BACKGROUND
hdlg, the human homologue of the drosophila dlg tumor suppressor, is an alternatively spliced protein that belongs to the membrane-associated guanylate kinase  protein family.
maguks are characterized by several protein interaction domains: three pdz domains, an sh <dig> domain, a guanylate kinase-like domain , and a l <dig> self-association domain  <cit> .
most pdz domains bind to the c-terminal portion of proteins often characterized by one of three consensus sequence classes: -x--x-Φ , -x-Φx-Φ , -x--x-Φ  , with all four terminal residues additively contributing to interaction specificity  <cit> .
the three pdz repeats of hdlg use this mechanism to bind to several proteins involved in cellular growth control including the adenomatous polyposis coli  tumor suppressor  <cit> , the human papillomavirus e <dig> protein  <cit> , the adenovirus e <dig> protein  <cit> , the mitotic ser/thr kinase pbk/topk  <cit> , and p38γ map kinase  <cit> .
the gk domain of hdlg also recruits several proteins into macromolecular complexes: gkap/sapap  <cit> , the pka-targeting protein akap79/ <dig>  <cit> , and the microtubule-associated protein map1a  <cit> .
the sh <dig> domain of hdlg forms an intramolecular interaction with the gk domain  <cit> .
finally, homo- and hetero-oligomers of maguk proteins form through their l <dig> domains; for example, hdlg and the maguk protein cask heterodimerize through their l <dig> domains  <cit> .
the degree of hdlg self-association depends also on the presence or absence of the alternatively spliced insertion i1a  <cit> .
i1a and b, two proline-rich alternatively spliced insertions upstream of the first pdz repeat in hdlg, recruit sh3-containing proteins  <cit> .
first described as a cytoplasmic protein localized at the membrane at regions of intercellular contacts  <cit> , hdlg is responsible for the recruitment of a variety of proteins forming a complex network at sites of epithelial cell-cell contact and in pre-synaptic densities.
for example, hdlg has been found to be closely associated with e-cadherin in human intestinal epithelial cells , and both i3- and i2- containing variants were reported to localize to the midbody of cells in cytokinesis  <cit> .
while the various localization sites of hdlg are known, it is unclear what its function is at those sites.
an important step in understanding the function of hdlg as a tumor suppressor is the identification of all of its binding partners.
here we describe the interaction of hdlg with the phosphorylated form of mek <dig>  a signaling protein found, like hdlg, at the midbody of cells undergoing cytokinesis.
importantly, our data also indicate that e-cadherin concentrates in the midbody during cytokinesis and is necessary for proper localization of hdlg, but not phosphorylated mek <dig>  at the midbody.
RESULTS
a c-terminal fragment of mek <dig> interacts with hdlg
like other members of the maguk family, hdlg plays an important role in clustering signaling molecules at sites of cell-cell contact.
most of the structural modules found in hdlg are known to function as protein-interaction domains.
in an effort to identify new signaling proteins that bind to hdlg, we performed a two-hybrid screen using full-length hdlg as bait.
this screen yielded many positives.
among the clones that most strongly activated the lacz reporter gene was a cell cycle-regulated kinase  <cit>  and a ~ <dig> bp sequence encoding the c-terminal  <dig> residues of mek <dig> ).
once isolated, this mek <dig> construct was retransformed in s. cerevisiae hf7c with pgbt9-hdlg to confirm that the interaction was not due to another co-transforming plasmid .
the mek <dig> construct was also co-transformed with a series of control plasmids to confirm that reporter gene activation was indeed dependent on a specific interaction with hdlg ; with this set of transformations we observed the same pattern of β-galactosidase activity that was found with a c-terminal clone of pbk, a previously identified hdlg-interacting protein  <cit> .
although only mek <dig> was isolated in our screen, mek <dig> and mek <dig> share 80% sequence similarity  <cit> .
notably, their c-terminal sequences differ: only mek <dig> is characterized by a conserved x--x-Φ motif matching the consensus sequence found at the c-terminus of class i pdz-binding proteins .
human, mouse and rat mek <dig> all contain this motif, while in chicken mek <dig>  the threonine residue in position p- <dig> is replaced by an alanine .
mek <dig> sequences from all four species are characterized by this same thr to ala substitution and by the addition of glycine or serine at the penultimate position .
the presence of a pdz-binding motif in mammalian mek <dig> proteins suggests that human mek <dig> interacts with hdlg through one of its three pdz repeats.
to test our hypothesis that the c-terminus of mek <dig> but not that of mek <dig> interacts with the hdlg pdz repeats, we designed a peptide binding assay.
in this assay, peptides corresponding to the last  <dig> residues of mek <dig>  or mek <dig> , or a randomized sequence of the mek <dig> peptide  were incubated with a gst fusion protein containing pdz1-pdz <dig> of hdlg , a fragment of hdlg that was previously identified as a super-motif  <cit> .
maldi-tof analyses consistently showed that only the mek <dig> peptide bound to gst-pdz1- <dig> .
the peptide recovered after elution had a mass identical to the mek <dig> peptide directly spotted on the proteinchip array .
when the same experiment was repeated with an unrelated gst fusion protein , the mek <dig> peptide did not bind, demonstrating the specificity of this peptide for pdz1- <dig> of hdlg .
binding of the mek <dig> peptide was also observed when each of the three pdz repeats of hdlg was expressed individually and used in our binding assay, although our results suggest that the binding is weakest with pdz <dig> .
