Rodolfo Murillas‡, Kimberly S. Simms‡, Shigetsugu Hatakeyama §, Allan M. Weissman and ...

38 Pages·2001·2.34 MB·English

JBC Papers in Press. Published on November 20, 2001 as Manuscript M110047200
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:6)(cid:8)(cid:9)(cid:5)(cid:6)(cid:10)(cid:4)(cid:11)(cid:10)(cid:7)(cid:11)(cid:2)(cid:3)(cid:12)(cid:3)(cid:13)(cid:10)(cid:14)(cid:15)(cid:3)(cid:4)(cid:5)(cid:9)(cid:13)(cid:13)(cid:16)(cid:11)(cid:3)(cid:17)(cid:14)(cid:18)(cid:3)(cid:19)(cid:19)(cid:3)(cid:2)(cid:11)(cid:14)(cid:18)(cid:10)(cid:5)(cid:3)(cid:6)(cid:4)(cid:19)(cid:11)(cid:5)(cid:20)(cid:9)(cid:5)(cid:11)(cid:7)(cid:21)(cid:4)(cid:8)(cid:5)(cid:6)(cid:10)(cid:4)(cid:9)(cid:13)(cid:13)(cid:16)(cid:11)(cid:6)(cid:4)(cid:5)(cid:3)(cid:18)(cid:9)(cid:8)(cid:5)
(cid:22)(cid:6)(cid:5)(cid:20)(cid:11)(cid:23)(cid:3)(cid:2)(cid:2)(cid:24)(cid:11)(cid:21)(cid:25)(cid:6)(cid:26)(cid:21)(cid:6)(cid:5)(cid:6)(cid:4)(cid:11)(cid:13)(cid:6)(cid:27)(cid:9)(cid:19)(cid:3)
Rodolfo Murillas‡, Kimberly S. Simms‡, Shigetsugu Hatakeyama§, Allan M. Weissman and
Michael R. Kuehn‡¶
From the ‡Experimental Immunology Branch, National Cancer Institute, National Institutes of D
o
w
n
lo
Health, Bethesda MD 20892-1360, USA, and Regulation of Protein Function Laboratory, ade
d
fro
m
National Cancer Institute, National Institutes of Health, Bethesda MD 20892-1152, USA http
://w
w
w
.jb
c
.o
rg
§ Current address: Department of Molecular and Cellular Biology, Medical Institute of b/
y
g
u
e
s
Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 t o
n
A
p
ril 1
2
, 2
0
1
9
¶ To whom correspondence should be addressed: Bldg. 10/Rm. 4B-36, 10 Center Dr., Bethesda,
MD 20892-1360. Tel: [phone]; Fax: [phone]; E-mail: [email protected].
Running Title: Nedd4 developmental targets
Nedd4 developmental targets
Summary
Nedd4 is a HECT domain containing ubiquitin ligase that mediates ubiquitylation and proteasome
degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates
lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a
developmentally expressed protein and may have a fundamental role to play in embryonic
processes. However, whether Nedd4 has such a function is currently unknown, in part because no
developmentally regulated ubiquitylation substrates have been identified or characterized. We have
D
o
carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation w
n
lo
a
d
e
d
embryo that are able to interact with Nedd4. Characterization of their functional interaction with fro
m
h
Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding ttp://w
w
w
partners, and two have the capacity to be ubiquitylation substrates. One of these is the first .jb
c
.o
rg
b/
identified non-viral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of y g
u
e
s
t o
these two ubiquitylated proteins interacts with Nedd4 through PY mediated mechanisms. For one n A
p
ril 1
2
of the three Nedd4 binding partners there was no discernable evidence of ubiquitylation. However, , 2
0
1
9
this protein clearly associates with Nedd4 through its PY domains and can alter the location of
Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.
