1 Fels Institute for Cancer Research and Molecular Biology, Temple University
School of Medicine, 3307 N. Broad Street, Philadelphia, PA 19140, USA
2 Hanson Centre for Cancer Research, Institute of Medical and Veterinary
Science, PO Box 14, Rundle Mall, Adelaide SA 5000, Australia
Author for correspondence (e-mail: dhaines{at}unix.temple.edu )
Accepted 28 November 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Nedd4, Rsp5, WW domain, ole1, Spt23
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Covalent attachment of ubiquitin to a protein substrate requires the
coordinated activity of numerous proteins. These include a
ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a
specificity factor that functions in substrate binding (an E3 or ubiquitin
ligase) (Hershko and Ciechanover,
1998; Pickart,
2001
). One major subclass of ubiquitin ligases is the HECT (for
Homologous to E6-AP C-Terminus)-domain-containing proteins
(Huibregtse et al., 1995
).
Unlike other types of ubiquitin ligases, substrate ubiquitination by
HECT-domain-containing enzymes requires the intermolecular transfer of
ubiquitin from the bound E2 to the E3 prior to attachment onto the target.
This transfer is dependent on the formation of a thioester bond between
ubiquitin and a conserved Cys residue that is localized at the C-terminus of
the HECT domain (Hershko and Ciechanover,
1998
; Pickart,
2001
).
A number of HECT domain ubiquitin ligases have been identified that possess
multiple WW domains and a Ca2+/lipid-binding (C2) domain
(Harvey and Kumar, 1999).
These proteins, termed the Nedd4 family, have been cloned from yeast,
Caenorhabditis elegans, Xenopus leavis, rodents and man and include
Rsp5, Pub1, Itch, AIP-4, WWP1/AIP5, WWP2/AIP2, Nedd4, Smurf-1, Smurf-2,
KIA0322 and KIAA0439. The WW domain is a protein-interacting module, and it
binds to at least four different types of sequence
(Sudol and Hunter, 2000
).
These are polypeptides with core amino acids Pro-Pro-X-Tyr or Pro-Pro-Leu-Pro,
polyproline residues that are flanked by Arg or Lys, and peptides that are
phosphorylated on Ser or Thr residues. A C2 domain mediates translocation of
proteins to phospholipid membranes in response to increased cytosolic
Ca2+ (Rizo and
Südhof, 1998
).
Orthologous Rsp5 and Nedd4 are the best studied of the Nedd4 family.
Although only a limited number of Nedd4 substrates have been identified
(Staub et al., 1996;
Abriel et al., 2000
;
Hamilton et al., 2001
;
Pham and Rotin, 2001
), Rsp5
has been implicated in the ubiquitination and degradation of many proteins
(Huibregtse et al., 1997
;
Erdeniz et al., 2000
;
Rotin et al., 2000
;
Andoh and et al., 2000
).
Recently, the essential function of Rsp5 has been linked to ole1 gene
transactivation (Hoppe et al.,
2000
). Rsp5 induces `regulated ubiquitin/proteasomal-dependent
processing' (termed RUP) of Spt23, one of two ER-membrane-bound proteins that
are positive regulators of ole1 gene expression
(Zhang et al., 1999
;
Hoppe et al., 2000
). Processed
Spt23 is released from the ER membrane and translocates to the nucleus, where
it enhances ole1 gene transcription
(Hoppe et al., 2000
;
Hitchcock et al., 2001
).
Although it is widely accepted that the C2 domain promotes membrane
localization of Rsp5 and Nedd4 (Plant et
al., 1997
; Plant et al.,
2000
; Wang et al.,
2001
, Wang et al.,
2001
) and their C-terminal WW domains mediate binding to
substrates (Staub et al.,
1996
; Harvey et al.,
1999
; Chang et al.,
2000
; Farr et al.,
2000
; Hoppe et al.,
2000
), the function of the highly conserved WW domain 1 remains
undefined.