this last result corresponds well with those of maiga et al.
who have also recently described an interaction of hdlg with mek <dig> and found, in a two-hybrid assay likely less sensitive than our peptide binding assay, that a c-terminal construct of mek <dig>  interacts with a pdz1- <dig> construct but not with pdz <dig>  overall, our peptide assay data confirmed that mek <dig>  but not mek <dig>  has a class i pdz-binding motif that interacts with the pdz repeats of hdlg.
hdlg interacts only with activated full-length mek2
to confirm that hdlg and mek <dig> interact in vivo, full-length hmek <dig> was co-expressed with a gst-hdlg fusion protein in insect cells.
although co-infected insect cells synthesized both proteins, our initial attempts to show a co-purification of mek <dig> and gst-hdlg failed.
the co-localization of hdlg with the phosphorylated form of mek <dig> during cytokinesis  prompted us to test for the interaction upon stimulation of raf/mek/map kinase pathway.
abdullah et al.
demonstrated that pma-treatment of sf <dig> insect cells activates this signaling pathway.
we co-infected high  <dig> insect cells with recombinant baculoviruses responsible for the expression of gst-hdlg and hmek <dig>  gst-hdlg was affinity purified from untreated and pma-treated insect cell lysates and mek <dig> co-purification was assayed by immunoblot.
while the amounts of gst-hdlg affinity-purified in untreated and pma-treated samples were comparable, hmek <dig> co-purified with hdlg only in pma-treated samples .
both treated and untreated lysates expressed hmek <dig> at similar levels although hmek <dig> phosphorylation level was significantly increased by pma treatment .
our data shows that mek <dig> is not pulled down non-specifically by glutathione beads and suggest that in this cellular context hdlg interacts very selectively with the activated form of mek <dig>  which is present only in pma-treated cells.
nevertheless, we cannot rule out that the activation of the raf/mek/erk pathway resulted in the phosphorylation of other proteins, including hdlg, and that this may also contribute to the observed interaction.
hdlg localizes to the midbody ring during cytokinesis
because activated mek <dig> is reported to localize to spindle poles  <cit> , kinetochores  <cit>  and to the central portion of the midbody in mitotic cells  <cit> , we next examined the localization of hdlg during various stages of mitosis.
mcf10a human breast carcinoma cells were stained with affinity purified anti-hdlg  and anti-β-tubulin antibodies.
the mitotic cells were staged based on the appearance of their spindle in the β-tubulin staining and that of their chromosomes stained by dapi, as well as from phase-contrast imaging of the cellular membrane.
from prophase to anaphase, hdlg was distributed at the surface of condensed chromosomes  and at the cell periphery .
i2-containing hdlg variants were previously observed in the interphase nuclei of a variety of cells types including mcf10a cells .
during mitosis, it appears that the nuclear variants of hdlg were not dispersed in the cytoplasm but, instead, remained associated with condensed chromosomes, distributed in discrete spots along their entire length .
a set of contiguous z-sections was used to create a three-dimensional rendering of the cell shown in figure 4c.
a 360° rotation of this rendering showed that there was no polarity in the distribution of hdlg at the surface of metaphase chromosomes; it was also present at the sides of the two sets of anaphase chromosome facing each other .
based on this staining distribution, we conclude that hdlg was not specifically associated with telomeric or kinetochore proteins in mitotic cells, but spread all over the chromosomes.
in cells in cytokinesis, midbody structures were easily detected by immunostaining with anti-β-tubulin .
co-staining with anti-nag showed that hdlg was located in the central portion of the midbody called the midbody ring, and was not distributed along the bundles of microtubules linking the two nascent daughter cells .
our data confirms similar observations of hdlg at the midbody in hacat skin keratinocytes, saos <dig> and u2os osteosarcoma cell lines by massimi et al.
and in u2os and hela cervical carcinoma cells by unno et al.
although we found that the localization of hdlg is restricted to the midbody ring.
a few proteins, such as β-tubulin, centriolin and activated mek <dig>  are known to associate specifically with the midbody ring while others, such as aurora b, are found adjacent to the ring structure .
this co-localization of activated mek <dig> and hdlg to the very limited structure of the midbody ring suggests that hdlg could recruit or anchor activated mek <dig> in a particular signaling complex at the midbody.
to verify that the distribution of hdlg that we observed did not simply reflect an unregulated or non-specific interaction with tubulin subunits or microtubule-associated proteins, we looked more closely at the co-staining of midbody structures with anti-hdlg and anti-β-tubulin antibodies in cells at late stages of cell division.
the distribution of hdlg during late anaphase or early cytokinesis showed that it localizes near microtubules plus ends .
before constriction of the cytoplasmic bridge, hdlg was distributed in a narrow line enriched in tubulin in the middle of the central spindle .
a cross-section of the midbody ring structure reconstituted from contiguous z-sections showed that, before membrane constriction, hdlg was concentrated at the center of the ring structure rather than distributed at its periphery .
the overlap between tubulin and hdlg staining was only partial and in most cases hdlg appeared juxtaposed to microtubules , clearly showing that the association of hdlg with the midbody ring is not simply due to an unregulated association with tubulin.
finally, when staining with an antibody that specifically recognizes the membrane-associated hdlg variants  we found that they localized to the midbody ring .
