Cell Cycle Restriction Is More Important Than Apoptosis Induction for RASSF1A Protein Tumor Suppression

Howard Donninger,Jennifer A Clark,Megan K Monaghan,M Lee Schmidt,Michele Vos,Geoffrey J Clark

doi:10.1074/jbc.m114.609537

Abstract

The Ras association domain family protein 1A (RASSF1A) is arguably one of the most frequently inactivated tumor suppressors in human cancer. RASSF1A modulates apoptosis via the Hippo and Bax pathways but also modulates the cell cycle. In part, cell cycle regulation appears to be dependent upon the ability of RASSF1A to complex with microtubules and regulate their dynamics. Which property of RASSF1A, apoptosis induction or microtubule regulation, is responsible for its tumor suppressor function is not known. We have identified a short conserved motif that is essential for the binding of RASSF family proteins with microtubule-associated proteins. By making a single point mutation in the motif, we were able to generate a RASSF1A variant that retains wild-type apoptotic properties but completely loses the ability to bind microtubule-associated proteins and complex with microtubules. Comparison of this mutant to wild-type RASSF1A showed that, despite retaining its proapoptotic properties, the mutant was completely unable to induce cell cycle arrest or suppress the tumorigenic phenotype. Therefore, it appears that the cell cycle/microtubule effects of RASSF1A are key to its tumor suppressor function rather than its apoptotic effects.

Highlights

The Ras association domain family protein 1A (RASSF1A) tumor suppressor regulates apoptosis and the cell cycle
By making a single point mutation in the motif, we were able to generate a RASSF1A variant that retains wild-type apoptotic properties but completely loses the ability to bind microtubule-associated proteins and complex with microtubules. Comparison of this mutant to wild-type RASSF1A showed that, despite retaining its proapoptotic properties, the mutant was completely unable to induce cell cycle arrest or suppress the tumorigenic phenotype. It appears that the cell cycle/microtubule effects of RASSF1A are key to its tumor suppressor function rather than its apoptotic effects
The crystal structure of NORE1A (RASSF5) bound to Ras has been solved [29], and the crystal structure of RASSF1A bound to Ras has not yet been elucidated, the residues in RASSF1A required for its interface with Ras have been determined [29], and the conserved FAL motif resides in both regions in RASSF1A and NORE1A required for their interactions with Ras [29]

Summary

Background

The RASSF1A tumor suppressor regulates apoptosis and the cell cycle. Results: Loss of microtubule association does not affect the proapoptotic function of RASSF1A but abolishes its ability to modulate the cell cycle and suppress transformation. By making a single point mutation in the motif, we were able to generate a RASSF1A variant that retains wild-type apoptotic properties but completely loses the ability to bind microtubule-associated proteins and complex with microtubules Comparison of this mutant to wild-type RASSF1A showed that, despite retaining its proapoptotic properties, the mutant was completely unable to induce cell cycle arrest or suppress the tumorigenic phenotype. A subsequent report suggested that association of RASSF1A with microtubules requires two regions within the protein, a five-amino stretch within the ATM phosphorylation site spanning residues 131–135 and a region in the C-terminal SARAH domain spanning residues 300 –305 [4] Both of these deletion mutants suffered defects in their tumor-suppressing activity. We show that it is the association with MAPs and the ability to regulate the cell cycle that is essential for the tumor suppressor properties of RASSF1A, not its apoptotic effects

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION