arrest.
Main Methods:
- In vitro and in vivo complex assembly assays.
- Co-expression of full-length RB and RB fragments (SE delta).
- Analysis of protein complex formation, protein expression, phosphorylation, and nuclear localization.
Main Results:
- RB's A/B and C pockets are functionally distinct and can be simultaneously occupied.
- A ternary complex of E2F, RB, and c-Abl can be formed.
- The RB fragment SE delta inhibits c-Abl tyrosine kinase activity but not RB-E2F or RB-D2 complex formation or RB function.
- SE delta acts as a dominant-negative inhibitor of RB function without intrinsic growth inhibitory activity.
Conclusions:
- RB's growth suppression activity depends on both target inhibition and the assembly of specific protein complexes.
- Inhibition of RB-binding proteins alone is insufficient for cell growth suppression.
- The assembly of RB-mediated protein complexes is critical for promoting cell-cycle arrest.