Biomedical Engineering Reference
In-Depth Information
CREI was detected in the nuclear fraction, while a strong signal was obtained
for the cytosolic fraction under all conditions. These data indicate that the
localisation of CREI is glucose-dependent, with CREI being translocated
from the cytosol to the nucleus when the fungus is grown on glucose thus
establishing repression of glucose-regulated genes. Under glucose deprived
conditions, CREI is capable of moving from the nucleus to the cytosol as
was shown by the weak detection of CREI in the nucleus of cells induced
on pectin (Vautard-Mey et al. 1999).
Phosphorylation of a serine residue has been shown to positively
regulate DNA-binding of the CREI proteins in both
Tr. reesei
and
Sc.
sclerotiorum
(Vautard-Mey and Fevre 2000, Cziferszky et al. 2002). Five
putative cyclic AMP-dependent protein kinase (AMPK) phosphorylation
motifs were identifi ed in the CREI protein of
Sc. sclerotiorum
and the Ser in
each motif was mutated to an Ala (Vautard-Mey and Fevre 2000). Mutation
of Ser
266
, which corresponds to Ser
241
in the
Tr. reesei
CREI, abolished
repressor activity suggesting that phosphorylation may play a role in the
repressor activity of the
Sc. sclerotiorum
CREI protein. Vautard-Mey and co-
workers suggested the potential involvement of an AMPK in the function
of the glucose repressor but the putative phosphorylation site does not fully
match the consensus motif for AMP-dependent protein kinases (Vautard-
Mey and Fevre 2000).
Subsequent fi ndings in
Tr. reesei
confi rmed that phosphorylation of
Ser
241
of CREI (Ser
266
in CREI of
Sc. sclerotiorum
) is important for its repressor
activity (Cziferszky et al. 2002). A region of the
cre1
gene from
Tr. reesei
containing several putative phosphorylation sites for protein kinases,
(protein kinase C, casein kinase II and cAMP- or cGMP-dependent protein
kinase), was expressed as a Glutatione-S-transferase (GST) fusion protein in
E. coli.
To identify the amino acid serving as an acceptor for phosphorylation,
C
-terminal truncated versions of the
cre1
fragment were generated also.
On CREI from
Tr. reesei
, Ser
241
was found to be phosphorylated (Cziferszky
et al. 2002). This observation contrasts with
S. cerevisiae
Mig1 where
phosphorylation was shown to have no effect on DNA-binding (Treitel
and Carlson 1995). In the same study, the effect of phosphorylation on the
ability of CREI to bind DNA was also investigated (Cziferszky et al. 2002).
Dephosphorylation of the CREI protein resulted in reduced DNA binding
in vitro
and loss of DNA binding in the presence of cell-free extracts.
Rephosphorylation of the dephosphorylated CREI protein restored its
DNA binding ability. However, when Ser
241
was mutated to an Ala a loss
of DNA binding was not observed. The mutant protein (Ser
241
to Ala
241
)
was capable of binding to its target sequence even though the protein could
not be phosphorylated. Cziferszky and co-workers have suggested that
CREI requires the phosphorylation of Ser
241
in order to bind DNA when
Ser
241
is present in the protein, but that the absence of Ser
241
allows CREI to
