Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1
Natalia Shcherbik1,
Sharad Kumar2 and
Dale S. Haines*1
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
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Fig. 1. Nedd4 WW domain 1 mutants block the growth of budding yeast. (A) The known
functional domains of human Nedd4 and consensus amino acids of the WW domain
are depicted. The amino acid underlined is the residue of WW domain 1 that was
mutated in the toxic Nedd4 clone as well as the one that was changed to an Ala
by site-directed mutagenesis. (B) Cells were transformed with pYes (V),
pYes-wt Nedd4 (wt) and the pYes constructs encoding the WW domain 1 point
(WW1) or deletion ( WW1) mutants. Single colonies were picked from
plates and grown overnight to equal densities in glucose media. Cells were
diluted to varying degrees and streaked onto glucose and galactose agar
plates. Cells were incubated for 3 days. It should be noted that multiple
clones from each transformation were analyzed and the results obtained were
identical to those presented. (C) Cells transformed with the same constructs
presented in Fig. 1B were grown
in glucose media overnight. Cells were pelleted and resuspended in glucose (-)
or galactose (+) media. Cultures were incubated for 8 hours. Cells were
pelleted, protein extract was prepared and the amount of Nedd4 was measured by
a western blot. Ponceau S staining of blots after transfer revealed equivalent
loading of total protein.
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Fig. 2. An ole1 cDNA or oleic acid suppresses the toxic activity of the WW
domain 1 mutant, and the WW domain 1 mutant inhibits ole1 gene
expression and Spt23 processing. (A) Cells containing pYes-WW1 were
transformed with the ole1 cDNA expression construct (ole1)
or an empty vector control (V). Cells were processed as described in the
legend of Fig. 1B. For the
oleic acid experiment, cells containing pYes-WW1 were plated onto either plain
galactose media (-) or galactose media that had been supplemented with 0.5 mM
of this unsaturated fatty acid. (B) Cells transformed with pYes (V) or
pYes-WW1 (WW1) were picked and processed as described in the Materials and
Methods. RNA was isolated from cells, separated on agarose gels containing
formaldehyde, transferred to nylon membrane and an ole1 northern blot
was performed. Also depicted is a picture of the ethidium-bromide-stained gel
prior to transfer showing equivalent loading of RNA. (C) Cells were
transformed first with the pESC-FLAG-Spt23 expression construct and then with
pYes (v) or pYes-WW1 (WW1). Transformed cells were grown in glucose media
overnight. Cells were pelleted and resuspended in galactose media. Cultures
were harvested 6 hours later for the preparation of protein extract.
Measurement of Nedd4 protein in the extract was performed by a western blot
using the anti-Nedd4 polyclonal antibody while the amount of membrane-bound
(MB) and processed Spt23 was measured by the anti-FLAG antibody M5. Ponceau S
staining of the blot after transfer revealed equivalent loading of total
protein.
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Fig. 3. The WW domain 1 Nedd4 mutant interacts with membrane-bound Spt23. (A) Cells
transformed with Spt23 and the indicated Nedd4 expression constructs or vector
alone control (V) were grown as described in the legend of
Fig. 2C and lysed in RIPA
buffer. The extract (600 µg per immunoprecipitation reaction) was
pre-cleared for 1 hour with protein-G sepharose. After a brief centrifugation,
the supernatant was transferred to a new tube containing 1 µg of the
anti-FLAG antibody M5 or an isotype control (C). After a 4 hour incubation,
protein-G sepharose was added and the mixtures were incubated for an
additional 2 hours. Protein complexes were washed three times with cold lysis
buffer, eluted from beads by resuspending in 1x SDS-PAGE loading buffer,
and Nedd4 westerns were performed as described above. Ponceau S staining of
the blot after transfer revealed equivalent amounts of immunoprecipitating
antibody. (B) Immunoprecipiations were performed as described in (A), except
that the Nedd4 polyclonal was used in the immunoprecipitations and the M5
antibody was used for the western blot.
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Fig. 4. Inhibition of Spt23 processing by the WW domain 1 mutants requires WW
domains 2 and 3, and both of these C-terminal WW domains are required for
Spt23 binding. (A) Cells were first transformed using the pESC-FLAG-Spt23
expression construct and then with pYes (v), pYes-wt Nedd4 (wt), pYes-WW1 (-)
and pYes-WW domain 1 mutant Nedd4 expression vectors harboring substitutions
in the indicated WW domains. Cells were processed as described in the legend
of Fig. 2C. Measurement of
Nedd4 protein in the extract was performed by a western blot using the
anti-Nedd4 polyclonal while the amount of membrane-bound (MB) and processed
Spt23 was measured by the anti-FLAG tag antibody M5. Ponceau S staining of the
blot after transfer revealed equivalent loading of total protein. (B) An equal
amount of extract prepared from cells producing FLAG-tagged Spt23 and the
indicated Nedd4 proteins were subjected to immunoprecipitation with the
anti-FLAG antibody M5 or an isotype control antibody (C). The amount of Nedd4
protein immunoprecipitated with Spt23 was then determined by a western blot.
Ponceau S staining of the blot after transfer revealed equivalent amounts of
immunoprecipitating antibody.
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Fig. 5. Spt23 processing is inhibited by WW-domain1- or ligase-defective Nedd4 and
Rsp5 mutants. (A) Cells were transformed first with the pESC-FLAG-Spt23
expression construct and then with the indicated Nedd4 and Rsp5 expression
constructs. Cells were processed as described in the legend of
Fig. 2C. The amount of
membrane-bound (MB) and processed Spt23 was measured by the anti-FLAG tag
antibody M5. Ponceau S staining of the blot after transfer revealed equivalent
loading of total protein. (B) Equal amount of extract prepared from cells
producing FLAG-tagged Spt23 and the indicated Rsp5 proteins were subjected to
immunoprecipitation with the anti-HA antibody 12CA5 (12) or an isotype control
antibody (C). The amount of Nedd4 protein immunoprecipitated with Spt23 was
then determined by a western blot. Ponceau S staining of the blot after
transfer revealed equivalent amounts of immunoprecipitating antibody.
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Fig. 6. The WW domain 1 is required for proteasome-dependent degradation of Spt23
in mammalian cells. H1299 cells were transfected with pCEP-FLAG-Spt23 and pCEP
(V), pCEP-wt Nedd4 (wt) or pCEP containing the indicated Nedd4 mutants by
Lipofectamine. After transfection, cells were re-fed with complete media (-)
or media containing the proteasome inhibitor MG115 (+). Cells were incubated
for 9 hours prior to harvesting. Extracts were prepared as described in the
Materials and Methods and western blots were performed with the Nedd4
polyclonal or the anti-FLAG antibody M5. Ponceau S staining of the blot after
transfer revealed equivalent loading of total protein.
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© The Company of Biologists Ltd 2002
