Reverse Mutational Analysis Reveals Threonine-373 as a Potentially Sufficient Phosphorylation Site for Inactivation of the Retinoblastoma Tumor Suppressor Protein (pRB)

Previous studies in our laboratory have shown that constitutive cyclin E expression can alleviate the requirement for cyclin Δ-CDK activity in the inactivation of the retinoblastoma protein (pRb). RbΔCDK, a mutant construct of pRb with 15 of the 16 CDK phosphorylation sites mutated to alanine represses activation of E2F by mitogen, despite cyclin E over-expression. However, restoration of the four cyclin E-CDK2 phosphorylation sites to RbΔCDK renders this construct sensitive to inactivation by CDK phosphorylation. In the present study, we engage a “reverse mutational analysis” by restoring cyclin E-CDK2 phosphorylation sites to RbΔCDK individually and in combinations in an attempt to discover phosphorylation sites on Rb that are critical for inactivation. Surprisingly, we report that, in both rodent and human cells, restoration of threonine-373 to RbΔCDK, alone or in combination with other phospho-resotrations, results in a loss of the constitutively repressive effect of this construct on E2F activation. Further, induction of endogenous cyclin A protein is blocked by RbΔCDK, but not by mutants of RbΔCDK containing a restored threonine-373. Finally, while S phase entry is blocked by expression of RbΔCDK, restoration of threonine-373 largely attenuates this effect. These findings reveal that phosphorylation of threonine-373 by CDK2-cyclin E represent a potentially crucial event in the inactivation of the pRb protein.