strikingly, staining with affinity purified antibodies raised against the alternatively spliced insertion i <dig>  present in nuclear variants of hdlg, failed to label this structure .
therefore we conclude that only the membrane-bound form of hdlg was recruited to the midbody, where it is found specifically at the midbody ring, near microtubule plus ends.
in contrast, the i2-containing variants remains associated with the chromosomes.
this isoform-specific localization of endogenously expressed hdlg contrasts with the non-specific localization of both i3- and i2-containing isoforms others have found for overexpressed full-length or truncated c-terminal forms of hdlg  <cit> .
e-cadherin is necessary for localization of hdlg but not phosphorylated mek <dig> to the midbody ring during cytokinesis
we have previously reported that hdlg is closely associated with e-cadherin adhesion complexes in epithelial cells  <cit> .
interestingly, staining for both e-cadherin and hdlg in subconfluent growing caco-2/ <dig> colorectal epithelial cells showed that both proteins co-localized at the midbody ring during cytokinesis .
to investigate whether e-cadherin controls the localization of hdlg and phosphorylated mek <dig> to the midbody, e-cadherin expression was knocked down with rna interference .
reduction in e-cadherin levels markedly attenuated hdlg staining to the midbody .
with both e-cadherin and hdlg missing from the midbody, it appeared as if its structure was altered, as we consistently observed that the width of β-tubulin staining was reduced along the central spindle .
although the midbody size in both control cells  and e-cadherin knockdown cells was variable, we found that on average, there was a clear, and highly statistically significant difference in the average midbody width .
somewhat surprisingly however, midbody staining for phosphorylated mek <dig> was not affected by e-cadherin downregulation .
from these results we conclude that while e-cadherin is necessary for the localization of the i3-containing isoform of hdlg to the midbody ring during cytokinesis, the presence of neither of these proteins at the midbody structure is necessary for the recruitment of phosphorylated mek <dig>
discussion
although hdlg is known to localize to sites of cell-cell contacts, to interphase nuclei, and to the midbody of cells in cytokinesis  <cit> , little is known about its binding partners at sites other than cell-cell contacts.
here we report that hdlg associates with activated mek <dig>  a protein specifically found at the midbody ring during cytokinesis  <cit> .
using in vitro binding assays, we found that the pdz domains of hdlg interact directly with the c-terminal peptide of mek <dig>  which contains a class i consensus pdz-binding motif.
interestingly, while others have very recently shown partial co-precipitation of hdlg and mek <dig> from asynchronous hek- <dig> and human vascular endothelial cell lysates  <cit> , in the cellular contexts we tested, the association between hdlg and full-length mek <dig> is only detectable when mek <dig> is activated.
several lines of evidence, including these data, suggest that the c-terminal pdz-binding motif of full-length mek <dig> may only be accessible to hdlg in the activated protein.
structural studies have shown that upon activation, mek <dig> likely undergoes a change of conformation common to other kinases  <cit> .
this change of conformation results in the relative rotation of the n- and c-terminal lobed-structures forming the catalytic domain.
it is unknown how this rotation affects the c-terminal pdz-binding sequence of mek <dig> because the c-terminal seven amino acids, including this region, were not well ordered in the mek <dig> structure  <cit> .
nevertheless, the crystallographic structure of mek <dig> bound to mg-atp and a small molecule non-competitive inhibitor, pd <dig>  clearly shows that in this active-conformation homodimer, the pdz-binding sequences should be accessible to the pdz repeats of hdlg.
although not structured, the c-terminal seven residues would extend at the periphery of the complex, near the atp binding site, and not be buried in the dimer interface  <cit> .
further supporting our hypothesis, we note that both the mek <dig> clone isolated in our two-hybrid screen and the clone identified in a similar screen by magai et al.
coded for only the c-terminal portion of mek <dig>  therefore in assays using a c-terminal fragment of mek <dig> , or a c-terminal mek <dig> peptide , the c-terminal pdz-binding motif is likely more exposed than in full-length mek <dig> clones, allowing for an interaction that is normally regulated by a phosphorylation-driven conformational change in the full-length protein.
while our results and those of maiga et al.
<cit>  provide some initial information as to the binding specificities and the mode of interaction between hdlg and mek <dig>  a better understanding of how, and in which context, these two proteins can interact will require further structural studies.
other unresolved questions include how the intramolecular interaction between the gk and sh <dig> domains of hdlg, the self-association of hdlg proteins via their n-terminal ends, and the identity of hdlg splice variants contribute to the regulation of its binding to mek <dig>
the role of mek/erk-dependent signaling in the control of cell-cycle has been extensively studied, particularly its role in g1/s phase transition.
recent studies have shown that activation of this signaling pathway is also required for mitotic progression.
during late anaphase and cytokinesis, the two nascent daughter cells are separated by a cytoplasmic bridge characterized by a bundle of antiparallel microtubules forming the central spindle.
at the half-point of the central spindle, a structure called the midbody determines the site of cell abscission and recruits proteins that are necessary for the completion of mitosis.
the activated forms of mek1/ <dig>  erk and the erk substrates rsk1- <dig> are found in the central spindle and in the midbody  <cit> .
inhibition of mek activity prevents the completion of mitosis in cells released from nocodazole-mediated mitotic arrest, thus suggesting the role of mek1/ <dig> and downstream substrates in cytokinesis  <cit> .
the interaction observed between hdlg and activated mek <dig> suggests that both proteins should co-distribute at specific stages of the cell cycle when mek <dig> is activated.
