Secondary literature sources for the KAP domain

For each of the hand selected primary papers associated with the KAP domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation.

Zumbrunn J, Kinoshita K, Hyman AA, Nathke IS
Curr Biol. 2001; 11: 44-9

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.

KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport.

Yamazaki H, Nakata T, Okada Y, Hirokawa N
J Cell Biol. 1995; 130: 1387-99

We cloned a new member of the murine brain kinesin superfamily, KIF3B, and found that its amino acid sequence is highly homologous but not identical to KIF3A, which we previously cloned and named KIF3 (47% identical). KIF3B is localized in various organ tissues and developing neurons of mice and accumulates with anterogradely moving membranous organelles after ligation of nerve axons. Immunoprecipitation assay of the brain revealed that KIF3B forms a complex with KIF3A and three other high molecular weight (approximately 100 kD)-associated polypeptides, called the kinesin superfamily-associated protein 3 (KAP3). In vitro reconstruction using baculovirus expression systems showed that KIF3A and KIF3B directly bind with each other in the absence of KAP3. The recombinant KIF3A/B complex (approximately 50-nm rod with two globular heads and a single globular tail) demonstrated plus end-directed microtubule sliding activity in vitro. In addition, we showed that KIF3B itself has motor activity in vitro, by making a complex of wild-type KIF3B and a chimeric motor protein (KIF3B head and KIF3A rod tail). Subcellular fractionation of mouse brain homogenates showed a considerable amount of the native KIF3 complex to be associated with membrane fractions other than synaptic vesicles. Immunoprecipitation by anti-KIF3B antibody-conjugated beads and its electron microscopic study also revealed that KIF3 is associated with membranous organelles. Moreover, we found that the composition of KAP3 is different in the brain and testis. Our findings suggest that KIF3B forms a heterodimer with KIF3A and functions as a new microtubule-based anterograde translocator for membranous organelles, and that KAP3 may determine functional diversity of the KIF3 complex in various kinds of cells in vivo.

Crystal structure of a beta-catenin/APC complex reveals a critical role for APC phosphorylation in APC function.

Xing Y, Clements WK, Le Trong I, Hinds TR, Stenkamp R, Kimelman D, Xu W
Mol Cell. 2004; 15: 523-33

The tumor suppressor adenomatous polyposis coli (APC) plays a critical role in the turnover of cytosolic beta-catenin, the key effector of the canonical Wnt signaling pathway. APC contains seven 20 amino acid (20 aa) beta-catenin binding repeats that are required for beta-catenin turnover. We have determined the crystal structure of beta-catenin in complex with a phosphorylated APC fragment containing two 20 aa repeats. Surprisingly, one single phosphorylated 20 aa repeat, together with its flanking regions, covers the entire structural groove of beta-catenin and may thus compete for beta-catenin binding with all other beta-catenin armadillo repeat partners. Our biochemical studies show that phosphorylation of the APC 20 aa repeats increases the affinity of the repeats for beta-catenin by 300- to 500-fold and the phosphorylated 20 aa repeats prevent beta-catenin binding to Tcf. Our work suggests that the phosphorylation of the APC 20 aa repeats could be a critical switch for APC function.

Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex.

Xing Y, Clements WK, Kimelman D, Xu W
Genes Dev. 2003; 17: 2753-64

The "beta-catenin destruction complex" is central to canonical Wnt/beta-catenin signaling. The scaffolding protein Axin and the tumor suppressor adenomatous polyposis coli protein (APC) are critical components of this complex, required for rapid beta-catenin turnover. We determined the crystal structure of a complex between beta-catenin and the beta-catenin-binding domain of Axin (Axin-CBD). The Axin-CBD forms a helix that occupies the groove formed by the third and fourth armadillo repeats of beta-catenin and thus precludes the simultaneous binding of other beta-catenin partners in this region. Our biochemical studies demonstrate that, when phosphorylated, the 20-amino acid repeat region of APC competes with Axin for binding to beta-catenin. We propose that a key function of APC in the beta-catenin destruction complex is to remove phosphorylated beta-catenin product from the active site.

A +TIP for a smooth trip.

Xiang X
J Cell Biol. 2006; 172: 651-4

Is there a cellular mechanism for preventing a depolymerizing microtubule track from "slipping out from under" its cargo? A recent study in budding yeast indicates that when a chromosome is transported to the minus end of a spindle microtubule, its kinetochore-bound microtubule plus end-tracking protein (+TIP) Stu2 may move to the plus end to promote rescue; i.e., to switch the depolymerizing end to a polymerizing end. The possibility that other +TIPs may play a similar role in sustaining a microtubule track during vesicular transport deserves investigation.

Sequences of sea urchin kinesin light chain isoforms.

Wedaman KP, Knight AE, Kendrick-Jones J, Scholey JM
J Mol Biol. 1993; 231: 155-8

We have deduced the amino acid sequences of four sea urchin (Strongylocentrotus purpuratus; SP) kinesin light chain (KLC) isoforms (SPKLC 1-4) and compared them to rat brain light chain sequences. Examination of the SPKLC open reading frames (SPKLC1, 649; SPKLC2, 677; SPKLC3, 686; and SPKLC4, 451 amino acid residues) reveals that the first 500 or so residues of the KLCs are highly conserved but the C-terminal ends of rat and sea urchin light chains are divergent; SPKLCs 1, 2 and 3 share a highly basic, 86 residue C-terminal segment that is missing from the shorter rat light chains and SPKLC4. The insertion of 28 and 37 residue segments at residue 563 of SPKLCs 2 and 3, respectively, gives rise to sequence heterogeneity at the C-terminal ends of the sea urchin KLCs. C-terminal sequence differences between light chains may provide inter- and intraspecies differences in the functional properties of the presumptive cargo attachment elements of kinesin.

Protein phosphatase Dusp26 associates with KIF3 motor and promotes N-cadherin-mediated cell-cell adhesion.

Tanuma N, Nomura M, Ikeda M, Kasugai I, Tsubaki Y, Takagaki K, Kawamura T, Yamashita Y, Sato I, Sato M, Katakura R, Kikuchi K, Shima H
Oncogene. 2009; 28: 752-61

Recent studies have demonstrated essential functions for KIF3, a microtubule-directed protein motor, in subcellular transport of several cancer-related proteins, including the beta-catenin-cadherin(s) complex. In this study, we report identification of the protein-phosphatase Dusp26 as a novel regulator of the KIF3 motor. Here we undertake yeast two-hybrid screening and identify Kif3a, a motor subunit of the KIF3 heterotrimeric complex, as a novel Dusp26-binding protein. Co-immunoprecipitation and colocalization experiments revealed that Dusp26 associates not only with Kif3a, but also with Kap3, another subunit of the KIF3 complex. Dephosphorylation experiments in vitro and analysis using mutant forms of Dusp26 in intact cells strongly suggested that Dusp26 is recruited to the KIF3 motor mainly by interaction with Kif3a, and thereby dephosphorylates Kap3. Forced expression of Dusp26, but not its catalytically inactive mutant, promoted distribution of beta-catenin/N-cadherin, an established KIF3 cargo, to cell-cell junction sites, resulting in increased cell-cell adhesiveness. We also showed that Dusp26 mRNA expression was downregulated in human glioblastoma samples. These results suggest previously unidentified functions of Dusp26 in intracellular transport and cell-cell adhesion. Downregulation of Dusp26 may contribute to malignant phenotypes of glioma.

KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells.

Tanaka K, Sugiura Y, Ichishita R, Mihara K, Oka T
J Cell Sci. 2011; 124: 2457-65

Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Balpha and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Balpha mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Balpha bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.

PSD-95 alters microtubule dynamics via an association with EB3.

Sweet ES, Previtera ML, Fernandez JR, Charych EI, Tseng CY, Kwon M, Starovoytov V, Zheng JQ, Firestein BL
J Neurosci. 2011; 31: 1038-47

Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology.

The ced-8 gene controls the timing of programmed cell deaths in C. elegans.

