Threonine phosphorylation of the MMAC1/PTEN PDZ binding domain both inhibits and stimulates PDZ binding

Two-hybrid searches with the tumor suppressor MMAC1/PTEN isolated the prote ins hDLG and kMAST205. Further two-hybrid analysis and microtiter plate bin ding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTE N-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. Th e recovered proteins were resolved by SDS-PAGE and immobilized on a nitroce llulose membrane, Treatment of this membrane with an anti-hDLG antibody ide ntified a M-r 140,000 band, consistent with the size of hDLG, Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single promine nt band of slightly larger than M-r 200,000 (M-r 200,000 kDa), Threonine ph osphorylation of the MMAC1/PTEN-PDZBD peptide inhibited both microtiter pla te binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the M-r 140,000 and >200,000 proteins, but promoted coprecipitation of proteins of approximately M-r 90,000 and M-r 120,000 from A431 cell lysate, This re sult suggests phosphorylation of the MMAC1/PTEN PDZ binding domain ran both inhibit and promote PDZ interactions.