Structural basis for recognition of the tumor suppressor protein PTPN14 by the oncoprotein E7 of human papillomavirus
Fig 5
Interaction with E7 is critical for the proteasomal degradation of PTPN14.
Lysates of cells transiently expressing the indicated proteins for 24 hours were subjected to IP and immunoblotting, with antibodies as marked. (A) Endogenous expressions of HPV18 E7 and PTPN14 in four cell types were verified. (B) Protein levels of PTPN14 transiently expressed in three cell types were quantified using Vilber Lourmat software with normalization to Hsp90. (C–D) Coimmunoprecipitation assay using WT and binding-defective mutant PTPN14 and HPV18 E7 C-terminal constructs in HeLa (C) or C33a (D) cells. (E–F) MG132 (top) or CHX (bottom) treatment. PTPN14 protein levels at the indicated time post 20 μM MG132 treatment for blocking proteasomal degradation (top) or 50 μg/mL CHX treatment for preventing protein synthesis (bottom) in HeLa (E) or C33a (F) cells were determined by immunoblotting. Relative protein amounts were quantified using Vilber Lourmat software and normalized to Hsp90. AA, R84A and L91A; CHX, cycloheximide; HPV, human papillomavirus; Hsp90, heat shock protein 90; IP, immunoprecipitation; PTPN14, nonreceptor-type protein tyrosine phosphatase 14; SQA, F1044S, G1055Q, and E1095A; WCL, whole-cell lysate; WT, wild type.