Control of the activity of WW-HECT domain E3 ubiquitin ligases by NDFIP proteins

Abstract

HECT domain E3 ubiquitin ligases of the NEDD4 family control many cellular processes, but their regulation is poorly understood. They contain multiple WW domains that recognize PY elements. Here, we show that the small PY-containing membrane proteins, NDFIP1 and NDFIP2 (NEDD4 family-interacting proteins), activate the catalytic activity of ITCH and of several other HECT ligases by binding to them. This releases them from an autoinhibitory intramolecular interaction, which seems to be characteristic of these enzymes. Activation of ITCH requires multiple PY-WW interactions, but little else. Binding of NDFIP proteins is highly dynamic, potentially allowing activated ligases to access other PY-containing substrates. In agreement with this, NDFIP proteins promote ubiquitination in vivo both of Jun proteins, which have a PY motif, and of endophilin, which does not.

Figure 1

NDFIP2 induces ubiquitination of HECT domain ligases and potential substrates. (A) HEK293T cells expressed His-tagged ubiquitin (His-Ub), HA-tagged E3 ligases as indicated and an empty vector control (lane 1), Myc–NDFIP2 (lane 2) or Myc–NDFIP2 with triple PY motif mutations (lane 3). (B) Cells expressed His-tagged ubiquitin, HA-tagged c-Jun or JunB and either an empty vector (1), Myc-tagged NDFIP2 (2) or the PY mutant NDFIP2 (3). (C) As in (B) but with HA–endophilin A1 instead of Jun proteins. Lane 0 is a control in which His–ubiquitin is omitted. (D) As in (B,C), with indicated proteins expressed. All samples are from the same experiment, but the panels to the right are from a separate gel. HA, hemagglutinin; HECT, homologous to E6-AP carboxy terminus; HEK, human embryonic kidney; NDFIP, NEDD4 family-interacting protein.

Figure 2

Recruitment of NEDD4 by NDFIP2. (A) COS-7 cells expressing both GFP-tagged NEDD4 and either Myc-NDFIP2 or the PY mutant form of this were imaged. (B) Cells expressing HA-ITCHΔC2 with or without NDFIP2 were lysed and centrifuged, and the supernatant (S100) and pellet (P100) were blotted. (C) Examples of FRAP experiments; time 0 is immediately after bleaching. (D) Quantitation of FRAP experiments. Each curve represents one bright dot in the bleached region. FRAP, fluorescence recovery after photobleaching; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; NDFIP, NEDD4 family-interacting protein.

Figure 3

NDFIPs are substrates that increase autoubiquitination of E3 ligases in vitro. (A) Coomassie-stained gels and immunoblots (IB) of assays containing the indicated proteins and the cytoplasmic domains of NDFIP1 (1cyt) or NDFIP2 (2cyt) or the same with all three PY motifs mutated (PY^*). Asterisks indicate the smaller ubiquitinated forms of the NDFIPs; X is a constitutively active fragment of NEDD4. (B) Assay with methylated ubiquitin. (C) Assays with NDFIP2 fragments. This experiment used His–ubiquitin, which reduces modification and allows a clear separation of the modified forms of NDFIP and ITCH. (D) Assays with Y to F changes in individual PY motifs of NDFIP2. Note that polyubiquitinated proteins sometimes transfer poorly to the blot. (E) Modification of NDFIP1 by various ITCH WW mutants. The schematic diagram summarizes the results. NDFIP, NEDD4 family-interacting protein; Ub, ubiquitin; WT, wild type.

Figure 4

Sequences required for autoinhibition of NEDD4 and ITCH. (A) Ubiquitination assays using His-ubiquitin were Coomassie stained. PR indicates the proline-rich region in ITCH. (B) Cartoon indicating proposed model; NDFIP, NEDD4 family-interacting protein.

Figure 5

NDFIP2 promotes the modification of endophilin by ITCH in vitro. Ubiquitination assays containing the indicated proteins were analysed by immunoblotting. GST, glutathione S-transferase; NDFIP, NEDD4 family-interacting protein; Ub, ubiquitin.