Abrogation of retinoblastoma protein function by c-Abl through tyrosine kinase-dependent and -independent mechanisms.

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.