Results presented here indicate that WW domain 1 binds cofactors involved in ubiquitin/proteasome-dependent proteolysis of substrates. We have found that Spt23 processing is inhibited in budding yeast by Nedd4 and Rsp5 mutants harboring alterations in WW domain 1 or the ligase domain. In transfected mammalian cells, wild-type (wt) Nedd4 promotes proteasome-mediated degradation of precursor Spt23 whereas WW-domain1- and ligase-defective Nedd4 mutants block degradation. Because we are able to easily detect an interaction between the WW domain 1 Nedd4/Rsp5 mutants and Spt23 in cells, it is likely that disruption of WW-domain1-binding function eliminates E3-induced degradation but not substrate-binding activity. These mutational studies point to the existence of Nedd4- and Rsp5 WW-domain1-binding proteins that are required for proteasome-dependent degradation and/or processing of substrates.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The FLAG-tagged Spt23 construct was generated by cloning PCR product (generated with primers that span the coding region of the gene, genomic DNA prepared from the InvSc1 strain and PFU) inframe with the FLAG tag present (N-terminal to Spt23) in the pESC-LEU2 vector. The Rsp5 constructs were generated in a similar manner in pYes-BBV-HA using oligodeoxynucleotides amplifying full length Rsp5 or truncated Rsp5 starting immediately C-terminal to WW domain 1. The C-terminal pYes-Rsp5 mutant contains a stop codon upstream of the C- terminal Cys residue.
Yeast techniques
Standard protocols were followed for preparation of yeast media and yeast
manipulations (Guthrie and Fink,
1991). For viability assays, yeast were transformed with plasmids
by the lithium acetate method and plated onto glucose agar (all media used in
described assays included the appropriate drop-out supplements that allows for
the selection of transformed yeast with the indicated plasmids). Cells were
incubated at 30°C for 3 days. Colonies were picked and cells were grown
overnight to equal densities in glucose media. Cells were diluted to varying
degrees, streaked onto glucose or galactose agar and incubated at 30°C. To
determine the effects of oleic acid on Nedd4-induced toxicity, cells were
processed as described above and streaked onto galactose agar supplemented
with 0.5 mM oleic acid (made up in 1% tergitol (NP-35)). For the high copy
suppressor screen, pESC-LEU containing the Nedd4 WW domain 1 mutant was
transformed into the InvSc1 strain. Cells harboring this construct were
subsequently transformed with a yeast expression cDNA library (ATCC; library
number 87276) and plated onto galactose agar. Over 400,000 individual yeast
cDNAs clones were screened and approximately 100 visible colonies appeared on
these plates after 3 days at 30°C. DNA was isolated from yeast clones
retaining Nedd4 expression protein and transformed into ultracompetent XL-1
cells. Multiple colonies were picked from each transformation and grown
overnight in ampicillin-containing liquid broth media. Plasmid DNA was
prepared and those derived from the yeast cDNA library were identified by a
XhoI restriction endonuclease digestion. One clone was chosen from
each of the transformants and tested for its ability to reverse the
growth-inhibitory activity of the WW domain 1 mutant. Only 7 clones generated
a suppressible phenotype. These clones were sequenced and database searches
revealed that all clones were derived from the ole1 gene.
Western blots, coimmunoprecipitations and northern blots
Cells were grown under the appropriate conditions (described in detail in
the legend of the Figures), pelleted and stored at -80°C prior to lysis.
Cells were lysed in RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS,
0.5% deoxycholic acid and 1% Nonidet P-40) supplemented with 1 mM PMSF, 2
µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml soy trypsin inhibitor and
1 µg/ml pepstatin A. Acid-washed glass beads (425-600 microns) (Sigma) were
added to the lysate to give a 50% beads/lysate (v/v) mixture. Samples were
vortexed at 4°C for 20 minutes. Lysates were clarified by centrifugation,
the supernatant transferred to a new tube and the protein concentration
determined by the Bradford method (BioRad). Samples were mixed with 4x
SDS-PAGE sample loading buffer and boiled for 5 minutes. Approximately 10
µg of total protein was separated on SDS-polyacrylamide gels and
transferred onto nitrocellulose membranes. Blots were probed with rabbit
polyclonal antibodies raised against mouse Nedd4 or commercially available
mouse monoclonal antibodies against HA (12CA5, Roche Molecular Biochemicals)
or FLAG (M5, Sigma) epitope tags. Signals were visualized using
horseradish-peroxidase-conjugated secondary antibodies and enhanced
chemiluminescence (NEN). For coimmunoprecipitation assays, cells were grown
under the appropriate conditions, pelleted and lysed in RIPA buffer as
previously described. After clarification, protein concentration was measured
by the Bradford assay and 600 µg of protein was pre-cleared for 1 hour with
protein-G sepharose (Amersham Pharmacia) at 4°C. After a brief
centrifugation, the supernatant was transferred to a new tube containing 1
µg of the immunoprecipitating antibody. After a 4 hour incubation at
4°C, protein-G sepharose was added and the mixtures were incubated for an
additional 2 hours at 4°C. Protein complexes were washed three times with
cold lysis buffer and resuspended in 1x SDS-PAGE loading buffer. Samples
were placed in a boiling water bath for 5 minutes to elute protein from beads
and western blots were performed as described above.