Stanfield GM, Horvitz HR
Mol Cell. 2000; 5: 423-33

Loss-of-function mutations in the gene ced-8 lead to the late appearance of cell corpses during embryonic development in C. elegans. ced-8 functions downstream of or in parallel to-the regulatory cell death gene ced-9 and may function as a cell death effector downstream of the caspase encoded by the programmed cell death killer gene ced-3. In ced-8 mutants, embryonic programmed cell death probably initiates normally but proceeds slowly. ced-8 encodes a transmembrane protein that appears to be localized to the plasma membrane. The CED-8 protein is similar to human XK, a putative membrane transport protein implicated in McLeod Syndrome, a form of hereditary neuroacanthocytosis.

Wild-type but not mutant APC associates with the microtubule cytoskeleton.

Smith KJ, Levy DB, Maupin P, Pollard TD, Vogelstein B, Kinzler KW
Cancer Res. 1994; 54: 3672-5

The adenomatous polyposis coli protein (APC) is mutated in familial adenomatous polyposis patients as well as in sporadic colorectal tumors. In an attempt to further understand the function of APC, the subcellular localization of APC was examined. Wild-type and mutant forms of APC were expressed in mammalian cells and protein detected by immunofluorescence using monoclonal and polyclonal antibodies. Staining of wildtype APC protein revealed a filamentous network which extended throughout the cytoplasm and colocalized with microtubules. In striking contrast, mutant APC protein gave a diffuse cytoplasmic staining pattern. Treatment with a microtubule depolymerizing agent, nocodazole, caused APC as well as tubulin to become diffusely cytoplasmic. In addition, immunoperoxidase staining of transfected APC protein followed by transmission electron microscopy revealed staining of microtubules. These results suggest that wild-type but not mutant APC protein may be associated with the microtubule cytoskeleton.

Analysis of the roles of kinesin and dynein motors in microtubule-based transport in the Caenorhabditis elegans nervous system.

Signor D, Rose LS, Scholey JM
Methods. 2000; 22: 317-25

The heteromeric kinesins constitute a subfamily of kinesin-related motor complexes that function in several distinct intracellular transport events. The founding member of this subfamily, heterotrimeric kinesin II, has been purified and characterized from early sea urchin embryos, where it was shown using antibody perturbation to be required for the synthesis of motile cilia, presumably by driving the anterograde transport of raft complexes. To further characterize heteromeric kinesin transport pathways, and to attempt to identify cargo molecules, we are using the model organism Caenorhabditis elegans to exploit its well-characterized nervous system and simple genetics. Here we describe methods for large-scale nematode growth and partial purification of kinesin-related holoenzymes from C. elegans, and an in vivo transport assay that allows the direct labeling and visualization of motor complexes and putative cargo molecules moving in living C. elegans neurons. This transport assay is being used to characterize the in vivo transport properties of motor enzymes in living cells, and to exploit a number of existing mutations in C. elegans that may represent constituents of heteromeric kinesin-driven transport pathways, for example, the retrograde intraflagellar transport motor CHE-3 dynein, as well as cargo molecules and/or regulatory molecules.

APC and GSK-3beta are involved in mPar3 targeting to the nascent axon and establishment of neuronal polarity.

Shi SH, Cheng T, Jan LY, Jan YN
Curr Biol. 2004; 14: 2025-32

In developing hippocampal neurons in culture, the evolutionarily conserved polarity complex mPar3/mPar6/aPKC selectively accumulates at the tip of one, and only one, of the immature neurites of a neuron and thus specifies the axon and generates neuronal polarity. How mPar3/mPar6 is enriched at the tip of the nascent axon, but not the dendrites, is not fully understood. Here, we report that mPar3 forms a complex with adenomatous polyposis coli (APC) and kinesin superfamily (KIF) 3A, proteins that move along microtubules. In polarizing hippocampal neurons, APC selectively accumulates at the nascent axon tip and colocalizes with mPar3. Expression of dominant-negative C terminus deletion mutants of APC or ectopic expression of APC leads to dislocalization of mPar3 and defects in axon specification and neuronal polarity. In addition to spatial polarization of APC, the selective inactivation of the GSK-3beta activity at the nascent axon tip is required for mPar3 targeting and polarization and establishing neuronal polarity. These results suggest that mPar3 is polarized in developing neurons through APC- and kinesin-mediated transport to the plus ends of rapidly growing microtubules at the nascent axon tip, a process that involves a spatially regulated GSK-3beta activity.

Distinct and redundant functions of mu1 medium chains of the AP-1 clathrin-associated protein complex in the nematode Caenorhabditis elegans.

Shim J, Sternberg PW, Lee J
Mol Biol Cell. 2000; 11: 2743-56

In the nematode Caenorhabditis elegans, there exist two micro1 medium chains of the AP-1 clathrin-associated protein complex. Mutations of unc-101, the gene that encodes one of the micro1 chains, cause pleiotropic effects (). In this report, we identified and analyzed the second mu1 chain gene, apm-1. Unlike the mammalian homologs, the two medium chains are expressed ubiquitously throughout development. RNA interference (RNAi) experiments with apm-1 showed that apm-1 and unc-101 were redundant in embryogenesis and in vulval development. Consistent with this, a hybrid protein containing APM-1, when overexpressed, rescued the phenotype of an unc-101 mutant. However, single disruptions of apm-1 or unc-101 have distinct phenotypes, indicating that the two medium chains may have distinct functions. RNAi of any one of the small or large chains of AP-1 complex (sigma1, beta1, or gamma) showed a phenotype identical to that caused by the simultaneous disruption of unc-101 and apm-1, but not that by single disruption of either gene. This suggests that the two medium chains may share large and small chains in the AP-1 complexes. Thus, apm-1 and unc-101 encode two highly related micro1 chains that share redundant and distinct functions within AP-1 clathrin-associated protein complexes of the same tissue.

IQ-domain GTPase-activating protein 1 regulates beta-catenin at membrane ruffles and its role in macropinocytosis of N-cadherin and adenomatous polyposis coli.

Sharma M, Henderson BR
J Biol Chem. 2007; 282: 8545-56

Beta-catenin is an integral component of E-cadherin dependent cell-cell junctions. Here we show that beta-catenin co-localizes with IQ-domain GTPase-activating protein 1 (IQGAP1), adenomatous polyposis coli (APC), and N-cadherin at actin-positive membrane ruffles in NIH 3T3 fibroblasts. We used deletion mapping to identify the membrane ruffle-targeting region of beta-catenin, localizing it to amino acids 47-217, which overlap the IQGAP1 binding site. Knockdown by small interference RNA (siRNA) revealed IQGAP1-dependent membrane targeting of beta-catenin, APC, and N-cadherin. Transient overexpression of IQGAP1 or N-cadherin increased beta-catenin at membrane ruffles. IQGAP1/APC regulates cell migration, and using a wound healing assay we demonstrate that siRNA-mediated loss of beta-catenin also caused a modest reduction in the rate of cell migration. More significantly, we discovered that beta-catenin is internalized by Arf6-dependent macropinocytosis near sites of membrane ruffling. The beta-catenin macropinosomes co-stained for APC, N-cadherin, and to a lesser extent IQGAP1, and internalization of each binding partner was abrogated by siRNA-dependent knockdown of beta-catenin. In addition, beta-catenin macropinosomes co-localized with the lysosomal marker, lysosome associated membrane protein 1, consistent with their recycling by the late endosomal machinery. Our findings expand on current knowledge of beta-catenin function. We propose that in motile cells beta-catenin is recruited by IQGAP1 and N-cadherin to active membrane ruffles, wherein beta-catenin mediates the internalization and possible recycling of the membrane-associated proteins N-cadherin and APC.

Localization of the adenomatous polyposis coli tumour suppressor protein in the mouse central nervous system.