2
Nedd4 developmental targets
Introduction
A large number of recent findings have highlighted the importance of protein modification by
the covalent addition of small polypeptides (1,2). The prototype for this is ubiquitin, a 76 amino
acid polypeptide that when added in multiple units to form a chain (polyubiquitylation), targets
proteins for degradation by the 26S proteasome (3). In contrast, the addition of a single ubiquitin
moiety (monoubiquitylation) has been implicated in receptor endocytosis (4,5), activation of gene
expression through histone modification (6) and targeting of proteins to specific subnuclear
D
o
locations (7). In addition, protein modification by the ubiquitin-like proteins SUMO and Nedd8 w
n
lo
a
d
e
d
has been found to have a wide range of functions, from control of subcellular localization to fro
m
h
modulation of protein stability (8,9). ttp://w
w
w
Ubiquitylation occurs at lysine residues, either within target proteins or on ubiquitin already .jb
c
.o
rg
b/
attached to the target protein, and involves three distinct enzymatic activities. Ubiquitin activating y g
u
e
s
t o
enzyme (E1) activates ubiquitin to a high-energy state in an ATP-dependent manner and then n A
p
ril 1
2
transfers it to a ubiquitin conjugating enzyme (E2). The E2 either can transfer ubiquitin directly to , 2
0
1
9
the target substrate or interact with a ubiquitin protein ligase (E3), which then mediates the
transfer of ubiquitin to substrate proteins (10). There are two major classes of E3 ubiquitin ligases.
Those containing a RING finger motif act in concert with E2 enzymes in the direct transfer of
ubiquitin from the E2 to the target protein (11). Members of the second class of E3 ubiquitin
ligases accept activated ubiquitin from the E2 and transfer it to substrate proteins. The first
identified member of this class is E6-AP, which mediates polyubiquitylation of p53 in conjunction
with human papillomavirus E6 protein (12,13). Other members of this family include Nedd4 and
3
Nedd4 developmental targets
related Nedd4-like proteins (14). These all have a carboxyl terminal region known as the HECT
(homologous to E6-AP carboxyl terminus) domain, which provides the ubiquitin ligase enzymatic
activity.
Unlike E6-AP, members of the Nedd4 family have two to four tryptophan-based WW
domains, which bind certain proteins containing proline-rich motifs (Fig. 1A). The WW domains
of Nedd4 have been categorized as group I, which bind preferentially to the consensus sequence
PPXY (PY domain) (15). Group II WW domains preferentially bind to the sequence PPLP (16),
while a third class is able to bind other proline-rich consensus sequences (17). The fourth class of
D
o
w
n
WW domains binds in a proline-independent manner, depending instead on phosphoserine and/or loa
d
e
d
fro
phosphothreonine residues (18). The best-characterized Nedd4 substrate is the Epithelial Sodium m
h
ttp
://w
Channel (ENaC), a plasma membrane protein composed of several subunits, two of which are w
w
.jb
c
.o
ubiquitylated by Nedd4. The WW domains of Nedd4 bind to PY domains in the cytoplasmic rg
b/
y
g
u
region of these proteins. Truncations of these PY domains result in a reduced turnover rate of es
t o
n
A
p
ENaC and lead to Liddle’s syndrome, an inherited form of hypertension (19,20). ril 1
2
, 2
0
Nedd4 was identified originally as a gene expressed at high levels in neural precursor cells 1
9
during development and subsequently downregulated in the adult (21). Nedd4 also is expressed at
high levels in proliferating chondrocytes in mid-gestation embryos (22,23). These sites of
expression suggest an important role for Nedd4 in regulating developmentally important gene