For ole1 transcript analysis, single colonies transformed with a control or Nedd4 expression constructs were picked and grown in glucose media for approximately 36 hours at 30°C. Cells were pelleted and resuspended in 0.5 mM oleic acid supplemented glucose media and incubated overnight. Cells were again pelleted and resuspended in oleic acid supplemented galactose media. Cells were incubated for an additional 4 hours, pelleted and resuspended in non-supplemented galactose media. Cells were grown for 2 more hours before harvesting and isolation of RNA. RNA was separated on agarose gels containing formaldehyde and transferred to nylon membranes. Hybridizations were performed in Rapid-Hyb (Amersham-Pharmacia) using a 32P radiolabeled ole1 cDNA probe generated from plasmid DNA obtained via the high copy suppressor screen.
Spt23 expression analysis in mammalian cells
Nedd4 and Spt23 cDNAs were cloned from appropriate yeast expression vectors
into the pCEP mammalian expression vector (Invitrogen). 5 µg of each
construct were transfected in duplicate in the human lung adenocarcinoma H1299
cell line by Lipofectamine (Invitrogen) according to manufacturer's
instructions. One set of transfections was washed and re-fed with complete
media while the other was re-fed with complete media containing 10 µM of
the proteasome inhibitor MG115 (Calbiochem). Cells were harvested 9 hours
later, and the extract was prepared in RIPA buffer.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Although the mutation introduced within WW domain 1 should eliminate its
binding function (Lu et al.,
1999), it is possible that the toxic activity of the WW domain 1
mutant is dependent on de novo protein association mediated by the mutated WW
domain. Therefore, we wanted to determine if a WW domain 1 deletion mutant
suppresses the growth of yeast. Fig.
1B demonstrates that Nedd4 with a deleted WW domain 1 inhibits
proliferation. Fig. 1C shows a
result of a western blot depicting equivalent level of expression of wt Nedd4
and the WW domain 1 mutants after galactose induction. These results indicate
that disruption of WW-domain1-binding function induces a toxic activity of
human Nedd4 in budding yeast, and this is not associated with differential
expression of the mutant protein.
The WW domain 1 mutant inhibits ole1 gene expression and
Spt23 processing
To investigate a mechanism of WW domain 1 mutant action, we performed a
high copy suppressor screen to identify yeast cDNAs that block its toxic
function. Of over 400,000 yeast cDNAs screened, only seven suppressors were
identified. Sequence analysis of the clones revealed that all were derived
from the ole1 gene. This gene encodes 9 fatty acid desaturase,
an essential yeast enzyme involved in the synthesis of oleic acid
(Stukey et al., 1989
). To test
if oleic acid also suppresses the growth inhibitory activity of this mutant,
WW-domain1-transformed cells were plated onto oleic acid supplemented media.
As shown in Fig. 2A,
overexpression of an ole1 cDNA or oleic acid supplementation of
growth media blocks the toxic activity of the WW domain 1 mutant.
|
Rsp5's essential function has recently been linked to 9 fatty acid
desaturase production (Hoppe et al.,
2000
). It has been documented that rsp5-null cells
proliferate on oleic acid media and that Rsp5 is required for ole1
RNA synthesis when cells are grown in oleic-acid-free media
(Hoppe et al., 2000
). These
findings prompted us to determine if the Nedd4 WW domain 1 mutant inhibits
ole1 RNA production. Yeast transformed with empty vector or the WW
domain 1 mutant were grown in oleic-acid-containing media to repress
ole1 transcription. Then, cells were cultured in galactose media
lacking oleic acid to induce ole1 transcription and harvested 2 hours
later for isolation of RNA. As shown in
Fig. 2B, lower amounts of
ole1 transcripts were evident in cells that express the Nedd4 WW
domain 1 mutant when compared to yeast transformed with the vector control.
