Download ExoS binds its co-factor 14-3-3 through a non

A64
Biochemical Society Transactions (2002) Volume 30, Part 3
D10 IGF- 1 induces phosphorylation of two novel residues of
FKHR
Graham Rena, Yvonne L. Woods, Alan R. Prescott,
Mark Peggie, Terry G.Unterman, Michayla R. Williams and
Philip Cohen
M R C Protein Phosphorylation Unit, School of Life Sciences,
MSI/WTB Complex, University of Dundee, DOWStreet, Dundee.
DDl5 EH, Scotland
Please refer to abstract number 82. This abstract was originally
submitted as a poster, and on the basis of its scientific interest and
merit, was chosen by the colloquium organizers to be presented as
an oral communication, as well as a poster.
D11 Multiple roles for 14-3-3 proteins
in nucleo-cytoplasmic
transport
A. Brunet, F. Kanai, J. Stehn, M.E. Greenberg, M.B. Yaffe
Center for Cancer Research E18-580, Massachusetts Institute of
Technology, 77 Massachusetts Avenue, Cambridge M A 02139
14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling
molecules with which they interact. Although most 14-3-3
molecules in unsynchronized cells arc found within the cytoplasm,
the majority of potential 14-3-3 ligands are found within the
nucleus. In the absence of bound ligands, 14-3-3 itself relocalizes to
the nuclear compartment, and endogenous 14-3-3 proteins can be
trapped in the nucleus by treating cells with Leptomycin-B,
suggesting that 14-3-3 binding to some ligands may normally occur
within thc nucleus. In agreement with this, we show that phosphorylation of the transcription factor FKHRLl at its 14-3-3 binding
site occurs within the nucleus immediately prior to F K H R L l relocalization to the cytoplasm. Efficient nuclear export of F K H R L l
requires both intrinsic NES sequences within F K H R L l and phosphorylation/l4-3-3-binding.In addition,
phosphorylation/l4-3-3-bindingmay also prevent F K H R L l nuclear
re-import. The leucinc rich region within the C-terminal a-helix of
14-3-3, which had been proposed to function as an NES, does not
directly mediate nuclear transport, but instead functions globally in
ligand binding. Thus, 14-3-3 proteins likely function as phosphodependent molecular chaperones, facilitating exposure of ligand NES
sequences or masking ligand NLS sequences to regulate nucleocytoplasmic trafficking.
0 2002 Biochemical Society
D12 14-3-3 as cruciform-binding
D N A rcplication protein
M. ZannisHadjopoulos, 0.Novac, D. Alvarez, G.B. Price
McGill Cancer Centre, 3655 Promenade Sir William Osler,
Montreal, Quebec H3G 1 Y6, C A N A D A
We purified from human (HeLa) cells a 66 kDa cruciform binding
protein, CBP, with binding specificity for cruciform D N A
regardless of its sequence. C B P belongs to the 14-3-3 protein family,
consisting of isoforms E, p, y, 5 and u. D N A cruciforms have been
implicated in the regulation of initiation of D N A replication. Using
a chromatin immunoprecipitation (CHIP) assay and quantitative
PCR analysis, we found that CBP/14-3-3 associates in vivo with the
monkey replication origins or58 and on12 in a cell cycle dependent
manner, being maximal at the G l / S boundary. The CBP-14-3-3
cruciform binding activity was also maximal at GI/S. The involvement of 14-3-3 in mammalian D N A replication was analyzed by
studying the effect of anti-14-3-3~,p, y, 5 and (r antibodies on the in
vitro replication of p186, a plasmid containing the minimal rcplication origin of ors8. Anti-14-3-3~,y, 5 and u antibodies inhibited
p186 replication by approximately 50-80%, while anti-14-3-3P
antibodies had a lesser effect. All of these antibodies interfered with
CBP’s binding t o cruciform D N A . The results indicate that
CBP/14-3-3 is a replication origin binding protein, acting at the
initiation step of D N A replication by binding to cruciformcontaining D N A , and dissociates after origin firing.
D13 ExoS binds its co-factor
14-3-3 through a non-phosphorylated
motif
B. Hallberg
Dept. of Medical Biosciences, Umea University, S-901 87 Umea,
Sweden
The interactions between 14-3-3 and many signalling proteins, such
as Raf and KSR, have been described to occur in a phospho-specific
manner. Lately, non-phosphorylation dependent interactions have
been observed between 14-3-3 and several proteins, such as 5-phosphatase, p75NTR-associated cell death executor (NADE) and
Exoenzyme S (ExoS; a bacterial ADP-ribosyltransferase toxin).
The motif required for the ExoS interaction with 14-3-3 and its
resultant activity both in vitro and in vivo has not been defined. We
have approached this issue using deletion and substitution analysis
of ExoS both in vitro and of transfected ExoS constructs into cell
lines. Moreover, in vivo an bacterialy expressed ExoS protein lacking
the 14-3-3 binding site has reduced capacity to A D P ribosylatc
cytoplasmatic proteins, cg., Ras, and shows reduced capacity to
change the morphology of infected cells. Amino acid residues on
ExoS, which arc responsible for its specific interaction with 14-3-3,
will be discussed.