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Scientific Report 2005
Molecular Biology
Regulating Cell Proliferation: Flipping Transcriptional and Proteolytic Switches
C. Wittenberg, M. Ashe, R. de Bruin, M. Guaderrama, B.-K. Han, T. Kalashnikova,
N. Spielewoy
Cell proliferation is governed primarily by controlling the activities of positive and
negative regulators of cell-cycle transitions. Inhibitors of cyclin-dependent protein
kinase (CDK) and the positive regulatory subunits, cyclins, are critical in establishing
the proper timing of cell-cycle transitions and in imposing cell-cycle checkpoints.
The activities of those proteins are largely regulated via periodic transcriptional
activation coupled with regulated proteolysis. We focus primarily on those regulatory
mechanisms.
As in animal cells, initiation of the cell cycle in the budding yeast Saccharomyces cerevisiae
occurs during late G1 phase and is governed by the controlled accumulation
of G1 CDK activity. A large family of G1-specific genes, including
those for the G1 cyclins Cln1 and Cln2, are coordinately regulated by
2 transcription factors: SBF and MBF. As in metazoans, the transcriptional activation
of those genes depends on the activity of a distinct G1 cyclin, Cln3,
that acts on promoter-bound transcription factors to promote recruitment of components
of the RNA polymerase II complex.
By analogy with metazoan Rb, an inhibitor of the E2F transcription factor that is antagonized
by cyclin D/CDK, we predicted the existence of a G1-specfic transcriptional
repressor that is inactivated by Cln3/CDK. Using the combined application of molecular
genetics and mass spectrometry–based multidimensional protein identification
technology, we identified an SBF-specific transcriptional repressor, Whi5, that
is inactivated via phosphorylation by Cln3/CDK. This discovery provides a unifying
mechanism for initiation of the cell cycle in yeast and metazoans.
We also identified several other transcriptional regulators, including Nrm1, a novel cell cycle–dependent
repressor of MBF-dependent transcription. Rather than repressing expression early
in the cell cycle as Whi5 does, Nrm1 acts as cells pass into S phase, thereby limiting
MBF-dependent gene expression to the G1 phase. Because expression of
the gene for NRM1 depends on MBF, the gene cannot act until MBF becomes active.
Consequently, the gene confers negative autoregulation on
MBF. Additional factors associated with the 2 transcription factors are under investigation.
One of the primary roles of G1 cyclin-associated CDKs is to promote the ubiquitin-dependent
proteolysis of cell-cycle regulators, including the G1 cyclins themselves.
CDK-dependent phosphorylation of a number of proteins targets the proteins for recognition
by the Cdc34-SCF ubiquitin ligase complex. Grr1, one of several distinct F box proteins
that associate with that complex, confers recognition of specific phosphorylated
targets. We are interested in the molecular basis of that recognition. Previously,
we showed that the interaction between Grr1 and Cln2 requires basic residues residing
in the pocket of the leucine-rich repeat of Grr1 and defined a transferable “degron”
in the C terminus of Cln2 that is phosphorylated by the CDK. These findings, combined
with our understanding of the mechanisms that govern G1-specific transcription,
indicate that an integrated autoregulatory circuit governs the events of G1
phase and ensures the orderly progression of events in the cell cycle.
In addition to its role in cell-cycle control, SCFGrr1 plays a central role in regulating
the expression of genes induced by glucose and amino acids. We showed that the glucose
signal promotes ubiquitin-mediated proteolysis of Mth1, which is required for maintenance
of transcriptional repression of glucose-inducible genes. Glucose triggers phosphorylation
of Mth1 by casein kinase I, thereby promoting recognition by SCFGrr1.
Surprisingly, recognition of phosphorylated Mth1 requires properties of Grr1 distinct
from those required for recognition of phosphorylated G1 cyclins. The
same properties are also important for Grr1-dependent recognition of an as yet unknown
target required for the activation of amino acid–regulated genes via SPS signaling.
Efforts are under way to identify novel targets of Grr1 and to investigate the possibility
that Grr1 mediates the coordination of cell-cycle progression with the availability
of environmental nutrients.
Publications
Flick,
K., Wittenberg, C.
Multiple pathways for suppression of mutants affecting G1-specific transcription
in Saccharomyces cerevisiae. Genetics 169:37, 2005.
Wittenberg,
C. Cell cycle: cyclin
guides the way. Nature 434:34, 2005.
Wittenberg,
C., Reed, S.I. Cell
cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes.
Oncogene 24:2746, 2004.
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