These results show that the Nedd4 WW domain 1 mutant inhibits expression of
the essential yeast gene ole1.
Rsp5 influences ole1 gene expression by an interesting mechanism.
Rsp5 induces ubiquitin/proteasome-dependent processing of Spt23, an ER-bound
transcription factor that is a positive regulator of ole1 gene
transcription (Zhang et al.,
1999; Hoppe et al.,
2000
; Hitchcock, 2001). Processed Spt23 is released from the ER
membrane and translocates to the nucleus, where it presumably functions as an
ole1 gene transactivator. Given that the Nedd4 WW domain 1 mutant is
an inhibitor of ole1 gene expression, we next wanted to determine if
it alters ubiquitin/proteasome-dependent processing of Spt23. We measured the
levels of membrane-bound (larger molecular weight product) and processed
(smaller molecular weight product) Spt23 in cells expressing the WW domain 1
mutant by immunoblotting. Interestingly, cells expressing the WW domain 1
mutant have a higher amount of unprocessed Spt23 and a lower level of
processed Spt23 protein (Fig.
2C) when compared to vector-alone-transformed cells. These results
show that the Nedd4 WW domain 1 mutant inhibits ubiquitin/proteasome-dependent
processing of Spt23. Inhibition of processing probably results in lower
amounts of transcriptionally active Spt23 protein and reduced levels of
ole1 RNA.
The Nedd4 WW domain 1 mutant interacts with Spt23 via WW domains 2
and 3
As the WW domain 1 mutant promotes accumulation of membrane-bound SPT23,
the results above indicate that the WW domain 1 mutant associates with Spt23,
forming a complex that is resistant to Rsp5-induced processing. To determine
if the WW domain 1 mutant interacts with Spt23, coimmunoprecipitation studies
were carried out. Spt23 protein complexes were isolated by
immunoprecipitation, separated by SDS-PAGE, and immunoblots were performed
with a Nedd4 polyclonal antibody. In addition, we carried out the reciprocal
approach where immunoprecipitations used the Nedd4 polyclonal and western
blots employed an antibody that detects Spt23. As depicted in
Fig. 3A, Nedd4 protein was
detected in Spt23 immunoprecipitates derived from cells expressing the WW
domain 1 mutant. Unprocessed but not processed Spt23 was detected in the Nedd4
immunoprecipitations (Fig. 3B),
indicating that Nedd4 interacts preferentially with membrane-localized Spt23.
We were able to detect an interaction between wt Nedd4 and membrane-bound
Spt23 (Fig. 3B), although the
amount of Spt23 in the complex with wt Nedd4 was much less than the amount
present in complex with the WW domain 1 mutant. This could be due to the fact
that either there is more membrane-localized Spt23 in cells expressing the WW
domain 1 mutant or the wt Nedd4-Spt23 interaction is unstable. Nevertheless,
these results indicate that the WW domain 1 mutant is not defective at
interacting with Spt23.
|
It is known that the C-terminal WW domains of Nedd4 binds to proline-rich
sequences found within its targets (Staub
et al., 1996; Harvey et al.,
1999
; Abriel et al.,
2000
; Hamilton et al.,
2001
; Pham and Rotin,
2001
). Interestingly, Spt23 does not contain any proline-rich
sequences. To determine if Nedd4 binding to Spt23 requires its C-terminal
localized WW domains, we tested WW domain 1 combination mutants for their
ability to inhibit Spt23 processing and interact with Spt23.
Fig. 4A shows that the WW
domain 1/2, 1/3 and 1/2/3 mutants do not alter Spt23 processing, indicating
that WW domains 2 and 3 are both required for the dominant-negative activity
of the WW domain 1 mutant. WW domain 4 is unlikely to be required for the
activity of the WW domain 1 mutant as the WW domain 1/4 mutant induces
accumulation of higher molecular weight Spt23 and inhibits generation of
processed Spt23 (Fig. 4A).
