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Research
We study
posttranscriptional mechanisms of cellular control in the fission yeast
Schizosaccharomyces pombe and in mammalian
cancer cells. Our work focusses
on three major areas:
(1.) Function and
control
of fission yeast cullin/RING ubiquitin ligases (CRLs)
(2.) Mechanisms
controlling
mRNA translation in fission yeast
(3.) The role of
ubiquitin-dependent proteolysis of tumor suppressors in prostate cancer
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1.1. CRL Control by CSN
CRLs
represent an extensive class of multisubunit E3 ubiquitin ligases each
consisting
of a core module containing a member of the cullin family and the RING
domain
protein Rbx1p, which recruits the E2 ubiquitin conjugating enzymes
(UBCs)
to the ligase. This core is joined by one of several hundred adapter
proteins
each of which targets a distinct array of substrates for ubiquitylation
and
proteasomal degradation. The
COP9 signalosome
(CSN) complex removed the stimulatory
modification by the ubiquitin-related peptide Ned8p from cullins. We have found that fission yeast
Cul3p
is subject to the same regulation by the CSN. Importantly, we also
identified a family of BTB/POZ
domain
containing proteins as putative substrate adapters of Cul3p.
Surprisingly, CSN inhibits CRL in vitro ubiquitylation activity, but
stimulates CRL-dependent substrate degradation in vivo. Cul3p
inhibition is mediated by CSN-dependent deneddylation and by the
CSN-associated
deubiquitylating enzyme Ubp12p. From these findings
we developed the model that the CSN/Ubp12p assists in CRL assembly
by preventing autocatalytic adapter instability.
Our
current work focuses on further testing this model, using Cul3p/Btb3p
complexes as our system.
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1.2. Identification of CRL substrates
Although several
hundred putative CRLs were identified in the human genome, substrates
are known for only a handful of them. Whereas CRLs are relatively easy
to identify based on conserved motifs (F-box, BTB domains), their
substrates seem to have little more in common than critical lysine
residues. To date, no techniques have been established to
systematically identify CRL substrates. We are using a series of
genetic, biochemical, and proteomic techniques to identify substrates
of CRLs and other RING E3s. Our approaches include affinity
purification of E3s, two-hybrid interaction screens, and in vitro
ubiquitylation assays to screen for novel substrates. Several
candidate substrates for fission yeast and human CUL3 are currently
being validated.
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2. Control of mRNA Translation
Like proteolysis, translational control has been
widely
implicated in the regulation of gene expression. Translation is
controlled
by the complex process of the stepwise assembly of translation
initiation
factors (eIFs) with mRNA and ribosomes. Our present activities in
this area include
the isolation and subunit characterization of fission yeast eIF
complexes. We have obtained evidence that fission yeast encodes two
distinct eIF3 complexes that are distinguished by a different set of
PCI domain proteins. We are currently pursuing the hypothesis that
these complexes regulate the translation of a distinct set of mRNAs. We
are also analyzing other eIF complexes to determine whether they are
regulated by cellular stress or signal transduction pathways.
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3. Studies on Prostate Cancer
The expression
of the CDK inhibitor p27 is primarily regulated through proteolysis
mediated by the SCF-SKP2 ubiquitin ligase. Despite high levels of p27
RNA, p27 protein is greatly reduced in many prostate carcinomas,
indicating hyperactive proteolysis. The SCF-SKP2 ubiquitin ligase would
thus seem an ideal target to interfere with p27 depletion in these
cancers. We are therefore developing strategies to screen for small
molecules able to disrupt the SKP2/p27 complex.
Secondly, we are studying the regulation
of the putative prostate tumor suppressor NKX3.1. One allele
of this gene is frequently lost in prostate cancers, whereas mutations
of the remaining allele are rare. Nevertheless, NKX3.1 protein
expression is still missing from many prostate cancers. We found that
NKX3.1 is an unstable protein that is degraded by the proteasome. Our
current efforts are aimed at identifying the NKX3.1-specific ubiquitin
ligase. We will then determine whether the proteolytic mechanisms of
NKX3.1 control is hyperactive in prostate cancers.