indeed, we have found that certain variants of hdlg, like activated mek <dig>  localize specifically to the midbody ring structure during cytokinesis.
it is unclear how hdlg is targeted to the midbody, however, because the pdz domains of hdlg are insufficient to target the protein to the midbody  <cit> , it cannot rely on activated mek <dig>  others have shown that a protein fragment spanning only the c-terminal sh <dig> and gk domains of hdlg localized to the midbody when overexpressed, competing with endogenous hdlg and disrupting cell growth  <cit> .
the gk domain of hdlg alone localizes to the midbody and causes a mild cytokinesis defect when overexpressed  <cit> .
this suggests that while the n-terminal region of hdlg, including its pdz domains, may be dispensable for proper localization of hdlg at the midbody, it is essential to its function.
a possibility raised by our data is that membrane-bound i3-variants of hdlg are redistributed to the midbody.
i3-variants of hdlg were previously found to be localized to the membrane at sites of cell-cell junctions  <cit> .
membrane-bound hdlg was demonstrated to be closely associated with e-cadherin-containing adhesion complexes in human epithelial cells and cells deficient in hdlg expression fail to organize their cortical actin cytoskeleton and are unable to stabilize their adherens junctions  <cit> .
e-cadherin protein was also previously found in the cleavage furrow of mitotic epithelial cells, consistent with the observation that the daughter cells form adherens junctions with each other immediately following cytokinesis and are not separated by neighboring cells  <cit> .
herein, we demonstrate that e-cadherin expression is required for localization of hdlg to the midbody in the same epithelial cells.
e-cadherin depletion also led to a marked narrowing of the midbody structure.
while our data does not specifically address whether e-cadherin depletion itself or its effect on the central spindle are the direct cause of the mis-localization of hdlg, because e-cadherin and hdlg are known to physically interact in confluent epithelial cells  <cit> , we surmise that e-cadherin may directly recruit hdlg to the midbody.
taken together, these data suggest that hdlg may also be important for the formation of adherens junction between the two daughter cells.
membrane-bound i3-containing variants of hdlg could either relocalize to the midbody from existing adherens junction sites or be recruited to the midbody from intracellular membrane systems.
in this report, we show that the localization of hdlg to the midbody ring precedes the constriction of the intracellular cytoplasmic bridge, while post-golgi secretory vesicles are delivered to the site of abscission after membrane constriction, during the late stage of cytokinesis  <cit> , arguing against recruitment from intracellular membranes.
although only i3-variants of hdlg are preferentially associated with membrane systems  <cit> , others have shown that the transport and/or anchoring of overexpressed hdlg to the midbody is independent of i <dig> and i <dig>  <cit> .
taken together our findings and those from these previous reports are consistent with the idea that sh3- or gk-binding proteins are responsible for the transport and or anchoring of hdlg to the midbody, while alternative splicing of the i3- vs. i2-insertion may determine which pool of hdlg relocalizes to this site, perhaps by regulating the accessibility of the gk domain.
hdlg plays an important role in clustering signaling molecules at different intracellular sites.
in this report, we describe the interaction of hdlg with mek <dig> which was also reported recently by maiga et al.
<cit> .
in contrast to hdlg, however, the recruitment of phosphorylated mek <dig> to the midbody seems to occur normally in e-cadherin-depleted cells indicating that although other assays show that hdlg can bind directly to phosphorylated mek <dig>  hdlg is not required for mek <dig> recruitment to the midbody.
nevertheless, we cannot exclude that hdlg may serve as a scaffold to form a signalosome that is responsive to mek/erk signaling during cytokinesis.
previous studies have identified several other kinases that bind to hdlg.
first, pbk/topk, a kinase which is activated during mitosis by the cyclin b/cdk <dig> complex and then phosphorylates p38-mapk, interacts with the pdz repeats of hdlg  <cit> .
interestingly, an activated form of pbk/topk was recently found to be associated with the central spindle and to promote cytokinesis  <cit> ; this association with the central spindle may reflect its interaction with hdlg.
a second kinase interacting with hdlg is p85/pi3k, which it recruits to e-cadherin-mediated sites of cell-cell contact in human intestinal epithelial cells where hdlg plays a key role in the organization and stabilization of adherens junctions  <cit> .
importantly, this recruitment of p85/pi3k is dependent on a change in the phosphorylation pattern of hdlg triggered by a yet unidentified kinase  <cit> .
finally, the sapk3/p38γ mapk is also known to interact with the pdz repeats of hdlg and to phosphorylate hdlg.
phosphorylation of hdlg by sapk3/p38γ mapk results in its dissociation from the cytoskeleton, apparently by disrupting the interaction between the gk domain of hdlg and its binding partner gkap  <cit> .
therefore, not only is hdlg implicated in the recruitement of several kinases at specialized sites in cells but, in turn, the activity of hdlg, and potentially its localization, is modulated by kinases.
CONCLUSIONS
in summary, we show that specific variants of hdlg associate with the midbody ring structure during late anaphase and cytokinesis and that the midbody localization of hdlg is dependent on the expression of e-cadherin.
a clear understanding of the functions that hdlg performs at different intracellular sites requires the identification of all its interaction partners.
we demonstrate that hdlg directly interacts with a component of the map kinase pathway, mek <dig>  specifically in cells where mek <dig> is activated.
activated mek <dig> is known to also localize to the midbody ring structure during late mitosis.
future studies will reveal how hdlg and mek <dig> work together to regulate cell cycle events and cellular proliferation.