function at the level of protein half-life. Indeed, the actions of two Nedd4-like proteins,
Suppressor of Deltex and Smurf1, are consistent with such a role during development. Smurf1
mediates polyubiquitylation of Smad1, a mediator of Bone Morphogenetic Protein (BMP)
signaling (24), and Suppressor of Deltex regulates Drosophila Notch signaling (25). The Nedd4
4
Nedd4 developmental targets
family member, Itch, can also ubiquitylate Notch (26). However, the specific functions of Nedd4
itself in developmental processes are unknown, in part due to the fact that very few
developmentally expressed ubiquitylation substrates have been characterized (27,28), although a
number of potential binding partners have been identified (23). Therefore, we searched for
proteins expressed in the mid-gestation embryo that interact with Nedd4 in a yeast two-hybrid
screen, and further characterized these to determine their functional interaction with Nedd4 both in
vitro and in vivo. We have identified four proteins, which we designated as Nedd4 binding
partners 1 through 4 (N4BP1-4). We demonstrate here that N4BP1 and N4BP2, both novel
D
o
w
n
proteins, have the capacity to be ubiquitylation substrates. N4BP1 is a bona fide substrate for loa
d
e
d
fro
Nedd4 ubiquitin ligase activity in vivo, and the first identified non-viral target for Nedd4-mediated m
h
ttp
://w
monoubiquitylation. N4BP3, also a novel protein, is not a ubiquitylation substrate, but it can alter w
w
.jb
c
.o
the subcellular location of Nedd4 indicating a functional interaction. rg
b/
y
g
u
e
s
t o
n
A
p
ril 1
2
, 2
0
1
9
5
Nedd4 developmental targets
Experimental Procedures
Yeast two hybrid screening—A fragment containing amino acids 199-777 of mouse Nedd4 was
cloned into the GAL4 binding domain vector pGBT9, and an 11-day mouse embryo cDNA library
cloned into the GAL4 activation domain vector pGAD10, were sequentially used to transform
Hf7c yeast cells according to the MATCHMAKER two hybrid system protocol (Clontech, Palo
Alto, CA). Transformants were plated on selection media (lacking tryptophan, leucine and
histidine) containing 25 mM 3-aminotriazole. After incubating 10 days at 30° C, clones expressing
HIS3 and β-galactosidase (β-gal) were identified. Plasmids were recovered according to D
o
w
n
lo
a
d
MATCHMAKER protocols, and transformed into yeast containing either pGBT9-Nedd4 or the e
d
fro
m
pGBT9 empty vector. Inserts from plasmids that activated GAL4 transcription with pGBT9- http
://w
w
Nedd4 were excised from pGAD10 and subcloned into pcDNA3.1 (Invitrogen Life Technologies, w
.jb
c
.o
rg
Carlsbad, CA), in frame with the Xpress and 6-histidine (His) epitope tags. b/
y
g
u
e
s
t o
n
A
Plasmid construction and in vitro mutagenesis—A full-length N4BP1 expression vector was p
ril 1
2
made in pcDNA3 (Invitrogen Life Technologies) by assembling the original clone with mouse EST , 20
1
9
AA444325 and a fragment containing the 5’ part of the coding region, produced by PCR using a
N4BP1 genomic cosmid clone as template. KIAA0341, a human full-length cDNA homologous to
N4BP3, was obtained from Kazusa DNA Research Institute as a pBluescript clone. The insert
was removed and subcloned into pCDNA3.1. Site directed mutagenesis of the proline-rich sites in
full-length mouse N4BP1 and KIAA0341 was done using QuikChange (Stratagene, La Jolla, CA).
Recombinant Protein Expression—All Nedd4 GST fusion constructs used in this study were
6
Nedd4 developmental targets
described previously (29). They were generated using the pGEX GST Gene Fusion System
(Amersham Pharmacia, Piscataway, NJ), and expressed in E. coli BL21 (Novagen, Madison, WI).
