Abstract
Protein phosphatase 2A (PP2A) enzyme consists of a heterodimeric core (AC core) comprising a scaffolding subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). Earlier studies suggest that upon DNA damage, a specific B subunit, B56γ, bridges the PP2A AC core to p53, leading to dephosphorylation of p53 at Thr-55, induction of the p53 transcriptional target p21, and the inhibition of cell proliferation and transformation. In addition to dephosphorylation of p53, B56γ-PP2A also inhibits cell proliferation and transformation by an unknown mechanism. B56γ contains 18 α-helices that are organized into eight HEAT (Huntington-elongation-A subunit-TOR) repeat motifs. Although previous crystal structure study has revealed the residues of B56γ that directly contact the A and C subunits, the contribution of HEAT repeats to holoenzyme assembly and to B56γ-PP2A tumor-suppressive function remains to be elucidated. Here, we show that HEAT repeat 1 is required for the interaction of B56γ with the PP2A AC core and, more importantly, for B56γ-PP2A tumor-suppressive function. Within this region, we identified a tumor-associated mutation, C39R, which disrupts the interaction of B56γ with the AC core and thus was unable to mediate dephosphorylation of p53 by PP2A. Furthermore, due to its lack of AC interaction, C39R was also unable to promote the p53-independent tumor-suppressive function of B56γ-PP2A. This study provides structural insight into the PP2A holoenzyme assembly and emphasizes the importance of HEAT repeat 1 in B56γ-PP2A tumor-suppressive function.
Highlights
How HEAT repeats contribute to phosphatase 2A (PP2A) assembly and function was unknown
We show that the HEAT repeat 1 motif of B56␥ plays a critical role in assembly of the B56␥-PP2A holoenzyme
C39R lost its ability to support both p53-dependent and p53-independent B56␥PP2A tumor-suppressive function. These results suggest a novel mechanism behind a cancer-associated loss of function mutation in the PP2A B56␥ subunit gene and provide evidence for the importance of the HEAT repeat 1 motif in B56␥-PP2A tumor-suppressive function
Summary
How HEAT repeats contribute to PP2A assembly and function was unknown. Results: A tumor-associated mutation within HEAT repeat 1 disrupts PP2A function. B56␣-PP2A has been reported to dephosphorylate the c-Myc oncogene, resulting in c-Myc inactivation [6, 7], and B56␦-PP2A has been reported to dephosphorylate Cdc25c, blocking cell cycle progression [8, 9] These studies suggest that B56-PP2A exerts its tumor-suppressive function by bridging the PP2A AC core to the substrate proteins involved in cell growth and proliferation. We have reported two tumor-associated mutations in B56␥ gene that block interaction with p53 and p53-dependent, but not p53-independent, tumor-suppressive activity of B56␥PP2A [15] Together, those data suggest that interaction of the B56 subunit with either PP2A AC core or its substrate is critical for its tumor-suppressive function. We show that retaining binding to p53, all HEAT repeat 1 mutants tested fail to promote p53 Thr-55 dephosphorylation and transcriptional activation of the p21 gene As a consequence, they abolished the p53-dependent tumor-suppressive function of PP2A. This study provides structural insight into the PP2A holoenzyme assembly and suggests an additional mechanism to inactivate tumor-suppressive function of B56␥-PP2A
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