Coimmunoprecipitation studies were next performed to determine if mutations of
WW domains 2 and/or 3 abrogate an interaction between the WW domain 1 mutant
and Spt23 in cells. As shown in Fig.
4B, Nedd4 protein was present in Spt23 immunoprecipitates derived
from cells expressing the WW domain 1 mutant but not in immunoprecipitates
derived from cells producing the WW1/2, WW1/3 or WW1/2/3 mutants. Although
these experiments do not give an indication whether the interaction between
Nedd4 and Spt23 is direct, the results suggest that the formation of the
Nedd4-Spt23 protein complex in cells require the cooperative binding function
of WW domains 2 and 3.
|
Rsp5 proteins harboring mutations in either WW domain 1 or the HECT
domain block Spt23 processing
The results indicate that both WW domain 1 binding function and ubiquitin
ligase activity is required for Spt23 processing by Nedd4 and Rsp5 in budding
yeast. To determine whether this is the case, we tested Nedd4 and Rsp5 mutants
lacking ligase function (harboring a C-terminal Cys mutation) and a Rsp5 WW
domain 1 deletion mutant for their ability to alter Spt23 processing.
Interestingly, similar to the catalytically dead Nedd4 and Rsp5 mutants and
the WW domain 1 Nedd4 mutant, the Rsp5 mutant lacking WW domain 1 inhibits the
production of processed Spt23 and induces accumulation of precursor Spt23
(Fig. 5A). As with the WW
domain 1 Nedd4 mutant and Spt23, a protein complex comprising unprocessed
Spt23 and the WW domain 1 Rsp5 mutant was easily detected by
coimmunoprecipitation (Fig.
5B). These results indicate that similar to WW domain 1 of Nedd4,
WW domain 1 of Rsp5 is dispensable for binding but not
ubiquitin/proteasome-dependent processing of the membrane bound transcription
factor Spt23.
|
WW domain 1 and the C-terminal Cys are required for Nedd4-induced
proteasome-mediated degradation of Spt23 in mammalian cells
WW domain 1 is the most highly conserved of the WW domains between Rsp5 and
Nedd4, and it likely that it performs a similar function in higher eukaryotes.
Nedd4 has not yet been shown to induce ubiquitin/proteasome-dependent
processing of any mammalian targets. Therefore, we used Spt23 as a model
substrate to determine whether wt Nedd4 induces proteasome-dependent
proteolysis of the protein in transfected mammalian cells and whether an this
is dependent on WW domain 1 or the C-terminal Cys residue. Human H1299 cells
were transfected with a mammalian Spt23 expression vector alone or with
expression constructs encoding various Nedd4 proteins (performed in
duplicate). One set of transfections was left untreated while the other was
treated with the proteasome inhibitor MG115. Cells were harvested and an
immunoblot was performed to assess Spt23 expression status. It appears that
the mechanism regulating Spt23 processing is conserved since the expression
pattern of Spt23 in transfected mammalian cells is similar to that observed in
yeast (Fig. 6). Moreover,
treatment of cells with MG115 leads to an increase in unprocessed Spt23 and
reduces the amount of lower molecular weight product. Interestingly, wt Nedd4
induces degradation of unprocessed Spt23 and this is blocked by MG115
(Fig. 6). Fig. 6 also demonstrates that
Nedd4 mutants harboring substitutions in WW domain 1 or the ligase domain are
unable to induce Spt23 degradation. In addition, the level of unprocessed
Spt23 precursor is higher in cells transfected with these mutants, indicating
that they interact with Spt23 and prevent degradation by endogenous proteins.