The E1 from wheat cloned in pET3a and human UbcH5B cloned in pET15b were expressed in E.
coli BL21 using the pET expression system (Novagen) as previously described (29). In vitro
expression and radiolabeling of proteins was performed using coupled transcription and translation
using the TnT wheat germ extract kit (Promega, Madison, WI), in the presence of 35S methionine
(Amersham Pharmacia) according to the manufacturer's protocol. To produce antisera against
N4BP1 (anti-N4BP1), the insert was subcloned from pGAD10 into the EcoRI site of pGEX-4T-1
D
o
w
n
(Amersham Pharmacia) and expressed in BL21 cells. Recombinant fusion protein was purified by loa
d
e
d
fro
glutathione sepharose and approximately 100 µg was injected into rabbits at 3-week intervals. m
h
ttp
://w
w
w
In vitro binding—Nedd4 GST fusion proteins (GST-Nedd4, GST-Nedd4:N and GST- .jb
c
.o
rg
b/
Nedd4:C) were adsorbed to glutathione sepharose beads and then combined with each N4BP y
g
u
e
s
protein or E6-AP, which had been in vitro translated and 35S labeled, in binding buffer (150 mM t on
A
p
ril 1
NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM dithiothreitol, 0.5% Nonidet P-40) with 2 mg/ml BSA. 2, 2
0
1
9
Mixtures were incubated 20 h at 4° C with agitation and then washed four times with 5 volumes
binding buffer to remove unbound proteins. Bound protein was eluted by boiling in reducing
sample buffer, and analyzed by SDS-PAGE and autoradiography.
In vitro ubiquitylation—N4BP proteins, in vitro translated and radiolabeled as above, were
incubated in the presence of GST-Nedd4, BL21 bacterial extracts of wheat E1and human UbcH5b,
and ubiquitin, in 25 mM Tris-HCl (pH 7.6), 120 mM NaCl, 3 mM dithiothreitol, 1 mM MgCl , 1
2
mM phosphocreatine, 100 units creatine phosphokinase, 0.6 units/ml inorganic pyrophosphatase
7
Nedd4 developmental targets
and 5 mM ATP-γ-S (Roche Molecular Biochemicals, Indianapolis, IN) for 1 hr at 30° C.
Reactions were terminated with the addition of SDS containing reducing sample buffer, resolved
by SDS-PAGE, and visualized by autoradiography.
Cell Culture, Transfection, Western blotting and immunoprecipitation—HEK293 cells were
cultured in Dulbecco’s modified minimal essential medium (Invitrogen Life Technologies)
supplemented with 10% fetal bovine serum. Transfections were made using the calcium
phosphate method. After 24 to 48 h cells were lysed in buffer containing 50 mM Tris, pH 7.5,
D
o
150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin and w
n
lo
a
d
e
d
1mM phenylmethylsulfonyl fluoride. The insoluble fraction was removed by centrifugation and fro
m
h
the cleared supernatant was either analyzed directly by Western blotting or used for ttp://w
w
w
immunoprecipitations. For N4BP1 immunoprecipitation, lysates were incubated for 16 h at 4° C .jb
c
.o
rg
b/
with anti-N4BP1 polyclonal antisera. For N4BP2 and N4BP3 immunoprecipitation, lysates were y
g
u
e
s
t o
incubated with anti-Xpress or anti-HisG monoclonal antibody (Invitrogen Life Technologies). n A
p
ril 1
Immunoprecipitates were collected by incubating with protein A/G agarose (Santa Cruz 2, 2
0
1
9
Biotechnology, Santa Cruz, CA) for an additional 1h. After brief centrifugation, the complexes
were washed 3 times with ice-cold 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.1%
Triton X-100. Proteins were recovered by boiling in SDS sample buffer, fractionated by SDS-
PAGE and transferred to PVDF membranes (Novex, San Diego, CA). Membranes were blocked in
5% milk in TBS and incubated for 2 h with the different primary antibodies diluted in TBS.
Antibody dilutions used were: anti-N4BP1, 1:2000 dilution; anti-Nedd4, 1:1000; anti-Xpress and
anti-HisG, 1:2000; and anti-Myc monoclonal antibody (Invitrogen Life Technologies), 1:1000.