 hdlg, the human homologue of the drosophila dlg tumor suppressor, is an alternatively spliced protein that belongs to the membrane-associated guanylate kinase protein family. maguks are characterized by several protein interaction domains: three pdz domains, an sh domain, a guanylate kinase-like domain, and a l self-association domain. most pdz domains bind to the c-terminal portion of proteins often characterized by one of three consensus sequence classes: -x--x-Φ, -x-Φx-Φ, -x--x-Φ, with all four terminal residues additively contributing to interaction specificity. the three pdz repeats of hdlg use this mechanism to bind to several proteins involved in cellular growth control including the adenomatous polyposis coli tumor suppressor, the human papillomavirus e protein, the adenovirus e protein, the mitotic ser/thr kinase pbk/topk, and p38γ map kinase. the gk domain of hdlg also recruits several proteins into macromolecular complexes: gkap/sapap, the pka-targeting protein akap79/, and the microtubule-associated protein map1a. the sh domain of hdlg forms an intramolecular interaction with the gk domain. finally, homo- and hetero-oligomers of maguk proteins form through their l domains; for example, hdlg and the maguk protein cask heterodimerize through their l domains. the degree of hdlg self-association depends also on the presence or absence of the alternatively spliced insertion i1a. i1a and b, two proline-rich alternatively spliced insertions upstream of the first pdz repeat in hdlg, recruit sh3-containing proteins. first described as a cytoplasmic protein localized at the membrane at regions of intercellular contacts, hdlg is responsible for the recruitment of a variety of proteins forming a complex network at sites of epithelial cell-cell contact and in pre-synaptic densities. for example, hdlg has been found to be closely associated with e-cadherin in human intestinal epithelial cells, and both i3- and i2- containing variants were reported to localize to the midbody of cells in cytokinesis. while the various localization sites of hdlg are known, it is unclear what its function is at those sites. an important step in understanding the function of hdlg as a tumor suppressor is the identification of all of its binding partners. here we describe the interaction of hdlg with the phosphorylated form of mek a signaling protein found, like hdlg, at the midbody of cells undergoing cytokinesis. importantly, our data also indicate that e-cadherin concentrates in the midbody during cytokinesis and is necessary for proper localization of hdlg, but not phosphorylated mek at the midbody.  a c-terminal fragment of mek interacts with hdlg like other members of the maguk family, hdlg plays an important role in clustering signaling molecules at sites of cell-cell contact. most of the structural modules found in hdlg are known to function as protein-interaction domains. in an effort to identify new signaling proteins that bind to hdlg, we performed a two-hybrid screen using full-length hdlg as bait. this screen yielded many positives. among the clones that most strongly activated the lacz reporter gene was a cell cycle-regulated kinase and a ~ bp sequence encoding the c-terminal residues of mek ). once isolated, this mek construct was retransformed in s. cerevisiae hf7c with pgbt9-hdlg to confirm that the interaction was not due to another co-transforming plasmid. the mek construct was also co-transformed with a series of control plasmids to confirm that reporter gene activation was indeed dependent on a specific interaction with hdlg; with this set of transformations we observed the same pattern of β-galactosidase activity that was found with a c-terminal clone of pbk, a previously identified hdlg-interacting protein. although only mek was isolated in our screen, mek and mek share 80% sequence similarity. notably, their c-terminal sequences differ: only mek is characterized by a conserved x--x-Φ motif matching the consensus sequence found at the c-terminus of class i pdz-binding proteins. human, mouse and rat mek all contain this motif, while in chicken mek the threonine residue in position p- is replaced by an alanine. mek sequences from all four species are characterized by this same thr to ala substitution and by the addition of glycine or serine at the penultimate position. the presence of a pdz-binding motif in mammalian mek proteins suggests that human mek interacts with hdlg through one of its three pdz repeats. to test our hypothesis that the c-terminus of mek but not that of mek interacts with the hdlg pdz repeats, we designed a peptide binding assay. in this assay, peptides corresponding to the last residues of mek or mek, or a randomized sequence of the mek peptide were incubated with a gst fusion protein containing pdz1-pdz of hdlg, a fragment of hdlg that was previously identified as a super-motif. maldi-tof analyses consistently showed that only the mek peptide bound to gst-pdz1-. the peptide recovered after elution had a mass identical to the mek peptide directly spotted on the proteinchip array. when the same experiment was repeated with an unrelated gst fusion protein, the mek peptide did not bind, demonstrating the specificity of this peptide for pdz1- of hdlg. binding of the mek peptide was also observed when each of the three pdz repeats of hdlg was expressed individually and used in our binding assay, although our results suggest that the binding is weakest with pdz. this last result corresponds well with those of maiga et al. who have also recently described an interaction of hdlg with mek and found, in a two-hybrid assay likely less sensitive than our peptide binding assay, that a c-terminal construct of mek interacts with a pdz1- construct but not with pdz overall, our peptide assay data confirmed that mek but not mek has a class i pdz-binding motif that interacts with the pdz repeats of hdlg. hdlg interacts only with activated full-length mek2 to confirm that hdlg and mek interact in vivo, full-length hmek was co-expressed with a gst-hdlg fusion protein in insect cells. although co-infected insect cells synthesized both proteins, our initial attempts to show a co-purification of mek and gst-hdlg failed. the co-localization of hdlg with the phosphorylated form of mek during cytokinesis prompted us to test for the interaction upon stimulation of raf/mek/map kinase pathway. abdullah et al. demonstrated that pma-treatment of sf insect cells