We conclude from these experiments that the WW domain 1 of Nedd4 interacts
with a protein that promotes ubiquitin/proteasome-dependent degradation of
Spt23 in mammalian cells.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have found that ectopic expression of Nedd4 or Rsp5 mutants, which have
alterations in WW domains, blocks processing of the membrane-bound
transcription factor Spt23 in budding yeast. Considering that the WW domain 1
mutants are present in a Spt23 protein complex, it is likely that they are
able to interact with Spt23 (either directly or through adapter proteins) but
are unable to promote proteasome-dependent processing. Therefore, it is
reasonable to speculate on the basis of this data that the WW domain 1 binds
to a cofactor in yeast cells that is required for
ubiquitin/proteasome-dependent processing of Spt23 by Rsp5 and Nedd4. The
cofactor may promote the attachment of ubiquitin to a specific site on Spt23
that is needed for processing, control ubiquitin chain assembly on Spt23,
which confers a specific type of degradation signal or modulate the folding of
Spt23 so that it is processed in a limited manner. Past studies have shown
that Bul1 and Bul2 function in similar pathways to Rsp5
(Fisk et al., 1999;
Wolfe et al., 1999
;
Helliwell et al., 2001
;
Soetens et al., 2001
).
Interestingly, Bul1 interacts with Rsp5 but is not a substrate of the ligase
(Yashiroda et al., 1996
;
Yashiroda et al., 1998
). Also,
it has been demonstrated that Bul1 and Bul2 are involved in Rsp5-induced
degradation of Rog1 (Andoh et al.,
2000
), raising the possibility that they work either as adapter
proteins or as cofactors in the degradation of a subset of Rsp5 targets. We
are currently testing whether Bul1 and Bul2 bind to WW domain 1 of Rsp5 and
promote Spt23 processing. Obviously, future studies are warranted to identify
WW-domain1-binding factors and determine their mechanisms of action.
Although mutation in WW domain 1 of Rsp5 affects fluidphase endocytosis
(Dunn and Hicke, 2001;
Gajewska et al., 2001
), WW
domains 1 and 2 of Rsp5 are dispensable for its essential function under
normal growth conditions (Hoppe et al.,
2000
; Gajewska et al.,
2001
). Therefore, it is possible that WW domain 1 is not required
for Rsp5-induced processing of MGA2, the other membrane-bound transcription
factor that performs redundant functions with Spt23 and is involved in
ole1 transactivation (Zhang et
al., 1999
). Alternatively, the WW-domain1-binding protein may
interact with multiple Rsp5 WW domains. Considering that Rsp5 interacts with
itself (Dunn and Hicke, 2001
),
it is plausible that under conditions where WW domains 1 and 2 are not
operational, binding to the target is mediated via WW domain 3 of one molecule
of the Rsp5 dimer while binding to the cofactor occurs through the other.
Nevertheless, it will be interesting to determine if the WW domain 1 Rsp5
mutant possesses dominant negative activity on other Rsp5 targets, including
MGA2 and those that function in different endocytosis pathways
(Wang et al., 1999
;
Dunn et al., 2001
;
Gajewska et al., 2001
).
WW domain 1 of Nedd4 probably performs cofactor functions in mammalian
cells. Utilizing Spt23 as a model substrate, we found that wt Nedd4 induces
proteasome-dependent degradation of unprocessed Spt23, and this activity is
dependent on WW domain 1 and the C-terminal Cys. Moreover, because these
mutants induce stabilization of unprocessed Spt23, it is highly probable that
they form protein complexes with full-length Spt23 in transfected mammalian
cells. The abundance of ectopically expressed proteins in transfected H1299
cells is not nearly as high as in yeast, making it difficult to detect an
association by coimmunoprecipitation. Interestingly, wt Nedd4 does not promote
degradation of precursor Spt23 in yeast. Although we can not rule out the
possibility that Nedd4 induces partial proteolysis that leads to total
degradation of precursor, the results raise the possibility that the
WW-domain1-binding factor in H1299 cells promotes complete rather than partial
destruction of this substrate. Processed Spt23 is probably being generated by
another E3 ligase or a contranslational proteasome-dependent processing
mechanism (Lin et al., 1998)
that is not susceptible to the dominant-negative activity of the WW domain 1
or C-terminal Cys mutants. The WW domain 1 has been reported to be required
for downregulation of epithelial Na+ channel activity even though
it does not interact with channel subunits
(Harvey et al., 1999
). It has
been postulated that the WW domain 1 interacts with a protein that recruits
Nedd4 to the channel or stabilizes the interaction between Nedd4 and the
epithelial Na+ channel subunits
(Harvey et al., 1999
). On the
basis of our work with Spt23, we speculate that Nedd4 WW-domain1-binding
proteins function as cofactors in Nedd4-induced ubiquitination and degradation
of substrates.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abriel, H., Kamynina, E., Horisberger, J. D. and Staub, O. (2000). Regulation of the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4. FEBS Lett. 466,377 -380.[Medline]
Andoh, T., Hirata, Y. and Kikuchi, A. (2000). Yeast glycogen synthase kinase 3 is involved in protein degradation in cooperation with Bul1, Bul2, and Rsp5. Mol. Cell. Biol. 18,6712 -6720.
