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Abrogation of retinoblastoma protein function by c-Abl through tyrosine kinase-dependent and -independent mechanisms
1Department of Biology, University of California, San Diego, La Jolla 92093-0347, USA.
Abstract:
The decision to enter the cell division cycle is governed by the interplay between growth activators and growth inhibitors. The retinoblastoma protein (RB) is an example of a growth inhibitor whose main function appears to be the binding and inactivation of key cell cycle activators. One target of RB is a proto-oncoprotein, the c-Abl tyrosine kinase. RB binds to the ATP-binding lobe in the kinase domain and inhibits the nuclear pool of c-Abl in quiescent and G1 cells. Phosphorylation of RB at G1/S releases c-Abl, leading to the activation of this nuclear tyrosine kinase. In this report, we describe the construction of a mutant Abl, replacing the ATP-binding lobe of c-Abl with that of c-Src. The mutant protein AS2 is active as a tyrosine kinase and can phosphorylate Abl substrates, such as the C-terminal repeated domain of RNA polymerase II. AS2, however, does not bind to RB, and its activity is not inhibited by RB. As a result, the nuclear pool of AS2 is no longer cell cycle regulated. Excess AS2, but not its kinase-defective counterpart, can overcome RB-induced growth arrest in Saos-2 cells. Interestingly, wild-type c-Abl, in both its kinase-active and -inactive forms, can also overcome RB. Furthermore, overexpression of a kinase-defective c-Abl in rodent fibroblasts accelerates the transition from quiescence to S phase and cooperates with c-Myc to induce transformation. These effects, however, do not occur with the kinase-defective form of AS2. Thus, the growth-stimulating function of the kinase-defective c-Abl is dependent on the binding and the abrogation of RB function. That RB function can be abolished by the overproduction of one of its binding proteins is consistent with the hypothesis that RB induces cell cycle arrest by acting as a "molecular matchmaker" to assemble protein complexes. Exclusive engagement of RB by one of its many targets is incompatible with the biological function of this growth suppressor protein.
Insights
The retinoblastoma protein (RB) inhibits cell growth by binding c-Abl. A modified Abl (AS2) bypasses RB, promoting cell cycle entry, highlighting RB
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Oncogenesis
Background:
- Cell division cycle entry is controlled by growth activators and inhibitors.
- Retinoblastoma protein (RB) inhibits cell cycle progression by binding and inactivating key activators.
- c-Abl tyrosine kinase is a nuclear target of RB, inhibiting its activity in quiescent and G1 cells.
Purpose of the Study:
- To investigate the role of RB in regulating c-Abl activity.
- To construct and characterize a mutant Abl protein (AS2) that is resistant to RB inhibition.
- To explore the functional consequences of RB-independent c-Abl activity on cell growth and transformation.
Main Methods:
- Construction of a chimeric Abl protein (AS2) by replacing the ATP-binding lobe of c-Abl with that of c-Src.
- Assay of tyrosine kinase activity of AS2 and its ability to phosphorylate known Abl substrates.
- Assessment of AS2 binding to RB and its effect on RB-mediated growth arrest in Saos-2 cells.
- Evaluation of wild-type c-Abl and AS2 in overcoming RB-induced growth arrest and cooperating with c-Myc for cell transformation.
Main Results:
- The AS2 mutant is active as a tyrosine kinase but does not bind to RB, rendering its activity RB-independent.
- Overexpression of AS2 overcomes RB-induced growth arrest in Saos-2 cells.
- Wild-type c-Abl (both active and inactive forms) also overcomes RB-induced growth arrest.
- Kinase-defective c-Abl, but not kinase-defective AS2, accelerates quiescence to S phase transition and cooperates with c-Myc for transformation, indicating RB-binding is crucial for this function.
Conclusions:
- RB's growth-inhibitory function is mediated by binding and inactivating targets like c-Abl.
- Bypassing RB inhibition of c-Abl can promote cell cycle entry and transformation.
- RB likely functions as a 'molecular matchmaker,' and its disruption by excessive target binding can abolish its suppressor activity.