Senda T, Iino S, Matsushita K, Matsumine A, Kobayashi S, Akiyama T
Neuroscience. 1998; 83: 857-66

The adenomatous polyposis coli gene is mutated in familial adenomatous polyposis and in sporadic colorectal tumours. The adenomatous polyposis coli gene product is a 300,000 mol. wt cytoplasmic protein that binds to at least three other proteins; beta-catenin, a cytoplasmic E-cadherin-associated protein; hDLG, a human homologue of the Drosophila discs large tumour suppressor protein and glycogen synthase kinase 3 beta, a mammalian homologue of the Drosophila ZESTE WHITE 3 protein. The adenomatous polyposis coli gene is highly expressed in the brain, suggesting that it may be involved in nerve function. Here we show that adenomatous polyposis coli is localized in the pericapillary astrocytic endfeet throughout the mouse central nervous system. Adenomatous polyposis coli is also localized in the astrocytic processes in the cerebellar granular layer, and displays concentrated expression in the terminal plexuses of the basket cell fibres around Purkinje cells. Adenomatous polyposis coli is further expressed in neuronal cell bodies and/or nerve fibres in the olfactory bulb, hippocampus, brain stem, spinal cord and dorsal root ganglia. Adenomatous polyposis coli is demonstrated to be co-localized with beta-catenin and/or hDLG in neurons and nerve fibres, but not in astrocytes. From these results, adenomatous polyposis coli is suggested to participate in a signal transduction pathway in astrocytes which is independent of beta-catenin and hDLG, and also in regulation of neuronal functions in association with beta-catenin and hDLG.

Identification of BMP and activin membrane-bound inhibitor (BAMBI), an inhibitor of transforming growth factor-beta signaling, as a target of the beta-catenin pathway in colorectal tumor cells.

Sekiya T, Adachi S, Kohu K, Yamada T, Higuchi O, Furukawa Y, Nakamura Y, Nakamura T, Tashiro K, Kuhara S, Ohwada S, Akiyama T
J Biol Chem. 2004; 279: 6840-6

The Wnt signaling pathway is activated in most human colorectal tumors. Mutational inactivation in the tumor suppressor adenomatous polyposis coli (APC), as well as activation of beta-catenin, causes the accumulation of beta-catenin, which in turn associates with the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors and activates transcription of their target genes. Here we show that beta-catenin activates transcription of the BMP and activin membrane-bound inhibitor (BAMBI)/NMA gene. The expression level of BAMBI was found to be aberrantly elevated in most colorectal and hepatocellular carcinomas relative to the corresponding non-cancerous tissues. Expression of BAMBI in colorectal tumor cell lines was repressed by a dominant-negative mutant of TCF-4 or by an inhibitor of beta-catenin-TCF interaction, suggesting that beta-catenin is responsible for the aberrant expression of BAMBI in colorectal tumor cells. Furthermore, overexpression of BAMBI inhibited the response of tumor cells to transforming growth factor-beta signaling. These results suggest that beta-catenin interferes with transforming growth factor-beta-mediated growth arrest by inducing the expression of BAMBI, and this may contribute to colorectal and hepatocellular tumorigenesis.

Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells.

Rosin-Arbesfeld R, Ihrke G, Bienz M
EMBO J. 2001; 20: 5929-39

Adenomatous polyposis coli (APC) is mutated in most colorectal cancers. APC downregulates nuclear beta-catenin, which is thought to be critical for its tumour suppressor function. However, APC may have additional and separate functions at the cell periphery. Here, we examine polarized MDCK and WIF-B hepatoma cells and find that APC is associated with their lateral plasma membranes. This depends on the actin cytoskeleton but not on microtubules, and drug wash-out experiments suggest that APC is delivered continuously to the plasma membrane by a dynamic actin-dependent process. In polarized MDCK cells, APC also clusters at microtubule tips in their basal-most regions. Microtubule depolymerization causes APC to relocalize from these tips to the plasma membrane, indicating two distinct peripheral APC pools that are in equilibrium with each other in these cells. Truncations of APC such as those found in APC mutant cancer cells can neither associate with the plasma membrane nor with microtubule tips. The ability of APC to reach the cell periphery may thus contribute to its tumour suppressor function in the intestinal epithelium.

Nuclear export of the APC tumour suppressor controls beta-catenin function in transcription.

Rosin-Arbesfeld R, Cliffe A, Brabletz T, Bienz M
EMBO J. 2003; 22: 1101-13

The adenomatous polyposis coli (APC) protein is inactivated in most colorectal tumours. APC loss is an early event in tumorigenesis, and causes an increase of nuclear beta-catenin and its transcriptional activity. This is thought to be the driving force for tumour progression. APC shuttles in and out of the nucleus, but the functional significance of this has been controversial. Here, we show that APC truncations are nuclear in colorectal cancer cells and adenocarcinomas, and this correlates with loss of centrally located nuclear export signals. These signals confer efficient nuclear export as measured directly by fluorescence loss in photobleaching (FLIP), and they are critical for the function of APC in reducing the transcriptional activity of beta-catenin in complementation assays of APC mutant colorectal cancer cells. Importantly, targeting a functional APC construct to the nucleus causes a striking nuclear accumulation of beta-catenin without changing its transcriptional activity. Our evidence indicates that the rate of nuclear export of APC, rather than its nuclear import or steady-state levels, determines the transcriptional activity of beta-catenin.

APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation.

Poulton JS, Mu FW, Roberts DM, Peifer M
Development. 2013; 140: 4226-36

To ensure the accurate transmission of genetic material, chromosome segregation must occur with extremely high fidelity. Segregation errors lead to chromosomal instability (CIN), with deleterious consequences. Mutations in the tumor suppressor adenomatous polyposis coli (APC) initiate most colon cancers and have also been suggested to promote disease progression through increased CIN, but the mechanistic role of APC in preventing CIN remains controversial. Using fly embryos as a model, we investigated the role of APC proteins in CIN. Our findings suggest that APC2 loss leads to increased rates of chromosome segregation error. This occurs through a cascade of events beginning with incomplete centrosome separation leading to failure to inhibit formation of ectopic cleavage furrows, which result in mitotic defects and DNA damage. We test several hypotheses related to the mechanism of action of APC2, revealing that APC2 functions at the embryonic cortex with several protein partners, including Axin, to promote mitotic fidelity. Our in vivo data demonstrate that APC2 protects genome stability by modulating mitotic fidelity through regulation of the cytoskeleton.

Altered SK3/KCa2.3-mediated migration in adenomatous polyposis coli (Apc) mutated mouse colon epithelial cells.

Potier M, Tran TA, Chantome A, Girault A, Joulin V, Bougnoux P, Vandier C, Pierre F
Biochem Biophys Res Commun. 2010; 397: 42-7

Lost of adenomatous polyposis coli gene (Apc) disturbs the migration of intestinal epithelial cells but the mechanisms have not been fully characterized. Since we have demonstrated that SK3/KCa2.3 channel promotes cancer cell migration, we hypothesized that Apc mutation may affect SK3/KCa2.3 channel-mediated colon epithelial cell motility. We report evidence that SK3/KCa2.3 channel promotes colon epithelial cells motility. Following Apc mutation SK3/KCa2.3 expression is largely reduced leading to a suppression of the SK3/KCa2.3 channel mediated-cell migration. Our findings reveal a previously unknown function of the SK3/KCa2.3 channel in epithelial colonic cells, and suggest that Apc is a powerful regulator SK3/KCa2.3 channel.

Assaying processive movement of kinesin by fluorescence microscopy.

Pierce DW, Vale RD
Methods Enzymol. 1998; 298: 154-71

Follicle separation during Drosophila oogenesis requires the activity of the kinesin II-associated polypeptide Kap in germline cells.

Pflanz R, Peter A, Schafer U, Jackle H
EMBO Rep. 2004; 5: 510-4

Cellular localization of organelles, protein complexes and single mRNAs depends on the directed transport along microtubule tracks, a process mediated by ATP-driven molecular motor proteins of the dynein and kinesin superfamilies. Kinesin II is a heterotrimeric protein complex composed of two motor subunits and a unique nonmotor Kinesin-associated protein (Kap). Kap was shown to transport both particulate cargo, as axoneme components in rafts, and membrane-bounded organelles such as melanosomes. Drosophila Kinesin II was shown to be essential for the axonal transport of choline acetyltransferase in a specific set of neurons. We have generated Kap mutants and show that gene activity is not only required for neuronal function but also for separation of follicles during early oogenesis. The data suggest that Kap participates in the transport of signalling components required for instructive interactions between germline and soma cells.

Structural basis for kinesin-1:cargo recognition.