activates this signaling pathway. we co-infected high insect cells with recombinant baculoviruses responsible for the expression of gst-hdlg and hmek gst-hdlg was affinity purified from untreated and pma-treated insect cell lysates and mek co-purification was assayed by immunoblot. while the amounts of gst-hdlg affinity-purified in untreated and pma-treated samples were comparable, hmek co-purified with hdlg only in pma-treated samples. both treated and untreated lysates expressed hmek at similar levels although hmek phosphorylation level was significantly increased by pma treatment. our data shows that mek is not pulled down non-specifically by glutathione beads and suggest that in this cellular context hdlg interacts very selectively with the activated form of mek which is present only in pma-treated cells. nevertheless, we cannot rule out that the activation of the raf/mek/erk pathway resulted in the phosphorylation of other proteins, including hdlg, and that this may also contribute to the observed interaction. hdlg localizes to the midbody ring during cytokinesis because activated mek is reported to localize to spindle poles, kinetochores and to the central portion of the midbody in mitotic cells, we next examined the localization of hdlg during various stages of mitosis. mcf10a human breast carcinoma cells were stained with affinity purified anti-hdlg and anti-β-tubulin antibodies. the mitotic cells were staged based on the appearance of their spindle in the β-tubulin staining and that of their chromosomes stained by dapi, as well as from phase-contrast imaging of the cellular membrane. from prophase to anaphase, hdlg was distributed at the surface of condensed chromosomes and at the cell periphery. i2-containing hdlg variants were previously observed in the interphase nuclei of a variety of cells types including mcf10a cells. during mitosis, it appears that the nuclear variants of hdlg were not dispersed in the cytoplasm but, instead, remained associated with condensed chromosomes, distributed in discrete spots along their entire length. a set of contiguous z-sections was used to create a three-dimensional rendering of the cell shown in figure 4c. a 360° rotation of this rendering showed that there was no polarity in the distribution of hdlg at the surface of metaphase chromosomes; it was also present at the sides of the two sets of anaphase chromosome facing each other. based on this staining distribution, we conclude that hdlg was not specifically associated with telomeric or kinetochore proteins in mitotic cells, but spread all over the chromosomes. in cells in cytokinesis, midbody structures were easily detected by immunostaining with anti-β-tubulin. co-staining with anti-nag showed that hdlg was located in the central portion of the midbody called the midbody ring, and was not distributed along the bundles of microtubules linking the two nascent daughter cells. our data confirms similar observations of hdlg at the midbody in hacat skin keratinocytes, saos and u2os osteosarcoma cell lines by massimi et al. and in u2os and hela cervical carcinoma cells by unno et al. although we found that the localization of hdlg is restricted to the midbody ring. a few proteins, such as β-tubulin, centriolin and activated mek are known to associate specifically with the midbody ring while others, such as aurora b, are found adjacent to the ring structure. this co-localization of activated mek and hdlg to the very limited structure of the midbody ring suggests that hdlg could recruit or anchor activated mek in a particular signaling complex at the midbody. to verify that the distribution of hdlg that we observed did not simply reflect an unregulated or non-specific interaction with tubulin subunits or microtubule-associated proteins, we looked more closely at the co-staining of midbody structures with anti-hdlg and anti-β-tubulin antibodies in cells at late stages of cell division. the distribution of hdlg during late anaphase or early cytokinesis showed that it localizes near microtubules plus ends. before constriction of the cytoplasmic bridge, hdlg was distributed in a narrow line enriched in tubulin in the middle of the central spindle. a cross-section of the midbody ring structure reconstituted from contiguous z-sections showed that, before membrane constriction, hdlg was concentrated at the center of the ring structure rather than distributed at its periphery. the overlap between tubulin and hdlg staining was only partial and in most cases hdlg appeared juxtaposed to microtubules, clearly showing that the association of hdlg with the midbody ring is not simply due to an unregulated association with tubulin. finally, when staining with an antibody that specifically recognizes the membrane-associated hdlg variants we found that they localized to the midbody ring. strikingly, staining with affinity purified antibodies raised against the alternatively spliced insertion i present in nuclear variants of hdlg, failed to label this structure. therefore we conclude that only the membrane-bound form of hdlg was recruited to the midbody, where it is found specifically at the midbody ring, near microtubule plus ends. in contrast, the i2-containing variants remains associated with the chromosomes. this isoform-specific localization of endogenously expressed hdlg contrasts with the non-specific localization of both i3- and i2-containing isoforms others have found for overexpressed full-length or truncated c-terminal forms of hdlg. e-cadherin is necessary for localization of hdlg but not phosphorylated mek to the midbody ring during cytokinesis we have previously reported that hdlg is closely associated with e-cadherin adhesion complexes in epithelial cells. interestingly, staining for both e-cadherin and hdlg in subconfluent growing caco-2/ colorectal epithelial cells showed that both proteins co-localized at the midbody ring during cytokinesis. to investigate whether e-cadherin controls the localization of hdlg and phosphorylated mek to the midbody, e-cadherin expression was knocked down with rna interference. reduction in e-cadherin levels markedly attenuated hdlg staining to the midbody. with both e-cadherin and hdlg missing from the midbody, it appeared as if its structure was altered, as we consistently observed that the width of β-tubulin staining was reduced along the central spindle. although the midbody size in both control cells and e-cadherin knockdown cells was