FIGURE 1.

ITCH and LAPTM5 interact through WW domains and the PPxY motif. A, schematic representation of the motif for ITCH, LAPTM5, and respective deletion mutants. The mutant with C830A results in the production of an inactivated ITCH protein. Four deletion mutants for each WW domain were generated from the ITCH C830A expression vector. The expression vector with deletion within the PPxY motif of LAPTM5 was generated. B, significance of PPxY motif for LAPTM5-ITCH interaction. HEK293 cells were transiently co-transfected with Myc-tagged LAPTM5 alone, or 3×FLAG-ITCH and Myc-tagged LAPTM5 WT, or PPPY deletion mutant (PPPY del). After 24 h, cell lysates were prepared in lysis buffer and immunoprecipitated with anti-FLAG antibody. The whole cell lysates or immunoprecipitates were subjected to SDS-PAGE and immunoblotted (W.B) using the indicated antibodies. C, significance of the HECT and WW domains of ITCH for LAPTM5-ITCH interaction. HEK293 cells were transiently co-transfected with Myc-tagged LAPTM5 alone, or Myc-tagged LAPTM5 and 3×FLAG-ITCH, 3×FLAG-ITCH C830A, 3×FLAG-ITCH C830A with a deletion in each WW domain. After 24 h, cell lysates were prepared in lysis buffer and immunoprecipitated (I.P) with anti-FLAG antibody. The whole cell lysates or immunoprecipitates were subjected to SDS-PAGE and immunoblotted using the indicated antibodies. a.a., amino acids. CA, C830A.