Pernigo S, Lamprecht A, Steiner RA, Dodding MP
Science. 2013; 340: 356-9

Kinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Structural information on how kinesins recognize their cargoes is required for a molecular understanding of this fundamental and ubiquitous process. Here, we present the crystal structure of the tetratricopeptide repeat domain of kinesin light chain 2 in complex with a cargo peptide harboring a "tryptophan-acidic" motif derived from SKIP (SifA-kinesin interacting protein), a critical host determinant in Salmonella pathogenesis and a regulator of lysosomal positioning. Structural data together with biophysical, biochemical, and cellular assays allow us to propose a framework for intracellular transport based on the binding by kinesin-1 of W-acidic cargo motifs through a combination of electrostatic interactions and sequence-specific elements, providing direct molecular evidence of the mechanisms for kinesin-1:cargo recognition.

Functional coordination of intraflagellar transport motors.

Ou G, Blacque OE, Snow JJ, Leroux MR, Scholey JM
Nature. 2005; 436: 583-7

Cilia have diverse roles in motility and sensory reception, and defects in cilia function contribute to ciliary diseases such as Bardet-Biedl syndrome (BBS). Intraflagellar transport (IFT) motors assemble and maintain cilia by transporting ciliary precursors, bound to protein complexes called IFT particles, from the base of the cilium to their site of incorporation at the distal tip. In Caenorhabditis elegans, this is accomplished by two IFT motors, kinesin-II and osmotic avoidance defective (OSM)-3 kinesin, which cooperate to form two sequential anterograde IFT pathways that build distinct parts of cilia. By observing the movement of fluorescent IFT motors and IFT particles along the cilia of numerous ciliary mutants, we identified three genes whose protein products mediate the functional coordination of these motors. The BBS proteins BBS-7 and BBS-8 are required to stabilize complexes of IFT particles containing both of the IFT motors, because IFT particles in bbs-7 and bbs-8 mutants break down into two subcomplexes, IFT-A and IFT-B, which are moved separately by kinesin-II and OSM-3 kinesin, respectively. A conserved ciliary protein, DYF-1, is specifically required for OSM-3 kinesin to dock onto and move IFT particles, because OSM-3 kinesin is inactive and intact IFT particles are moved by kinesin-II alone in dyf-1 mutants. These findings implicate BBS ciliary disease proteins and an OSM-3 kinesin activator in the formation of two IFT pathways that build functional cilia.

Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos.

Morris RL, English CN, Lou JE, Dufort FJ, Nordberg J, Terasaki M, Hinkle B
Dev Biol. 2004; 274: 56-69

KAP is the non-motor subunit of the heteromeric plus-end directed microtubule (MT) motor protein kinesin-II essential for normal cilia formation. Studies in Chlamydomonas have demonstrated that kinesin-II drives the anterograde intraflagellar transport (IFT) of protein complexes along ciliary axonemes. We used a green fluorescent protein (GFP) chimera of KAP, KAP-GFP, to monitor movements of this kinesin-II subunit in cells of sea urchin blastulae where cilia are retracted and rebuilt with each mitosis. As expected if involved in IFT, KAP-GFP localized to apical cytoplasm, basal bodies, and cilia and became concentrated on basal bodies of newly forming cilia. Surprisingly, after ciliary retraction early in mitosis, KAP-GFP moved into nuclei before nuclear envelope breakdown, was again present in nuclei after nuclear envelope reformation, and only decreased in nuclei as ciliogenesis reinitiated. Nuclear transport of KAP-GFP could be due to a putative nuclear localization signal and nuclear export signals identified in the sea urchin KAP primary sequence. Our observation of a protein involved in IFT being imported into the nucleus after ciliary retraction and again after nuclear envelope reformation suggests KAP115 may serve as a signal to the nucleus to reinitiate cilia formation during sea urchin development.

Abnormalities of the APC/beta-catenin pathway in endometrial cancer.

Moreno-Bueno G, Hardisson D, Sanchez C, Sarrio D, Cassia R, Garcia-Rostan G, Prat J, Guo M, Herman JG, Matias-Guiu X, Esteller M, Palacios J
Oncogene. 2002; 21: 7981-90

The activation of the APC/beta-catenin signalling pathway due to beta-catenin mutations has been implicated in the development of a subset of endometrial carcinomas (ECs). However, up to 25% of ECs have beta-catenin nuclear accumulation without evidence of beta-catenin mutations, suggesting alterations of other molecules that can modulate the Wnt pathway, such as APC, gamma-catenin, AXIN1 and AXIN2. We investigated the expression pattern of beta- and gamma-catenin in a group of 128 endometrial carcinomas, including 95 endometrioid endometrial carcinomas (EECs) and 33 non-endometrioid endometrial carcinomas (NEECs). In addition, we evaluated the presence of loss of heterozygosity and promoter hypermethylation of the APC gene and mutations in the APC, beta- and gamma-catenin, AXIN1, AXIN2, and RAS genes, and phospho-Akt expression. No APC mutations were detected but LOH at the APC locus was found in 24.3% of informative cases. APC promoter 1A hypermethylation was observed in 46.6% of ECs, and was associated with the endometrioid phenotype (P=0.034) and microsatellite instability (P=0.008). Neither LOH nor promoter hypermethylation of APC was associated with nuclear catenin expression. Nuclear beta-catenin expression was found in 31.2% of EECs and 3% of NEECs (P=0.002), and was significantly associated with beta-catenin gene exon 3 mutations (P<0.0001). beta-catenin gene exon 3 mutations were associated with the endometrioid phenotype, and were detected in 14 (14.9%) EECs, but in none of the NEECs (P=0.02). gamma-catenin nuclear expression was found in 10 ECs; it was not associated with the histological type but was associated with more advanced stages (P=0.042). No mutations in gamma-catenin, AXIN1 and 2 genes were detected in this series. Neither RAS mutations nor phospho-Akt expression, which were found in 16 and 27.6% of the cases, respectively, were associated with beta-catenin nuclear expression. Our results demonstrated a high prevalence of alterations in molecules of the APC/beta-catenin pathway, but only mutations in beta-catenin gene are associated with aberrant nuclear localization of beta-catenin.

The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.

Mimori-Kiyosue Y, Shiina N, Tsukita S
Curr Biol. 2000; 10: 865-8

Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under restricted conditions.

Novel self-association of the APC molecule affects APC clusters and cell migration.

Li Z, Kroboth K, Newton IP, Nathke IS
J Cell Sci. 2008; 121: 1916-25

Truncation mutations in the adenomatous polyposis coli (APC) gene are responsible for familial and sporadic colorectal cancer. APC is a multifunctional protein involved in cell migration, proliferation and differentiation. The APC protein forms specific clusters in the cell periphery that correlate with sites of active cell migration. Little is known about the molecular mechanisms that govern these clusters. Here, we identify a novel interaction of an N-terminal region of APC with the extreme C-terminal 300 amino acids of APC and also with itself. The latter interaction is phospho-sensitive and is enhanced by 14-3-3 (YWHA) protein. These interactions modulate the clustering of APC at the ends of membrane protrusions. Overexpressing this domain or inhibiting 14-3-3 proteins disperses APC clusters and leads to decreased cell migration. Moreover, deleting this domain from full-length APC results in less-dynamic clusters compared with wild-type APC. Our data indicate that this newly identified regions in the N-terminal third of APC contributes to the regulation of APC clusters, thus providing a molecular clue for how locally regulated phosphorylation events could mediate the dynamics of APC clusters and contribute to cell migration.

New approach to capture and characterize synaptic proteome.

Liu XA, Kadakkuzha B, Pascal B, Steckler C, Akhmedov K, Yan L, Chalmers M, Puthanveettil SV
Proc Natl Acad Sci U S A. 2014; 111: 16154-9

Little is known regarding the identity of the population of proteins that are transported and localized to synapses. Here we describe a new approach that involves the isolation and systematic proteomic characterization of molecular motor kinesins to identify the populations of proteins transported to synapses. We used this approach to identify and compare proteins transported to synapses by kinesin (Kif) complexes Kif5C and Kif3A in the mouse hippocampus and prefrontal cortex. Approximately 40-50% of the protein cargos identified in our proteomics analysis of kinesin complexes are known synaptic proteins. We also found that the identity of kinesins and where they are expressed determine what proteins they transport. Our results reveal a previously unappreciated role of kinesins in regulating the composition of synaptic proteome.

[Dynamic changes of adenomatous polyposis coli protein and glycogen synthase kinase 3beta in the repair of the injured airway epithelial cells in smoking mice].