variable, we found that on average, there was a clear, and highly statistically significant difference in the average midbody width. somewhat surprisingly however, midbody staining for phosphorylated mek was not affected by e-cadherin downregulation. from these results we conclude that while e-cadherin is necessary for the localization of the i3-containing isoform of hdlg to the midbody ring during cytokinesis, the presence of neither of these proteins at the midbody structure is necessary for the recruitment of phosphorylated mek discussion although hdlg is known to localize to sites of cell-cell contacts, to interphase nuclei, and to the midbody of cells in cytokinesis, little is known about its binding partners at sites other than cell-cell contacts. here we report that hdlg associates with activated mek a protein specifically found at the midbody ring during cytokinesis. using in vitro binding assays, we found that the pdz domains of hdlg interact directly with the c-terminal peptide of mek which contains a class i consensus pdz-binding motif. interestingly, while others have very recently shown partial co-precipitation of hdlg and mek from asynchronous hek- and human vascular endothelial cell lysates, in the cellular contexts we tested, the association between hdlg and full-length mek is only detectable when mek is activated. several lines of evidence, including these data, suggest that the c-terminal pdz-binding motif of full-length mek may only be accessible to hdlg in the activated protein. structural studies have shown that upon activation, mek likely undergoes a change of conformation common to other kinases. this change of conformation results in the relative rotation of the n- and c-terminal lobed-structures forming the catalytic domain. it is unknown how this rotation affects the c-terminal pdz-binding sequence of mek because the c-terminal seven amino acids, including this region, were not well ordered in the mek structure. nevertheless, the crystallographic structure of mek bound to mg-atp and a small molecule non-competitive inhibitor, pd clearly shows that in this active-conformation homodimer, the pdz-binding sequences should be accessible to the pdz repeats of hdlg. although not structured, the c-terminal seven residues would extend at the periphery of the complex, near the atp binding site, and not be buried in the dimer interface. further supporting our hypothesis, we note that both the mek clone isolated in our two-hybrid screen and the clone identified in a similar screen by magai et al. coded for only the c-terminal portion of mek therefore in assays using a c-terminal fragment of mek, or a c-terminal mek peptide, the c-terminal pdz-binding motif is likely more exposed than in full-length mek clones, allowing for an interaction that is normally regulated by a phosphorylation-driven conformational change in the full-length protein. while our results and those of maiga et al. provide some initial information as to the binding specificities and the mode of interaction between hdlg and mek a better understanding of how, and in which context, these two proteins can interact will require further structural studies. other unresolved questions include how the intramolecular interaction between the gk and sh domains of hdlg, the self-association of hdlg proteins via their n-terminal ends, and the identity of hdlg splice variants contribute to the regulation of its binding to mek the role of mek/erk-dependent signaling in the control of cell-cycle has been extensively studied, particularly its role in g1/s phase transition. recent studies have shown that activation of this signaling pathway is also required for mitotic progression. during late anaphase and cytokinesis, the two nascent daughter cells are separated by a cytoplasmic bridge characterized by a bundle of antiparallel microtubules forming the central spindle. at the half-point of the central spindle, a structure called the midbody determines the site of cell abscission and recruits proteins that are necessary for the completion of mitosis. the activated forms of mek1/ erk and the erk substrates rsk1- are found in the central spindle and in the midbody. inhibition of mek activity prevents the completion of mitosis in cells released from nocodazole-mediated mitotic arrest, thus suggesting the role of mek1/ and downstream substrates in cytokinesis. the interaction observed between hdlg and activated mek suggests that both proteins should co-distribute at specific stages of the cell cycle when mek is activated. indeed, we have found that certain variants of hdlg, like activated mek localize specifically to the midbody ring structure during cytokinesis. it is unclear how hdlg is targeted to the midbody, however, because the pdz domains of hdlg are insufficient to target the protein to the midbody, it cannot rely on activated mek others have shown that a protein fragment spanning only the c-terminal sh and gk domains of hdlg localized to the midbody when overexpressed, competing with endogenous hdlg and disrupting cell growth. the gk domain of hdlg alone localizes to the midbody and causes a mild cytokinesis defect when overexpressed. this suggests that while the n-terminal region of hdlg, including its pdz domains, may be dispensable for proper localization of hdlg at the midbody, it is essential to its function. a possibility raised by our data is that membrane-bound i3-variants of hdlg are redistributed to the midbody. i3-variants of hdlg were previously found to be localized to the membrane at sites of cell-cell junctions. membrane-bound hdlg was demonstrated to be closely associated with e-cadherin-containing adhesion complexes in human epithelial cells and cells deficient in hdlg expression fail to organize their cortical actin cytoskeleton and are unable to stabilize their adherens junctions. e-cadherin protein was also previously found in the cleavage furrow of mitotic epithelial cells, consistent with the observation that the daughter cells form adherens junctions with each other immediately following cytokinesis and are not separated by neighboring cells. herein, we demonstrate that e-cadherin expression is required for localization of hdlg to the midbody in the same epithelial cells. e-cadherin depletion also led to a marked narrowing of the midbody structure. while our data does not specifically address whether e-cadherin depletion itself or its effect on the central spindle