Boyd, M. T., Vlatkovic, N. and Haines, D. S.
(2000). A novel cellular protein (MTBP) binds to MDM2 and induces
a G1 arrest that is suppressed by MDM2. J. Biol. Chem.
275,31883
-31890.
Chang, A., Cheng, S., Espanel, X. and Sudol, M.
(2000). Rsp5 WW domains interact directly with the
carboxyl-terminal domain of RNA polymerase II. J. Biol.
Chem. 275,20556
-20571.
Dunn, R. and Hicke, L. (2001). Domains of the
Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase
endocytosis. Mol. Biol. Cell
12,421
-435.
Erdeniz, N. and Rothstein, R. (2000). Rsp5, a
ubiquitin-protein ligase, is involved in degradation of the
single-stranded-DNA binding protein rfal in Saccharomyces cerevisiae.
Mol. Cell. Biol. 20,224
-232.
Farr, T. J., Coddington-Lawson, S. J., Snyder, P. M. and McDonald, F. J. (2000). Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+ channel subunits. Biochem. J. 345,503 -509.[Medline]
Fisk, H. A. and Yaffe, M. P. (1999). A role for
ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae.J. Cell Biol. 145,1199
-1208.
Gajewska, B., Kaminska, J., Jesionowska, A., Martin, N. C.,
Hopper, A. K. and Zoladek, T. (2001). WW domains of Rsp5p
define different functions: determination of roles in fluid phase and uracil
permease endocytosis in Saccharomyces cerevisiae.Genetics 157,91
-101.
Guthrie, C. and Fink, G. R. (1991).Guide to Yeast Genetics and Molecular Biology , Academic Press, London.
Hamilton, M. H., Tcherepanova, I., Huibregtse, J. M. and
McDonnell, D. P. (2001). Nuclear import/export of hRPF1/Nedd4
regulates the ubiquitin-dependent degradation of its nuclear substrates.
J. Biol. Chem. 276,26324
-26331
Harvey, K. F. and Kumar, S. (1999). Nedd4-like proteins: an emerging family of ubiquitin protein ligases implicated in diverse cellular functions. Trends Cell Biol. 9, 166-169.[Medline]
Harvey, K. F., Dinudom, A., Komwatana, P., Jolliffe, C. N., Day,
M. L., Parasivam, G., Cook, D. I. and Kumar, S. (1999). All
three WW domains of murine Nedd4 are involved in the regulation of epithelial
sodium channels by intracellular Na+. J. Biol. Chem.
274,12525
-12530.
Helliwell, S. B., Losko, S. and Kaiser, C. A.
(2001). Components of a ubiquitin ligase complex specify
polyubiquitination and intracellular trafficking of the general amino acid
permease. J. Cell Biol.
153,649
-662.
Hershko, A. and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67,425 -479.[Medline]
Hicke, L. (1999). Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels. Trends Cell Biol. 9,107 -112.[Medline]
Hitchcock, A. L., Krebber, H., Frietze, S., Lin, A., Latterich,
M. and Silver, P. A. (2001). The conserved npl4 protein
complex mediates proteasome-dependent membrane-bound transcription factor
activation. Mol. Biol. Cell
12,3226
-3241.
Hoppe, T., Matuschewski, K., Rappe, M., Schlenker, S., Ulrich, H. D. and Jentsch, S. (2000). Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome dependent processing. Cell 102,577 -586.[Medline]
Huibregtse, J. M., Scheffner, M., Beaudenon, S. and Howley, P. M. (1995). A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. USA 92,2563 -2567.[Abstract]
Huibregtse, J. M., Yang, J. C. and Beaudenon, S. L.
(1997). The large subunit of RNA polymerase II is a substrate of
the Rsp5 ubiquitin-protein ligase. Proc. Natl. Acad. Sci.