Liu MG, Li NP, Wu RL, Ma Y, Hong YZ, Tian D, Zhu M
Sheng Li Xue Bao. 2006; 58: 255-61

To investigate the roles of adenomatous polyposis coli (APC) protein and glycogen synthase kinase 3beta (GSK3beta) of smoking murine model in the repair of the injured airway epithelial cells (AECs) in different stages, 30 male Kun-Ming mice were randomly divided into two groups, the control group and the smoking group. There were 24 mice in smoking group, and 6 animals were separately killed at the end of the 1st, 4th, 8th and 12th week after smoking. Then the following tests were undertaken: (1) HE staining of lung section to observe the morphological changes of the bronchi in the smoking mice. (2) Immunohistochemical staining of APC protein and GSK3beta in the AECs. (3) Western blot was used to detect the levels of APC protein, GSK3beta and phosphorated GSK3beta (p-GSK3beta) in pulmonary tissue. (4) Observing the localizations of APC protein and GSK3beta in the AECs by immunofluorescence technique. The results showed: (1) AECs showed changes of predominant injury (1-, 4-week), repair (8-week) and reinjury (12-week) along with smoking time prolonged. The experimental results indicated that the model of smoking mice was duplicated successfully. (2) Immunohistochemical results showed that the expression of APC protein in the AECs increased after 1-week smoking (0.458 +/- 0.062 vs 0.399 +/- 0.060, P< 0.05 vs control), but was significantly decreased at the end of the 4th week (0.339+/- 0.056, P<0.01 vs control) and increased at the end of the 8th and 12th week (0.387 +/- 0.041, 0.378 +/- 0.037, P<0.05 vs 4-week). The expression of GSK3beta in the AECs of smoking mice obviously decreased (P<0.01 or P<0.05 vs control). (3) Western blot showed that the expressions of APC protein and GSK3beta in lung tissue were consistent with the results of immunohistochemistry; and the levels of p-GSK3beta in all smoking models were higher than that in control. (4) The results of immunofluorescence showed that APC protein was localized mainly near the regions of epithelial cell membrane at the end of the 1st and 8th week after smoking, which were dissimilar with the localization in control, and this change was not seen in the location of GSK3beta. Taken together, these results demonstrate that the expressions and localizations of APC protein, GSK3beta and the activity of GSK3beta are dynamically changed in the AECs with experimental smoking injury at different phases, suggesting that APC protein and GSK3beta may be involved in the regulation of migration and proliferation of AECs, and play an important role in the process of repair of airway epithelium injury.

Intermediate filaments: not just for structure anymore.

Liem RK
Curr Biol. 2013; 23: 3224-3224

A recent paper has identified the tumor suppressor APC as a linker protein between intermediate filaments and microtubules. In the absence of APC, intermediate filaments collapse and the cells are no longer polarized and fail to migrate.

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1.

Li B, Zhuang L, Trueb B
J Biol Chem. 2004; 279: 20401-10

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.

Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex.

Kunttas-Tatli E, Roberts DM, McCartney BM
Mol Biol Cell. 2014; 25: 3424-36

The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3beta, and CK1 that targets beta-catenin/Armadillo (beta-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles we termed the destructosome. Whereas APC functions in the complex through its ability to bind both beta-cat and Axin, we hypothesize that APC proteins play an additional role in destructosome assembly through self-association. Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates beta-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in beta-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.

Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention.

Krieghoff E, Behrens J, Mayr B
J Cell Sci. 2006; 119: 1453-63

beta-catenin is the central signalling molecule of the canonical Wnt pathway, where it activates target genes in a complex with LEF/TCF transcription factors in the nucleus. The regulation of beta-catenin activity is thought to occur mainly on the level of protein degradation, but it has been suggested that beta-catenin nuclear localization and hence its transcriptional activity may additionally be regulated via nuclear import by TCF4 and BCL9 and via nuclear export by APC and axin. Using live-cell microscopy and fluorescence recovery after photobleaching (FRAP), we have directly analysed the impact of these factors on the subcellular localization of beta-catenin, its nucleo-cytoplasmic shuttling and its mobility within the nucleus and the cytoplasm. We show that TCF4 and BCL9/Pygopus recruit beta-catenin to the nucleus, and APC, axin and axin2 enrich beta-catenin in the cytoplasm. Importantly, however, none of these factors accelerates the nucleo-cytoplasmic shuttling of beta-catenin, i.e. increases the rate of beta-catenin nuclear import or export. Moreover, the cytoplasmic enrichment of beta-catenin by APC and axin is not abolished by inhibition of CRM-1-dependent nuclear export. TCF4, APC, axin and axin2 move more slowly than beta-catenin in their respective compartment, and concomitantly decrease beta-catenin mobility. Together, these data indicate that beta-catenin interaction partners mainly regulate beta-catenin subcellular localization by retaining it in the compartment in which they are localized, rather than by active transport into or out of the nucleus.

Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1.

Kita K, Wittmann T, Nathke IS, Waterman-Storer CM
Mol Biol Cell. 2006; 17: 2331-45

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remained bound to MTs stabilized with low nocodazole, whereas EB1 did not. Thus, we demonstrate that the association of endogenous APC with MT ends correlates directly with their increased growth stability, that this can occur independently of its association with EB1, and that APC and EB1 can associate with MT plus ends by distinct mechanisms.

Asef, a link between the tumor suppressor APC and G-protein signaling.

Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T
Science. 2000; 289: 1194-7

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. Here the APC gene product is shown to bind through its armadillo repeat domain to a Rac-specific guanine nucleotide exchange factor (GEF), termed Asef. Endogenous APC colocalized with Asef in mouse colon epithelial cells and neuronal cells. Furthermore, APC enhanced the GEF activity of Asef and stimulated Asef-mediated cell flattening, membrane ruffling, and lamellipodia formation in MDCK cells. These results suggest that the APC-Asef complex may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function.

Mutated APC and Asef are involved in the migration of colorectal tumour cells.

Kawasaki Y, Sato R, Akiyama T
Nat Cell Biol. 2003; 5: 211-5

The tumour suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC binds to beta-catenin, a key component of the Wnt signalling pathway, and induces its degradation. APC interacts with microtubules and accumulates at their plus ends in membrane protrusions, and associates with the plasma membrane in an actin-dependent manner. In addition, APC interacts with the Rac-specific guanine nucleotide exchange factor Asef and stimulates its activity, thereby regulating the actin cytoskeletal network and cell morphology. Here we show that overexpression of Asef decreases E-cadherin-mediated cell-cell adhesion and promotes the migration of epithelial Madin-Darby canine kidney cells. Both of these activities are stimulated by truncated APC proteins expressed in colorectal tumour cells. Experiments based on RNA interference and dominant-negative mutants show that both Asef and mutated APC are required for the migration of colorectal tumour cells expressing truncated APC. These results suggest that the APC-Asef complex functions in cell migration as well as in E-cadherin-mediated cell-cell adhesion, and that truncated APC present in colorectal tumour cells contributes to their aberrant migratory properties.

The adenomatous polyposis coli-associated guanine nucleotide exchange factor Asef is involved in angiogenesis.

Kawasaki Y, Jigami T, Furukawa S, Sagara M, Echizen K, Shibata Y, Sato R, Akiyama T
J Biol Chem. 2010; 285: 1199-207

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of beta-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef(-/-) mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.

Down-regulation of beta-catenin by the colorectal tumor suppressor APC requires association with Axin and beta-catenin.

Kawahara K, Morishita T, Nakamura T, Hamada F, Toyoshima K, Akiyama T
J Biol Chem. 2000; 275: 8369-74

The tumor suppressor adenomatous polyposis coli (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. APC forms a complex with beta-catenin, Axin, and glycogen synthase kinase-3beta and induces the degradation of beta-catenin. In the present study, we examined whether APC association with Axin is required for degradation of beta-catenin. We found that a fragment of APC that induces beta-catenin degradation was rendered inactive by disruption of its Axin-binding sites. Also, overexpression of an Axin fragment spanning the regulator of the G-protein signaling domain inhibited APC-mediated beta-catenin degradation. An APC fragment with mutated beta-catenin-binding sites but intact Axin-binding sites also failed to induce degradation of beta-catenin. These results suggest that APC requires interaction with Axin and beta-catenin to down-regulate beta-catenin.

