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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structure of cdc42 in a complex with the gtpase-Binding domain of the cell polarity protein, Par6.
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Authors
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S.M.Garrard,
C.T.Capaldo,
L.Gao,
M.K.Rosen,
I.G.Macara,
D.R.Tomchick.
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Ref.
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EMBO J, 2003,
22,
1125-1133.
[DOI no: ]
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PubMed id
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Abstract
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Cdc42 is a small GTPase that is required for cell polarity establishment in
eukaryotes as diverse as budding yeast and mammals. Par6 is also implicated in
metazoan cell polarity establishment and asymmetric cell divisions. Cdc42.GTP
interacts with proteins that contain a conserved sequence called a CRIB motif.
Uniquely, Par6 possesses a semi-CRIB motif that is not sufficient for binding to
Cdc42. An adjacent PDZ domain is also necessary and is required for biological
effects of Par6. Here we report the crystal structure of a complex between Cdc42
and the Par6 GTPase-binding domain. The semi-CRIB motif forms a beta-strand that
inserts between the four strands of Cdc42 and the three strands of the PDZ
domain to form a continuous eight-stranded sheet. Cdc42 induces a conformational
change in Par6, detectable by fluorescence resonance energy transfer
spectroscopy. Nuclear magnetic resonance studies indicate that the semi-CRIB
motif of Par6 is at least partially structured by the PDZ domain. The structure
highlights a novel role for a PDZ domain as a structural scaffold.
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Figure 2.
Figure 2 Crystal structure of Par6B(126 -253) bound to
Cdc42(Q61L)  GMPPNP
at 2.1 Å resolution. Ribbon diagram with Cdc42 colored blue and
Par6 yellow (left and center images). A structure of the WASP
-Cdc42 complex (Abdul-Manan et al., 1999) is also shown on the
right, for comparison. The Switch I and II regions of Cdc42 are
colored red, the semi-CRIB motif of Par6 and the CRIB motif of
WASP are colored black, and the GMP-PNP/Mg2+(H[2]O)[2] moiety is
presented in ball-and-stick representation. A short region of
the PDZ domain of Par6 (residues 204 -208) that makes contact
with Cdc42 is highlighted in magenta. The center image is of the
complex rotated by 90° about the vertical axis. The black arrow
indicates the location of the PDZ ligand-binding pocket.
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Figure 6.
Figure 6 Overlaid 1H/15N HSQC spectra of the Par6 PDZ domain
(residues 152 -253, red) and CRIB -PDZ elements (residues 126
-253, black).
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2003,
22,
1125-1133)
copyright 2003.
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Secondary reference #1
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Title
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Assembly of epithelial tight junctions is negatively regulated by par6.
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Authors
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L.Gao,
G.Joberty,
I.G.Macara.
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Ref.
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Curr Biol, 2002,
12,
221-225.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. The N-Terminal Domain of aPKC but Not of Par6B
Inhibits TJ Assembly(A) MDCK cells were transiently transfected
with plasmids encoding Par6B (1-125), aPKCĪ¶ (1-126), or aPKCĪ¶
(1-126, V[62,66]A). Cells were fixed and stained for the myc
epitope and ZO-1.(B) Quantification of TJ assembly defect,
showing percentages of cell-cell contacts between transfected
cells still incomplete 6 hr after calcium readdition.(C)
Alignment of OPR motifs. Stars show aPKC residues that were
mutated. Hs, Homo sapiens; Mm, Mus musculus, Rn, Rattus
norvegicus; Sc, Saccharomyces cerevisiae.(D) aPKCĪ¶ (1-126,
V[62,66]A) does not coimmunoprecipitate with Par6B. COS cells
were cotransfected with plasmids encoding HA[3]-Par6B or
HA[3]-Par6B (102-373) and myc-aPKCĪ¶ (1-126) or aPKCĪ¶ (1-126,
V[62,66]A) as indicated. Par6B was immunoprecipitated and
blotted for the myc epitope. CL = cell lysate (1/10 of total).
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Figure 4.
Figure 4. Cdc42 Activation Is Not Required for TJ
Assembly(A) MDCK cells expressing myc-Cdc42 (T17N) under control
of doxycycline (DC) were grown ± 20 ng/ml DC till
confluence and then subjected to calcium switch. Cells were
fixed 6 hr later and stained for myc-epitope and ZO-1.(B)
Transient expression of the CRIB domain of Ī³PAK has no effect
on TJs. Cells were fixed and stained for the myc epitope and
ZO-1 24 hr posttransfection.(C) Par6ĪPro[136] does not
coimmunoprecipitate Cdc42. COS cells were transfected with
plasmids encoding HA[3]-Cdc42 (Q61L) and/or myc-Par6B or
myc-Par6BĪPro[136] as indicated. Par6B was immunoprecipitated,
and bound Cdc42 was detected by immunoblot. CL = cell lysate
(1/10 of total).(D) Effect of myc-Par6BĪPro[136] on TJ
formation.(E) Percentage of cells lacking complete TJs between
cells that express myc-Par6B or myc-Par6BĪPro[136] at 6 hr
postcalcium addition.
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #2
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Title
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The cell-Polarity protein par6 links par3 and atypical protein kinase c to cdc42.
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Authors
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G.Joberty,
C.Petersen,
L.Gao,
I.G.Macara.
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Ref.
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Nat Cell Biol, 2000,
2,
531-539.