USA 94,3656
-3661.
Kaiser, P., Flick, K., Wittenberg, C. and Reed, S. I. (2000). Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4. Cell 102,303 -314.[Medline]
Lin, L., DeMartino, G. N. and Greene, W. C. (1998). Cotranslational biogenesis of NF-kappaB p50 by the 26S proteasome. Cell 92,819 -828.[Medline]
Lu, P. J., Zhou, X. Z., Shen, M. and Lu, K. P.
(1999). Function of WW domains as phosphoserine- or
phosphothreonine-binding modules. Science
283,1325
-1328.
Palombella, V. J., Rando, O. J., Goldberg, A. L. and Maniatis, T. (1994). The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell 78,773 -785[Medline]
Pham, N. and Rotin, D. (2001). Nedd4 regulates ubiquitination and stability of the guanine nucleotide exchange factor CNrasGEF. J. Biol. Chem. [epub ahead of print].
Pickart, C. M. (2001) Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70,503 -533.[Medline]
Plant, P. J., Yeger, H., Staub, O., Howard, P. and Rotin, D.
(1997). The C2 domain of the ubiquitin protein ligase Nedd4
mediates Ca2+-dependent plasma membrane localization. J. Biol.
Chem. 272,32329
-32336.
Plant, P.J., Lafont, F., Lecat, S., Verkade, P., Simons, K. and
Rotin, D. (2000). Apical membrane targeting of Nedd4 is
mediated by an association of its C2 domain with annexin XIIIb. J.
Cell Biol. 149,1473
-1484.
Rizo, J. and Südhof, T. C.
(1998). C2-domains, structure and function of a universal Ca2+
binding domain. J. Biol. Chem.
273,15879
-15882.
Rotin, D., Staub, O. and Haguenauer-Tsapis, R. (2000). Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J. Membr. Biol. 176,1 -17.[Medline]
Soetens, O., De Craene, J. O. and Andre, B. (2001). Ubiquitin is required for sorting to the vacuole of the yeast Gap1 permease. J. Biol Chem. Aug 10 [epub ahead of print].
Staub, O., Dho, S., Henry, P., Correa, J., Ishikawa, T., McGlade, J. and Rotin, D. (1996). WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. EMBO J. 15,2371 -2380.[Abstract]
Stukey, J. E., McDonough, V. M. and Martin, C. E.
(1989). Isolation and characterization of OLE1, a gene affecting
fatty acid desaturation from Saccharomyces cerevisiae. J. Biol.
Chem. 264,16537
-16544.
Sudol, M. and Hunter, T. (2000). NeW wrinkles for an old domain. Cell 103,1001 -1004.[Medline]
Wang, G., Yang, J. and Huibregtse, J. M.
(1999). Functional domains of the Rsp5 ubiquitin protein ligase.
Mol. Cell. Biol. 19,342
-352.
Wang, G., McCaffery, J. M., Wendland, B., Dupre, S.,
Haguenauer-Tsapis, R. and Huibregtse, J. M. (2001).
Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within
the endocytic pathway. Mol. Cell. Biol.
21,3564
-3575.
Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J. and Chen, Z. J. (2001). TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412,346 -351.[Medline]
Wolfe, D., Reiner, T., Keeley, J. L., Pizzini, M. and Keil, R.
L. (1999). Ubiquitin metabolism affects cellular response to
volatile anesthetics in yeast. Mol. Cell. Biol.
19,8254
-8262.
Yashiroda, H., Oguchi, T., Yasuda, Y., Toh-E., A. and Kikuchi, Y. (1996). Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae. Mol. Cell. Biol. 16,3255 -3263.[Abstract]
Yashiroda, H., Kaida, D., Toh-E., A. and Kikuchi, Y. (1998). The PY-motif of Bul1 protein is essential for growth of Saccharomyces cerevisiae under various stress conditions. Gene 225,39 -46.[Medline]
Zhang, S., Skalsky, Y. and Garfinkel, D. J.
(1999). MGA2 or SPT23 is required for transcription of the delta9
fatty acid desaturase gene, OLE1, and nuclear membrane integrity in
Saccharomyces cerevisiae. Genetics
151,473
-483.