Dimerization of the PTEN Tumor Suppressor and its Structural Characterization by SAXS

Abstract

The PTEN tumor suppressor is a dual-specificity phosphatase whose main target is the phosphatidylinositoltriphosphate (PI(3,4,5)P3) pool in the inner plasma membrane. It acts as the PI3K antagonist in the PI3K/Akt signaling pathway that controls cell growth and apoptosis and is the second most frequently mutated protein in human cancers. Because the propensity for tumor formation depends on PTEN dose in a way that is inconsistent with the “two-hit hypothesis”, it was postulated that PTEN multimers may form the active species of the phosphatase, and recently strong evidence has been presented for the formation of functional PTEN dimers in the cell (Papa et al., Cell 157, 2014, 595). Here, we use SAXS to investigate the multimerization of PTEN in buffer and show that it indeed forms dimers following elution from a size-exclusion column as a monomer. Electron density envelopes for the PTEN monomer and dimer obtained from SAXS could clearly be distinguished and were assigned by placing the PTEN crystal structure which was earlier determined for a truncated protein. In addition, the monomer envelope was validated by μs-long all-atom MD simulations of full-length PTEN. In these simulations, the auto-inhibitory, flexible C-terminal tail associates closely with the PTEN core domains while hopping between different bound conformations. The equilibrium ensemble of the resulting structures is in excellent agreement with the SAXS data. A structure prediction using the Rosetta docking protocol revealed a putative dimer arrangement that fits the dimer envelope derived from SAXS very well and is consistent with neutron reflectometry results for membrane-bound PTEN.