Regulation of cell migration by dynamic microtubules.

Kaverina I, Straube A
Semin Cell Dev Biol. 2011; 22: 968-74

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.

The tumor suppressor adenomatous polyposis coli and caudal related homeodomain protein regulate expression of retinol dehydrogenase L.

Jette C, Peterson PW, Sandoval IT, Manos EJ, Hadley E, Ireland CM, Jones DA
J Biol Chem. 2004; 279: 34397-405

Development of normal colon epithelial cells proceeds through a systematic differentiation of cells that emerge from stem cells within the base of colon crypts. Genetic mutations in the adenomatous polyposis coli (APC) gene are thought to cause colon adenoma and carcinoma formation by enhancing colonocyte proliferation and impairing differentiation. We currently have a limited understanding of the cellular mechanisms that promote colonocyte differentiation. Herein, we present evidence supporting a lack of retinoic acid biosynthesis as a mechanism contributing to the development of colon adenomas and carcinomas. Microarray and reverse transcriptase-PCR analyses revealed reduced expression of two retinoid biosynthesis genes: retinol dehydrogenase 5 (RDH5) and retinol dehydrogenase L (RDHL) in colon adenomas and carcinomas as compared with normal colon. Consistent with the adenoma and carcinomas samples, seven colon carcinoma cell lines also lacked expression of RDH5 and RDHL. Assessment of RDH enzymatic activity within these seven cell lines showed poor conversion of retinol into retinoic acid when compared with normal cells such as normal human mammary epithelial cells. Reintroduction of wild type APC into an APC-deficient colon carcinoma cell line (HT29) resulted in increased expression of RDHL without affecting RDH5. APC-mediated induction of RDHL was paralleled by increased production of retinoic acid. Investigations into the mechanism responsible for APC induction of RDHL indicated that beta-catenin fails to repress RDHL. The colon-specific transcription factor CDX2, however, activated an RDHL promoter construct and induced endogenous RDHL. Finally, the induction of RDHL by APC appears dependent on the presence of CDX2. We propose a novel role for APC and CDX2 in controlling retinoic acid biosynthesis and in promoting a retinoid-induced program of colonocyte differentiation.

Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.

Huang TG, Suhan J, Hackney DD
J Biol Chem. 1994; 269: 32708-32708

Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein.

Homma MK, Li D, Krebs EG, Yuasa Y, Homma Y
Proc Natl Acad Sci U S A. 2002; 99: 5959-64

Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2alpha- and beta-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G(2)/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2.

Synaptic scaffolding molecule interacts with axin.

Hirabayashi S, Nishimura W, Iida J, Kansaku A, Kishida S, Kikuchi A, Tanaka N, Hata Y
J Neurochem. 2004; 90: 332-9

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl-d-aspartate receptor subunits, neuroligin, and beta-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3beta-binding region of Axin. S-SCAM formed a complex with beta-catenin and Axin, but competed with GSK3beta for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of beta-catenin by GSK3beta.

Casein kinase 2- and protein kinase A-regulated adenomatous polyposis coli and beta-catenin cellular localization is dependent on p38 MAPK.

Hildesheim J, Salvador JM, Hollander MC, Fornace AJ Jr
J Biol Chem. 2005; 280: 17221-6

Skin cancer is the most common form of malignancy in the world with epidemic proportions. Identifying the biochemical and molecular mechanisms underlying the events leading to tumors is paramount to designing new and effective treatments that may aid in treating and/or preventing skin cancers. Herein we identify p38 MAPK, along with its positive modulator, Gadd45a, as important regulators of nucleocytoplasmic shuttling of the adenomatous polyposis coli (APC) tumor suppressor. APC normally functions to block beta-catenin from promoting cell proliferation and migration/invasion. Keratinocytes lacking proper p38 MAPK activation, either due to lack of Gadd45a or through the use of p38 MAPK-specific inhibitors, are unable to effectively transport APC into the nucleus. We also show that p38 MAPK is able to directly associate with and modulate both casein kinase 2 (CK2) and protein kinase A (PKA), which promote and block APC nuclear import, respectively. We demonstrate that p38 MAPK is able to not only enhance CK2 kinase activity but also suppress PKA kinase activity. Moreover, lack of normal p38 MAPK activity in either Gadd45a-null keratinocytes or in p38 MAPK inhibitor treated keratinocytes leads to decreased CK2 activity and increased PKA activity. In either case, disruption of APC nuclear import results in elevated levels of free cellular, and potentially oncogenic, beta-catenin. Numerous tumors, including skin cancers, are associated with high levels of beta-catenin, and our data indicate that p38 MAPK signaling, along with Gadd45a, may provide tumor suppressor-like functions in part by promoting APC nuclear localization and effective beta-catenin regulation.

Gadd45a regulates matrix metalloproteinases by suppressing DeltaNp63alpha and beta-catenin via p38 MAP kinase and APC complex activation.

Hildesheim J, Belova GI, Tyner SD, Zhou X, Vardanian L, Fornace AJ Jr
Oncogene. 2004; 23: 1829-37

The p53-regulated growth arrest and DNA damage-inducible gene product Gadd45a has been recently identified as a key factor protecting the epidermis against ultraviolet radiation (UVR)-induced skin tumors by activating p53 via the stress mitogen-activated protein kinase (MAPK) signaling pathway. Herein we identify Gadd45a as an important negative regulator of two oncogenes commonly over-expressed in epithelial tumors: the p53 homologue DeltaNp63alpha and beta-catenin. DeltaNp63alpha is one of the several p63 isoforms and is the predominant species expressed in basal epidermal keratinocytes. DeltaNp63alpha lacks the N-terminal transactivation domain and behaves as a dominant-negative factor blocking expression of several p53-effector genes. DeltaNp63alpha also associates with and blocks activation of the adenomatous polyposis coli (APC) destruction complex that targets free cytoplasmic beta-catenin for degradation. While most beta-catenin protein is localized to the cell membrane and is involved in cell-cell adhesion, accumulation of free cytoplasmic beta-catenin will translocate into the nucleus where it functions in a bipartite transcription factor complex, whose targets include invasion and metastasis promoting endopeptidases, matrix metalloproteinases (MMP). We show that Gadd45a not only directly associates with two components of the APC complex, namely protein phosphatase 2A (PP2A) and glycogen synthase kinase 3beta (GSK3beta) but also promotes GSK3beta dephosphorylation at Ser9, which is essential for GSK3beta activation, and resultant activation of the APC destruction complex. We demonstrate that lack of Gadd45a not only prevents DeltaNp63alpha suppression and GSK3beta dephosphorylation but also prevents free cytoplasmic beta-catenin degradation after UV irradiation. The inability of Gadd45a-null keratinocytes to suppress beta-catenin may contribute to the resulting observation of increased MMP expression and activity along with significantly faster keratinocyte migration in Matrigel in vitro and accelerated wound closure in vivo. Furthermore, epidermal keratinocytes treated with p38 MAPK inhibitors, both in vivo and in vitro, behave very similarly to Gadd45a-null keratinocytes after UVR. Similarly, Trp53-null mice are unable to attenuate DeltaNp63alpha expression in epidermal keratinocytes after such stress. These findings demonstrate a dependence on Gadd45a-mediated p38 MAPK and p53 activation for proper modulation of DeltaNp63alpha, GSK3beta, and beta-catenin after irradiation. Taken together, our results indicate that Gadd45a is able to repress DeltaNp63alpha, beta-catenin, and consequently MMP expression by two means: by maintaining UVR-induced p38 MAPK and p53 activation and also by associating with the APC complex. This implicates Gadd45a in the negative regulation of cell migration, and invasion.

Xenopus axin interacts with glycogen synthase kinase-3 beta and is expressed in the anterior midbrain.

Hedgepeth CM, Deardorff MA, Klein PS
Mech Dev. 1999; 80: 147-51

Axin is encoded by the fused locus in mice and is required for normal vertebrate axis formation. It has recently been shown that axin associates with APC, beta-catenin and glycogen synthase kinase-3 (GSK-3) in a complex that appears to regulate the level of cytoplasmic beta-catenin. We have identified the Xenopus homologue of axin through its interaction with GSK-3b. Xenopus axin (Xaxin) is expressed maternally and throughout early development with a low level of ubiquitous expression. Xaxin also shows remarkably high expression in the anterior mesencephalon adjacent to the forebrain-midbrain boundary.