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Publications
Schmidt, M.W., Houseman, E.A.,
Ivanov, A.R, Wolf, D.A. (2007) Comparative proteomic and transcriptomic
profiling of the fission yeast Schizosaccharomyces pombe. Molecular Systems Biology, 3:79
[PDF File]
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Zhou,
C., Arslan, F., Wee, S., Krishnan, S., Ivanov, A.R., Oliva, A.,
Leatherwood,
J., Wolf, D.A. (2005) PCI proteins eIF3e and eIF3m define
distinct translation initiation factor 3 complexes. BMC
Biology, 3:14
[PDF File]
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Wee,
S., Geyer, R., Toda, T., Wolf, D.A. (2005) CSN facilitates cullin-RING
ubiquitin ligase function by counteracting autocatalytic adapter
instability. Nature
Cell Biology 7, 387-391
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Doud, M.K.,
Schmidt, M.W., Hines, D., Naumann, C., Kocourek, A., Kashani-Poor, N.,
Zeidler, R., Wolf,
D.A. (2004) Rapid prefractionation of complex protein lysates with
centrifugal membrane adsorber units improves the resolving power of
2D-PAGE-based proteome analysis. BMC
Genomics 5:25 [PDF File]
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Wolf, D.A.,
Wee, S., Zhou, C. (2003)
The COP9 signalosome: an assembly and maintenance
platform for cullin ubiquitin ligases? Nature
Cell Biology 5, 1029-1033
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Schmidt, M.,
Jain, A., Wolf. D.A. (2003) Multidimensional proteomic analysis of
proteolytic pathways involved in cell cycle control. In: Cell
Cycle Checkpoint Control Protocols. Lieberman H. B. ed. New
York: Humana Press (2003) Vol. 241: 235-245
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Geyer, R.,
Wee, S., Anderson, S., Yates J.R.III, Wolf. D.A. (2003) BTB/POZ domain
proteins are putative substrate adaptors for cullin 3 ubiquitin
ligases. Molecular Cell 12, 783-790
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Wolf, D.A., Geyer, R. (2003)
Dynamic release of Cdc34 from SCF: The hand that rocks the cradle. Cell 114, 532-533
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Zhou, C.;
Wee, S.; Rhee, E.; Naumann, M.; Dubiel, W.; Wolf, D.A. (2003) Fission
yeast COP9/signalosome suppresses cullin activity through recruitment
of the deubiquitylating enzyme Ubp12p. Molecular
Cell 11, 927-938
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Wee, S., Hetfeld, B., Dubiel, W., Wolf, D.A.
(2002) Conservation of the COP9/signalosome in budding yeast. BMC
Genetics 3:15 [PDF
File]
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Lu, L., Schulz, H., Wolf, D.A. (2002)
The F-box protein SKP2 mediates androgen control of p27 stability in
LNCaP human prostate cancer cells. BMC Cell Biology
3:22 [PDF File]
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Seibert, V., Prohl, C., Schoultz, I., Rhee, E.,
Lopez, R., Abderazzaq, K., Zhou, C., Wolf, D.A. (2002) Combinatorial
diversity of fission yeast SCF ubiquitin ligases by homo- and
heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p. BMC
Biochemistry 3:22 [PDF File]
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Zhou, C.; Seibert, V.; Geyer, R.; Rhee,
E.; Lyapina, S.; Cope, G.; Deshaies, R.J.; Wolf, D.A. (2001) The
fission yeast COP9/signalosome is involved in cullin modification by
ubiquitin-related Ned8p. BMC
Biochemistry 2:7 [PDF File]
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| Lyapina, S.;
Cope, G.; Shevchenko, A.; Serino, G.; Tsuge, T.; Zhou, C.; Wolf, D. A.;
Wei, N.; Shevchenko, A.; Deshaies, R. J. (2001). Promotion of
NEDD8-CUL1 conjugate cleavage by COP9 signalosome. Science 292, 1382-1385 |
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