Investigating the Crosstalk between Nedd4 Ubiquitin Ligases and PIAS3 SUMO Ligase
Abstract
Previously it has been shown that Rsp5p, a member of Nedd4 ubiquitin ligases in
yeast, is modified by the ubiquitin-like protein SUMO and that this modification
is performed by Siz1p, a member of PIAS SUMO ligases that are in turn
substrates of Rsp5p-dependent ubiquitylation, thus defining a previously
unidentified system of crosstalk between the ubiquitin and SUMO systems in
yeast. This project aims to identify whether similar crosstalk pattern exists in
human cells.
In vitro ubiquitylation assays showed that some of the human Nedd4 family
members (Nedd4.1, Nedd4.2, WWP1) are capable of ubiquitylating the human
SUMO ligase PIAS3, while in contrast, Smurf2 does not appear to be able to
modify this protein. This modification is partially WW-PY-motif-dependent as
ubiquitylation level of PIAS3 mutants with altered PY motifs conducted by
Nedd4.1 or Nedd4.2 was reduced, but not completely disrupted. Interestingly, in
vitro SUMOylation assay revealed that Nedd4.1 is SUMOylated even in the
absence of SUMO E3 ligases and an apparent interaction between the SUMO E2
(Ubc9) and Nedd4.1 was observed both in vitro and in vivo. I show that auto-
SUMOylation of Nedd4.1 is accompanied with the formation of thioester-linked
conjugates between Nedd4.1 and SUMO, but these do not involve cysteine
residues (C867, C778, and C627) within the HECT domain itself and is not
occurring at a predicted SUMOylation consensus site (K357).
Furthermore, I have shown that Nedd4.1 and SUMO1/2 colocalize in HeLa cells,
and that overexpression of epitope tagged Nedd4 and SUMO1/2, followed by
denaturing pull-downs demonstrates that both Nedd4.1 and Nedd4.2 can be
SUMOylated in vivo. Meanwhile, I have generated a SUMO trap based on SUMO
interacting motifs (SIMs) and confirmed its ability of capturing SUMOylated
proteins both in vivo and in vitro. Its use reveals that Nedd4 SUMO conjugates
could be captured by SUMO trap when Nedd4 and SUMO were co-expressed in
HeLa cells, again confirming Nedd4.1 as a substrate for SUMO1 or SUMO2.
In conclusion, I show that SUMOylation of Nedd4.1 does exist in HeLa cells, and
on the other hand, some of Nedd4 family members are responsible for PIAS3
ubiquitylation in vitro, providing evidence of a crosstalk between Nedd4 family
of ubiquitin ligases and PIAS family of SUMO ligases in mammals.
Authors
Fan, JunCollections
- Theses [4125]