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification.

Farr GH 3rd, Ferkey DM, Yost C, Pierce SB, Weaver C, Kimelman D
J Cell Biol. 2000; 148: 691-702

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

Nuclear accumulation of full-length and truncated adenomatous polyposis coli protein in tumor cells depends on proliferation.

Fagman H, Larsson F, Arvidsson Y, Meuller J, Nordling M, Martinsson T, Helmbrecht K, Brabant G, Nilsson M
Oncogene. 2003; 22: 6013-22

The adenomatous polyposis coli (APC) tumor suppressor is a nucleocytoplasmic protein. The nuclear accumulation of APC was recently found to vary depending on cell density, suggesting that putative APC function(s) in the nucleus is controlled by the establishment of cell contacts. We report here that the density-dependent redistribution of APC between nucleus and cytoplasm prevails in 6/6 thyroid and colorectal carcinoma cell lines. Moreover, mutated APC lacking known nuclear localization sequences had the similar distribution pattern as the full-length protein. APC invariably accumulated in the nuclei of Ki-67 expressing cells, but was largely cytoplasmic when cell cycle exit was induced by serum starvation or at high cell density. APC colocalized with beta-catenin in the nucleus only in one cell line (SW480). Also, APC maintained a predominantly nuclear position in early confluent states when cytoplasmic beta-catenin was recruited to newly formed adherens-like junctions. The results indicate that nuclear targeting of APC is driven by cell cycle entry rather than altered cell-cell contact. The ability of C-terminally truncated APC to accumulate in the nucleus suggests that nuclear import signals other than NLS1(APC) and NLS2(APC) are functionally important. Residual function(s) of N-terminal APC fragments in tumor cells carrying APC mutations might be beneficial to tumor growth and survival.

APC in cell migration.

Etienne-Manneville S
Adv Exp Med Biol. 2009; 656: 30-40

Adenomatous Polyposis Coli (APC) is a tumor suppressor protein involved in the initiation and progression of colon cancer. The most widely accepted function ofAPC is to participate to the Wnt signaling pathway, by downregulating beta-catenin and thereby controlling gene transcription and cell proliferation. However, APC is clearly a multifunctional protein whose loss contributes to tumor formation in multiple ways. Regulation of APC localization during cell migration and the ability of APC to bind multiple polarity proteins and microtubule-associated molecules support the idea that APC plays a key role in directed cell migration and that this function may contribute to its tumour suppressor activity.

The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain.

Erdmann KS, Kuhlmann J, Lessmann V, Herrmann L, Eulenburg V, Muller O, Heumann R
Oncogene. 2000; 19: 3894-901

Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying five PDZ protein-protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (K(D)) of 8.1 x 10(-9) M. We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC protein. The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as beta-catenin playing a major role in the regulation of cell division, migration and cell adhesion.

The adenomatous polyposis coli protein: in the limelight out at the edge.

Dikovskaya D, Zumbrunn J, Penman GA, Nathke IS
Trends Cell Biol. 2001; 11: 378-84

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial and sporadic colonic tumours. APC is best known for its role in regulating beta-catenin, an important mediator of cell adhesion and a transcriptional activator. However, recent studies indicate that APC has additional roles in cytoskeletal regulation. It binds to microtubules directly and indirectly. Furthermore, indirect connections between APC and the actin cytoskeleton have also been described. Here, we integrate recent information describing the association between APC and the cytoskeleton to illustrate how this multifaceted protein might link different cytoskeletal elements to each other and to cellular signaling pathways.

The adenomatous polyposis coli protein is required for the formation of robust spindles formed in CSF Xenopus extracts.

Dikovskaya D, Newton IP, Nathke IS
Mol Biol Cell. 2004; 15: 2978-91

Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correlate with chromosomal instability. Here, we show that depletion of APC from cystostatic factor (CSF) Xenopus extracts leads to a decrease in microtubule density and changes in tubulin distribution in spindles and asters formed in such extracts. Addition of full-length APC protein or a large, N-terminally truncated APC fragment to APC-depleted extracts restored normal spindle morphology and the intact microtubule-binding site of APC was necessary for this rescue. These data indicate that the APC protein plays a role in the formation of spindles that is dependent on its effect on microtubules. Spindles formed in cycled extracts were not sensitive to APC depletion. In CSF extracts, spindles predominantly formed from aster-like intermediates, whereas in cycled extracts chromatin was the major site of initial microtubule polymerization. These data suggest that APC is important for centrosomally driven spindle formation, which was confirmed by our finding that APC depletion reduced the size of asters nucleated from isolated centrosomes. We propose that lack of microtubule binding in cancer-associated mutations of APC may contribute to defects in the assembly of mitotic spindles and lead to missegregation of chromosomes.

The APC protein binds to A/T rich DNA sequences.

Deka J, Herter P, Sprenger-Haussels M, Koosch S, Franz D, Muller KM, Kuhnen C, Hoffmann I, Muller O
Oncogene. 1999; 18: 5654-61

The tumor suppressor protein APC (Adenomatous Polyposis Coli) is localized in the cytosol and in the nucleus. In this study, we demonstrate that the nuclear APC protein level is high in cells in the basal crypt region of the normal colorectal epithelium. Strikingly, the APC protein staining resembles the staining pattern of a nuclear proliferation marker. As a first step towards a possible role of the nuclear APC protein, we provide data showing the direct interaction of the nuclear APC protein with DNA. A nuclear APC isoform precipitates with matrix-immobilized DNA. Vice versa, the immunoprecipitation of APC from nuclear lysates results in co-precipitation of genomic DNA. Using recombinant APC fragments we mapped three DNA binding domains: one within the beta-catenin binding and regulatory domain, and two in the carboxyterminal third of the APC protein. All these three domains contain clusters of repetitive S(T)PXX sequence motifs that were described to mediate the DNA interaction of many other DNA binding proteins. In analogy to S(T)PXX proteins, the APC protein binds preferentially to A/T rich DNA sequences rather than to a single DNA sequence motif.

Dynamic properties of APC-decorated microtubules in living cells.

Dayanandan R, Butler R, Gordon-Weeks PR, Matus A, Kaech S, Lovestone S, Anderton BH, Gallo JM
Cell Motil Cytoskeleton. 2003; 54: 237-47

The adenomatous polyposis coli (APC) tumour suppressor protein is a component of the Wnt signalling pathway in which it plays a major role in controlling nuclear accumulation of beta-catenin and hence in the modulation of beta-catenin-regulated gene transcription. APC also associates with microtubules at the ends of cytoplasmic extensions in epithelial cells, a distribution that can be reproduced in COS cells ectopically expressing APC. To examine the effect of APC on microtubule properties, we monitored directly the behaviour of APC and of APC-decorated microtubules by time-lapse imaging of cytoplasmic extensions in live COS cells expressing APC tagged with a green fluorescent protein. On the proximal part of microtubules, APC was visualised as particulate material moving unidirectionally towards the plus end of microtubules. The distal parts of microtubules were uniformly decorated by APC and were animated by a motile behaviour in the form of aperiodic bending. This behaviour is likely to be the consequence of compression forces acting on microtubules encountering obstacles while elongating. The majority of APC-decorated microtubules in transfected COS cells was sensitive to depolymerisation by nocodazole, but they contained detyrosinated and acetylated alpha-tubulin, suggesting a reduction in the rate of subunit exchange at their growing end. Taken together, these results demonstrate that microtubule domains uniformly decorated by APC display dynamic and motile properties that may be significant for the postulated role of APC in targeting microtubules to specialised membrane sites.

Density-dependent location and interactions of truncated APC and beta-catenin.

