Ee. Traverso et al., Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast, J BIOL CHEM, 276(24), 2001, pp. 21924-21931
In the budding yeast Saccharomyces cerevisiae, the multifunctional protein
Net1 is implicated in regulating the cell cycle function of the Cdc14 prote
in phosphatase. Genetic and cell biological data suggest that during interp
hase and early mitosis Net1 holds Cdc14 within the nucleolus where its acti
vity is suppressed. Upon its transient release from Net1 at late anaphase,
active Cdc14 promotes exit from mitosis by dephosphorylating targets in the
nucleus and cytoplasm. In this paper we present evidence supporting the pr
oposed role of Net1 in regulating Cdc14 and exit from mitosis, We show that
the NH2-terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is
a highly specific competitive inhibitor of its activity (K-i = 3 nM) with
five different substrates including the physiologic targets Swi5 and Sic1.
An analysis of truncation mutants indicates that the Cdc14 binding site is
located within a segment of Net1 containing residues 1-341. We propose that
Net1 inhibits by occluding the active site of Cdc14 because it acts as a c
ompetitive inhibitor, binds to a site located within the catalytic domain (
residues 1-374), binds with reduced affinity to a Cdc14 C283S mutant in whi
ch an active site Cys is replaced, and is displaced by tungstate, a transit
ion state analog known to bind in the catalytic site of protein-tyrosine ph
osphatases.