8
Nedd4 developmental targets
After incubation with primary antibodies, membranes were washed with TBST and incubated for
1 h with the appropriate peroxidase-coupled secondary antibodies (Pierce, Rockford, IL). After
washing with TBST, chemiluminescent detection was performed using Supersignal (Pierce). For
proteasome inhibition, stock solutions of MG101 (calpain inhibitor I), lactacystin, MG132 and
epoxomycin (Calbiochem, San Diego, CA) were prepared in DMSO and diluted immediately
before use in DMEM to final concentrations of 100 µM for MG101, 5 µM for lactacystin, 10
µM for MG132 and 5 µM for epoxomycin.
D
o
Subcellular localization—HEK293 cells were plated on glass coverslips, transiently w
n
lo
a
d
e
transfected and 24 h later, fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X- d fro
m
h
100 in PBS. Cells were then washed with 0.1% PBS/BSA, incubated for 2 h with primary ttp
://w
w
w
antibodies diluted in 0.1% PBS/BSA, washed in PBS and further incubated with FITC-conjugated .jb
c
.o
rg
b/
anti-rabbit antibody (Vector Laboratories, Burlingame, CA) or rhodamine-conjugated anti-mouse y
g
u
e
s
t o
antibody (Pierce) for 1 h. N4BP1 was detected with anti-N4BP1 antiserum used at 1:400 dilution. n
A
p
ril 1
His-tagged N4BP2 and N4BP3 were detected with anti-HisG monoclonal antibody used at 1:5000 2, 2
0
1
9
dilution. HA-tagged Nedd4 was detected with either anti-HA monoclonal antibody (Covance
Research Products, Denver, PA) at 1:1000 dilution or anti-Nedd4 polyclonal antiserum at 1:1000
dilution. After incubation with secondary antibodies, samples were mounted in ProLong
(Molecular Probes, Eugene, OR) plus DAPI (4', 6-diamidino-2-phenylindole), observed with a
Zeiss Axioplan fluorescence microscope and imaged with a SPOT2 digital camera.
9
Nedd4 developmental targets
Results
Isolation of developmentally expressed Nedd4 interacting proteins—To identify potential
Nedd4 ubiquitylation substrates that might play a role in embryo development, a fragment of
Nedd4 containing WW domains 2 and 3 and the HECT domain (Fig. 1B) was used as bait in a
yeast two-hybrid screen of a mid-gestation mouse embryo cDNA library. From 7x106 clones
screened, four interacting clones were identified. These were designated Nedd4 binding partners 1
through 4 (N4BP1-4; Fig. 2). While N4BP4 encodes an amino-terminally truncated form of PLIC-
D
o
2 (Fig. 2D), a protein recently characterized as a regulator of the interaction between the w
n
lo
a
d
e
d
proteasome and ubiquitin ligases (30), N4BP1-3 show no similarity to proteins with known fro
m
h
functions. Taking advantage of overlapping ESTs and, in the case of N4BP1, a genomic clone that ttp://w
w
w
we isolated encoding the 5' region of the gene, cDNAs for the entire open reading frames encoded .jb
c
.o
rg
b/
by N4BP1 and N4BP3 were deduced (Fig. 2A and 2C). For clarity these are referred to as y
g
u
e
s
t o
flN4BP1 and flN4BP3 respectively. For N4BP1-3 amino acid sequences of full-length human n A
p
ril 1
orthologs were found or deduced. For flN4BP1, an 893 amino acid human protein KIAA0615 was 2, 2
0
1
9
found to have 89% similarity. N4PB2 was found to be 92% similar to the carboxyl region of a full-
length open reading frame encoding 1770 amino acids assembled from KIAA1413 and EST
AK001542. flN4BP3 was found to be 95% similar to KIAA0341.
N4BP1-3 bind Nedd4 in vitro—To confirm the yeast two-hybrid results, N4BP1-4 were in
vitro translated and radiolabeled with 35S methionine. These were then evaluated for binding to
glutathione sepharose-immobilized bacterially expressed GST-Nedd4 fusion proteins that included
either both the WW and HECT domains (GST-Nedd4; Fig. 1C), the WW domain containing region
10