Davies ML, Roberts GT, Spiller DG, Wakeman JA
Oncogene. 2004; 23: 1412-9

Adenomatous polyposis coli (APC) is a multifunctional tumour suppressor protein, central to development and the mature organism. It is mutated in most cases of colorectal cancer, rendering it ineffective in mediating beta-catenin degradation. We show that localization of full-length APC in colon carcinoma and noncancer cell lines is independent of cell density. However, the location of truncated APC is a function of cell density and in high-density cells truncated APC is predominantly not nuclear. Although the distribution of truncated APC and beta-catenin is closely linked in subconfluent SW480 cells, at high cell density they are not colocalized. We postulated that in this cell line this could be due to an increase in beta-catenin bound to E-cadherin with formation of adherens junctions at high cell density. However, while in coimmunoprecipitation assays we observe an increase in binding between beta-catenin and E-cadherin and a corresponding decrease in binding between beta-catenin and APC at high cell density, we did not observe a strict colocalization of beta-catenin and E-cadherin at the membrane of all cells.

Ectopic expression of a Haemonchus contortus GATA transcription factor in Caenorhabditis elegans reveals conserved function in spite of extensive sequence divergence.

Couthier A, Smith J, McGarr P, Craig B, Gilleard JS
Mol Biochem Parasitol. 2004; 133: 241-53

Comparative analysis between Caenorhabditis elegans and other nematode species offers a powerful approach to study gene function. C. elegans also has great potential as a surrogate expression system to study the function of genes from parasitic nematode species where transgenic methodologies are unavailable. However there is little information on the extent to which the biology of C. elegans is conserved with other nematode species and very few parasitic nematode genes have yet been functionally expressed in C. elegans. We have identified and characterised a homologue of the C. elegans GATA transcription factor elt-2, a central regulator of endoderm development, from the parasitic nematode Haemonchus contortus. The H. contortus ELT-2 polypeptide is present in endoderm nuclei throughout embryonic and post-embryonic development, except for in the infective L3 stage, and our experiments reveal that the development of the H. contortus endodermal lineage is strikingly similar to that of C. elegans. Sequence conservation between the H. contortus and C. elegans ELT-2 polypeptides broadly reflects function since the major region of sequence identity corresponds to the DNA binding domain. However, the overall level of sequence identity is remarkably low with the only other major region of identity corresponding to an unusual zinc finger domain. In spite of this, ectopic expression of the H. contortus elt-2 gene in transgenic C. elegans is sufficient to activate a programme of endodermal differentiation demonstrating that function is highly conserved. This approach of ectopic expression using an inducible promoter provides an effective way in which to use C. elegans for the in vivo functional analysis of parasitic nematode genes.

Mitogen-activated protein kinase (MAPK/ERK) regulates adenomatous polyposis coli during growth-factor-induced cell extension.

Caro-Gonzalez HY, Nejsum LN, Siemers KA, Shaler TA, Nelson WJ, Barth AI
J Cell Sci. 2012; 125: 1247-58

Regulation of the microtubule- and actin-binding protein adenomatous polyposis coli (APC) is crucial for the formation of cell extensions in many cell types. This process requires inhibition of glycogen synthase kinase-3beta (GSK-3beta), which otherwise phosphorylates APC and decreases APC-mediated microtubule bundling. Although it is assumed, therefore, that APC phosphorylation is decreased during initiation of cell extensions, the phosphorylation state of APC has never been analyzed directly. We show here that NGF- and EGF-induced initial cell extensions result in APC phosphorylation by the MAPK/ERK pathway, which, in parallel with inhibition of GSK-3beta, promotes localization of APC to the tip of cell extensions. Whereas GSK-3beta inhibition promotes APC binding and stabilization of microtubules, we show that phosphorylation by ERK inhibits the interaction of APC with F-actin, and APC-mediated F-actin bundling, but not APC-mediated microtubule bundling, in vitro. These results identify a previously unknown APC regulatory pathway during growth-factor-induced cell extension, and indicate that the GSK-3beta and ERK pathways act in parallel to regulate interactions between APC and the cytoskeleton during the formation of cell extensions.

Dauer pheromone and G-protein signaling modulate the coordination of intraflagellar transport kinesin motor proteins in C. elegans.

Burghoorn J, Dekkers MP, Rademakers S, de Jong T, Willemsen R, Swoboda P, Jansen G
J Cell Sci. 2010; 123: 2077-84

Cilia length and function are dynamically regulated by modulation of intraflagellar transport (IFT). The cilia of C. elegans amphid channel neurons provide an excellent model to study this process, since they use two different kinesins for anterograde transport: kinesin-II and OSM-3 kinesin together in the cilia middle segments, but only OSM-3 in the distal segments. To address whether sensory signaling modulates the coordination of the kinesins, we studied IFT protein motility in gpa-3 mutant animals, since dominant active mutation of this sensory Galpha protein GPA-3QL) affects cilia length. In addition, we examined animals exposed to dauer pheromone, since dauer formation, which involves gpa-3, induces changes in cilia morphology. Live imaging of fluorescently tagged IFT proteins showed that in gpa-3 mutants and in larvae exposed to dauer pheromone, kinesin-II speed is decreased and OSM-3 speed is increased, whereas structural IFT proteins move at an intermediate speed. These results indicate that mutation of gpa-3 and exposure to dauer pheromone partially uncouple the two kinesins. We propose a model in which GPA-3-regulated docking of kinesin-II and/or OSM-3 determines entry of IFT particles into the cilia subdomains, allowing structural and functional plasticity of cilia in response to environmental cues.

APC: the plot thickens.

Bienz M
Curr Opin Genet Dev. 1999; 9: 595-603

Adenomatous polyposis coli (APC) is an important tumour suppressor in the human colon. It is conserved between human and flies, and promotes, together with Axin and glycogen synthase kinase 3 (GSK3), the degradation of the Wnt-signalling effector beta-catenin. Recent experiments have shaped our understanding of how Axin and GSK3 function but the role of APC in this process remains elusive.

Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta.

Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D, Birchmeier W
Science. 1998; 280: 596-9

Control of stability of beta-catenin is central in the wnt signaling pathway. Here, the protein conductin was found to form a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC). Conductin induced beta-catenin degradation, whereas mutants of conductin that were deficient in complex formation stabilized beta-catenin. Fragments of APC that contained a conductin-binding domain also blocked beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfered with wnt-induced axis formation.

Role of adenomatous polyposis coli (APC) and microtubules in directional cell migration and neuronal polarization.

Barth AI, Caro-Gonzalez HY, Nelson WJ
Semin Cell Dev Biol. 2008; 19: 245-51

In response to extracellular signals during embryonic development, cells undergo directional movements to specific sites and establish proper connections to other cells to form organs and tissues. Cell extension and migration in the direction of extracellular cues is mediated by the actin and microtubule cytoskeletons, and recent results have shed new light on how these pathways are activated by neurotrophins, Wnt or extracellular matrix. These signals lead to modifications of microtubule-associated proteins (MAPs) and point to glycogen synthase kinase (GSK) 3beta as a key regulator of microtubule function during directional migration. This review will summarize these results and then focus on the role of microtubule-binding protein adenomatous polyposis coli (APC) in neuronal polarization and directed migration, and on its regulation by GSK3beta.

Fatty acylation of polypeptides in the nematode Caenorhabditis elegans.

Aspbury RA, Fisher MJ, Rees HH
Biochim Biophys Acta. 1998; 1382: 111-9

Covalent modification of eucaryotic proteins, involving addition of fatty acyl groups, is a widespread phenomenon. Here we describe the occurrence of this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of either [3H]-myristic acid or [3H]-palmitic acid, specific C. elegans polypeptides became labelled. Chemical analysis revealed that following incubation of C. elegans with [3H]-myristic acid, polypeptides became labelled with myristoyl, palmitoyl or stearoyl moieties; after incubation with [3H]-palmitic acid, palmitoyl or stearoyl moieties were incorporated into polypeptides. Fatty acyl groups were linked to target polypeptides, predominantly through alkali-labile thioester or ester linkages and acid-labile amide linkages. Where myristoylation involved an amide linkage, the modified amino acid was usually glycine. Preliminary immunological evidence indicated that heterotrimeric GTP-binding protein alpha subunit(s) are possible target(s) for acylation in C. elegans.

Lipid modification of signal-transducing polypeptides in the free-living nematode Caenorhabditis elegans.

Aspbury RA, Fisher MJ, Rees HH
Biochem Soc Trans. 1995; 23: 3-3

Catch and pull a microtubule: getting a grasp on the cortex.

Allan V, Nathke IS
Nat Cell Biol. 2001; 3: 2268-2268