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 proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding 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/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibited both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and > 200,000 proteins, but promoted coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions.
Adey
N B
NB
Myriad Genetics Inc., Salt Lake City, Utah 84108, USA. nils@myriad.com
Huang
L
L
Ormonde
P A
PA
Baumgard
M L
ML
Pero
R
R
Byreddy
D V
DV
Tavtigian
S V
SV
Bartel
P L
PL
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D000074288:Discs Large Homolog 1 Protein; D051528:Guanylate Kinases; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D013912:Threonine / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; C000614189:Dlg1 protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D013912:Threonine; D051528:Guanylate Kinases; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human