[DOI no: ]
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PubMed id
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Figure 5.
Figure 5. Par6 binds directly and specifically to atypical
PKCs. a, The PDZ-domain mutant of Par6B can efficiently
immunoprecipitate PKC  /
 .
HA[3] -Par6B proteins were immunoprecipitated (IP) from cell
lysates and the immunoprecipitates were blotted for endogenous
PKC /
and
Par3 (upper panel), and for HA[3] -Par6B (middle panel). In the
control, no antibody was added during immunoprecipitation. The
PDZ mutant (mut.PDZ) harbours KPLG167 -170AAAA mutations.
Immunoblots of the cell lysates are shown in the lower panel. b,
Conjugation assays. HF7c transformed with pVP16 encoding the
N-terminal region of PKC /
or
with pVP16 encoding Cdc42(L61) were mated to W303 transformed
with pGBT9 plasmids encoding Par6A, Par6B, Par6B(1 -271),
Par6B(102 -271) or Par6C. c, Par 6B(102 -271) does not
co-precipitate with PKC /
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HA[3] -Par6B proteins were immunoprecipitated from COS cells and
immunoblotted for associated endogenous PKC /
(upper
panel) and for HA[3] -Par6B (middle panel). In the control, no
antibody was added during immunoprecipitation. Immunoblots of
cell lysates are shown in the lower panel. d, Par6 binds
directly to atypical PKCs. Recombinant S -Par6B(1 -271) attached
to S-protein -agarose beads was incubated with GST or the
indicated recombinant atypical PKC GST-fusion fragments.
Complexes were immunoblotted for GST (left and middle panels).
One-tenth of the protein input was also immunoblotted (right
panel). e, Par6 binds to atypical PKCs but not to novel or
conventional PKCs. HA[3] -Par6B(1 -271) was immunoprecipitated
from transfected COS cells and immunoblotted for PKC /
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PKC  ,
PKC  and
PKC  .
L, cell lysates; IP, immunoprecipitation. In the control, no
antibody was used in the immunoprecipitation.
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Figure 6.
Figure 6. Expression of Par6 disrupts tight junctions but not
adherens junctions. MDCK cells were mock-transfected (a, b)
or transfected with vector encoding Myc-tagged Par6B (c -o).
Cells were fixed and stained for Myc -Par6B (red; c, e, g, i, j,
l, n), and for the following endogenous proteins (all green):
ZO-1 (b, i), Par3 (a, d, f, h), PKC (k),
E-cadherin (m) and  -catenin
( o). Nuclei were labelled with DAPI (blue). For images l and n,
a polyclonal anti-Myc antibody was used to show the localization
of Myc -Par6B. PDZ-mutant Par6B (g, h) harbours KPLG167 -170AAAA
mutations.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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Secondary reference #3
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Title
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A mammalian par-3-Par-6 complex implicated in cdc42/rac1 and apkc signalling and cell polarity.
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Authors
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D.Lin,
A.S.Edwards,
J.P.Fawcett,
G.Mbamalu,
J.D.Scott,
T.Pawson.
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Ref.
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Nat Cell Biol, 2000,
2,
540-547.
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PubMed id
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Secondary reference #4
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Title
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A human homolog of the c. Elegans polarity determinant par-6 links rac and cdc42 to pkczeta signaling and cell transformation.
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Authors
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R.G.Qiu,
A.Abo,
G.Steven martin.
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Ref.
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Curr Biol, 2000,
10,
697-707.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Par-6 is conserved in evolution. (a) Full-length
hPar-6 and the 2HP-4 clone (hPar-2HP) are shown schematically.
CRIB, Cdc42/Rac interactive binding motif; PDZ, PDZ domain. The
residue numbers indicate the approximate limits of each domain.
(b) Tissue distribution of hPar-6 mRNA. The size markers (in kb)
are shown at the left. The blot was also probed with actin cDNA
as an RNA loading control (bottom panel). (c) Sequence
alignments of Par-6 from C. elegans (cPar-6), Drosophila
(dPar-6), Xenopus (xPar-6), mouse (mPar-6) and human (hPar-6),
and clone CAB44747. The sequences corresponding to the CRIB
motif in hPar-6 are boxed and those corresponding to the PDZ
domain are underlined.
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Figure 7.
Figure 7. The hPar-6/PKCĪ¶ pathway is distinct from the
RafCAAX-mediated pathway. (a) Results of focus assays showing
the effect of PKCĪ¶ on the transforming synergy between Rac1 and
RafCAAX and on transformation induced by Ras. The amount of DNA
used in each transfection was: 100 ng for Rac1(G12V) or RafCAAX;
2 Ī¼g for KD-Ī¶ or WT-Ī¶; 10 ng for Ras(G12V). RafCAAX-H
designates the higher amount of DNA (1 Ī¼g) that was used to
obtain a higher number of foci. Data are from two independent
experiments performed in duplicate. (b) Morphologies of
different foci transformed by the protein(s) indicated in each
panel.
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #5
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Title
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Par-6, A gene involved in the establishment of asymmetry in early c. Elegans embryos, Mediates the asymmetric localization of par-3.
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Authors
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J.L.Watts,
B.Etemad-Moghadam,
S.Guo,
L.Boyd,
B.W.Draper,
C.C.Mello,
J.R.Priess,
K.J.Kemphues.
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Ref.
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Development, 1996,
122,
3133-3140.
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PubMed id
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