are the direct cause of the mis-localization of hdlg, because e-cadherin and hdlg are known to physically interact in confluent epithelial cells, we surmise that e-cadherin may directly recruit hdlg to the midbody. taken together, these data suggest that hdlg may also be important for the formation of adherens junction between the two daughter cells. membrane-bound i3-containing variants of hdlg could either relocalize to the midbody from existing adherens junction sites or be recruited to the midbody from intracellular membrane systems. in this report, we show that the localization of hdlg to the midbody ring precedes the constriction of the intracellular cytoplasmic bridge, while post-golgi secretory vesicles are delivered to the site of abscission after membrane constriction, during the late stage of cytokinesis, arguing against recruitment from intracellular membranes. although only i3-variants of hdlg are preferentially associated with membrane systems, others have shown that the transport and/or anchoring of overexpressed hdlg to the midbody is independent of i and i. taken together our findings and those from these previous reports are consistent with the idea that sh3- or gk-binding proteins are responsible for the transport and or anchoring of hdlg to the midbody, while alternative splicing of the i3- vs. i2-insertion may determine which pool of hdlg relocalizes to this site, perhaps by regulating the accessibility of the gk domain. hdlg plays an important role in clustering signaling molecules at different intracellular sites. in this report, we describe the interaction of hdlg with mek which was also reported recently by maiga et al.. in contrast to hdlg, however, the recruitment of phosphorylated mek to the midbody seems to occur normally in e-cadherin-depleted cells indicating that although other assays show that hdlg can bind directly to phosphorylated mek hdlg is not required for mek recruitment to the midbody. nevertheless, we cannot exclude that hdlg may serve as a scaffold to form a signalosome that is responsive to mek/erk signaling during cytokinesis. previous studies have identified several other kinases that bind to hdlg. first, pbk/topk, a kinase which is activated during mitosis by the cyclin b/cdk complex and then phosphorylates p38-mapk, interacts with the pdz repeats of hdlg. interestingly, an activated form of pbk/topk was recently found to be associated with the central spindle and to promote cytokinesis; this association with the central spindle may reflect its interaction with hdlg. a second kinase interacting with hdlg is p85/pi3k, which it recruits to e-cadherin-mediated sites of cell-cell contact in human intestinal epithelial cells where hdlg plays a key role in the organization and stabilization of adherens junctions. importantly, this recruitment of p85/pi3k is dependent on a change in the phosphorylation pattern of hdlg triggered by a yet unidentified kinase. finally, the sapk3/p38γ mapk is also known to interact with the pdz repeats of hdlg and to phosphorylate hdlg. phosphorylation of hdlg by sapk3/p38γ mapk results in its dissociation from the cytoskeleton, apparently by disrupting the interaction between the gk domain of hdlg and its binding partner gkap. therefore, not only is hdlg implicated in the recruitement of several kinases at specialized sites in cells but, in turn, the activity of hdlg, and potentially its localization, is modulated by kinases.  in summary, we show that specific variants of hdlg associate with the midbody ring structure during late anaphase and cytokinesis and that the midbody localization of hdlg is dependent on the expression of e-cadherin. a clear understanding of the functions that hdlg performs at different intracellular sites requires the identification of all its interaction partners. we demonstrate that hdlg directly interacts with a component of the map kinase pathway, mek specifically in cells where mek is activated. activated mek is known to also localize to the midbody ring structure during late mitosis. future studies will reveal how hdlg and mek work together to regulate cell cycle events and cellular proliferation. 
the human homologue of the drosophila discs-large tumor suppressor protein, hdlg, is a multi-domain cytoplasmic protein that localizes to the membrane at intercellular junction sites. we show that both proteins localize to a sub-structure of the midbody, the midbody ring, and that the interaction between the pdz domains of hdlg and the c-terminal portion of mek is dependent on the phosphorylation of mek finally, we found that e-cadherin also localizes to the midbody and that its expression is required for the isoform-specific recruitment of hdlg, but not activated mek to that structure. at both synaptic junctions and epithelia cell-cell junctions, hdlg is known to recruit several signaling proteins into macromolecular complexes. our results suggest that like at other cell-cell junction sites, hdlg is part of a macromolecular complex of structural and signaling proteins at the midbody. hdlg is also found at the midbody, a small microtubule-rich structure bridging the two daughter cells during cytokinesis, but its function at this site is not clear.
 the human homologue of the drosophila discs-large tumor suppressor protein, hdlg, is a multi-domain cytoplasmic protein that localizes to the membrane at intercellular junction sites. at both synaptic junctions and epithelia cell-cell junctions, hdlg is known to recruit several signaling proteins into macromolecular complexes. hdlg is also found at the midbody, a small microtubule-rich structure bridging the two daughter cells during cytokinesis, but its function at this site is not clear.  here we describe the interaction of hdlg with the activated form of mek of the canonical raf/mek/erk pathway, a protein that is found at the midbody during cytokinesis. we show that both proteins localize to a sub-structure of the midbody, the midbody ring, and that the interaction between the pdz domains of hdlg and the c-terminal portion of mek is dependent on the phosphorylation of mek finally, we found that e-cadherin also localizes to the midbody and that its expression is required for the isoform-specific recruitment of hdlg, but not activated mek to that structure.  our results suggest that like at other cell-cell junction sites, hdlg is part of a macromolecular complex of structural and signaling proteins at the midbody. 