Cell Junctions, Structure, Function, and Regulation
367
paracellular permeability to 4-kDa dextran,
whereas depletion of this GEF by siRNA
decreased the permeability. Interestingly,
changes in the level of GEF-H1/Lfc did
not change TER or the permeability of
higher molecular weight molecules. In
addition, no change in the morphology
of the tight junction was noted, suggest-
ing that local activation of Rho produces
only subtle changes in tight junction ac-
tivity. Identi±cation of other GEFs and
GAPs associated with the tight junction
along with identi±cation of signaling path-
ways that activate these molecules will
be important for elucidating the role of
small GTPases in the regulation of tight
junctions.
As is the case with AJ proteins, the
plaque proteins found in tight junctions
are also phosphorylated on serine, threo-
nine, and tyrosine. Occludin, ZO-1 and
ZO-2 have all been found to be phos-
phorylated on all three residues under
various experimental conditions; however,
no clear association between phosphoryla-
tion of these proteins and TJ assembly
and/or function has been ±rmly estab-
lished. A number of kinases have been
identi±ed as residing in the tight junction
complex including occludin-associated ki-
nase, ZO-1-associated kinase, and several
PKC
isoforms.
Of
the
PKC
isoforms
localized to the TJs, only the atypical
PKCs (aPKCs) have been shown to play
a de±nitive role in junction assembly.
The aPKCs have been shown to form
a complex with PAR3 (also known as
ASIP, a PKC-speci±c interacting protein),
PAR6, and two PDZ proteins that are
found in the tight junction complex. A
number of studies support a role for
aPKC/PAR3/PAR6 in the formation of the
tight junction. For example, expression of
kinase-de±cient aPKC was found to pre-
vent the reforming of tight junction in the
Ca
++
switch assay. In addition, the ex-
pression of wild-type PAR3 has also been
shown to increase the rate of tight junc-
tion formation. Phosphorylation of PAR
on Ser827 by aPKC was shown to de-
crease aPKC binding to PAR3 and increase
junction formation. PAR6 was found to
complex with PAR3 and aPKC and to
link this complex with Cdc42, which, as
explained in the following section, may
play an important role in tight junction
formation.
The formation of AJs has been demon-
strated to trigger the formation of TJs.
Although the exact order of arrival for the
components of AJs and TJs is not known,
the following model was proposed. Ini-
tial contact between two epithelial cells
has been shown to occur when the tip
o
fa±
lopod
iaf
romonece
l
lcon
t
a
c
t
san
adjacent cell. This allows E-cadherin and
nectin to form microclusters at this rel-
atively small point of cell–cell contact.
The cadherin in this cluster begins to re-
cruit
α
-catenin, while the nectins recruit
afadin, resulting in the association of this
microcluster with the actin cytoskeleton.
The junction expands as actin polymer-
ization is stimulated through activation of
Rac and possibly through recruitment of
Arp 2/3 by E-cadherin and nectin trans-
dimerization. Nectin has also been shown
to activate Cdc42, which, in conjunction
with Rac, will also stimulate actin poly-
merization, thereby increasing junction
stability. In addition, Cdc42 also activates
aPKC resulting in the phosphorylation
of PAR3. PAR3 can associate with JAM,
which is recruited to the apical side of
the AJ. JAM and PAR3 and possibly ZO-
1, which can associate with
α
-catenin,
may then serve as an assembly site for
the tight junction, which matures into
a distinct junction complex as shown
in Fig. 15. The knockdown of speci±c
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