Посещений:
Wnt Сигнальные Пути

Выбор с Помощью Dishevelled

Dishevelled and Wnt signaling: is the nucleus the final frontier?
Raymond Habas and Igor B Dawid
Journal of Biology 2005, 4:2 doi:10.1186/jbiol22

The phosphoprotein Dishevelled (Dsh) is an essential component of Wnt signaling pathways and transduces signals into three separate branches, the canonical, non-canonical and Ca2+ pathways. How Dsh focuses signaling into these branches remains mysterious, but a new study reveals the importance of nuclear localization of Dsh for pathway-specific activation.


Рис.1.  | A schematic representation of the Wnt signal transduction cascade



Wnt signaling


Wnt белки представляют собой большое семейство секретируемых гликопротеинов, которые регулируют ключевые онтогенетические процессы, включая детерминацию клеточных судеб. пролиферацию, подвижность и установление первичной оси во время эмбриогенеза позвоночных [1-3]. Дефекты передачи сигналов Wnt вызывают патологические состояния у хозяев, включая рак и дефекты нервной трубки. Wnt лиганды могут трансформировать клетки, а мутации компонентов сигнального пути Wnt, таких как β-catenin, являются причиной рака толстой кишки у люде, тогда как мутации Dishevelled (Dsh) участвуют в нарушениях закрытия нервной трубки [1,4]. Кстати, идентифицировано 18 Wnt лигандов у людей [5,6]. Такое большое количество лигандов обнаруживает параллелизм со столь же впечатляющим количеством рецепторов и ко-рецепторов, которые кодируются генами семейства Frizzled и low-density-related lipoprotein receptor 5/6 (LRP5/6), которые содержат 10 и 2 члена,соотв., в геноме человека [1,6].
Известны молекулярные пути передачи сигналов Wnt (Figure 1). После связывания Wnt со своим рецептором, или Frizzled или комплексом из Frizzled и LRP5/6, сигнал передается цитоплазматическому фосфобелку Dsh. Имеется три Dsh белка у млекопитающих (Dsh-1, Dsh-2 и Dsh-3), a члены семейства Dsh у всех организмов представлены тремя высоко законсервированными доменами: N-терминальным DIX доменом (назван по Dsh and Axin), центральным PDZ доменом (назван по Postsynaptic density-95, Discs-large and Zonula occludens-1), и С-терминальным DEP доменом(по Dsh, Egl-10 and Pleckstrin) [7]. На уровне Dsh сигнал Wnt разветвляется на три отдельных пути, т.наз. canonical, non-canonical или planar cell polarity (PCP) и Wnt-Ca2+ пути (Figure 1) [1,8,9]. Во всех трех путях Dsh является ключевым передатчиком сигнала Wnt, который оперирует на плазматической мембране или в цитоплазме. Новое исследование [10] указывает на то, что Dsh функционирует также и внутри ядра.
Для канонической передачи сигналов, которая обеспечивает события индукции генов (Figure 1a), передача сигналов Wnt использует DIX и PDZ домены Dsh чтобы индуцировать стабилизацию цитозольного β-catenin; это делает возможным накопление в цитоплазме и последующую транслокацию β-catenin в ядро [1]. Регуляция стабильности β-catenin обеспечивается посредством комплекса белков, включая Axin, glycogen synthase kinase 3 (GSK3), GSK3-binding protein (GBP) и casein kinase 1 (CK1). В отсутствие стимуляции Wnt β-catenin отправляется на деградацию посредством протеосомного пути через β-transducin repeat containing protein (β-TrCP), но β-catenin стабилизируется если получает сигнал Wnt [1,6,11]. В ядре β-catenin формирует комплексы со членами семейства LEF/TCF транскрипционных факторов и др. факторами и обеспечивает транскрипцию генов мишеней для Wnt [1].
Не-канонический или PCP путь обеспечивает полярность клеток, клеточную подвижность во время гаструляции и др. процессы, путем передачи сигналов через PDZ и DEP домены Dsh, приводя к модификации актинового цитоскелета (Figure 1b) [8,12]. На уровне Dsh, два независимых и параллельных пути ведут к активации малых GTPases Rho и Rac. Активация Rho нуждается в formin-homology белке Daam1, который соединяется с PDZ доменом Dsh, приводя к активации Rho-associated kinase ROCK и обусловливая ре-организацию цитоскелета [8,13,14]. Rac активация не зависит от Daam1, нуждается в DEP домене Dsh и стимулирует активность Jun kinase (JNK) [8,15,16]. Др. связывающиеся с Dsh молекулы, которые влияют на PCP путь, включают Strabismus и Prickle, но механизмы их действия пока изучены не полностью [8,12,17].
Wnt-Ca2+ путь (Figure 1c) , как полагают, влияет на оба канонический и PCP пути [9]. Передача сигналов Wnt через рецепторы Frizzled ведет к высвобождению внутриклеточного Ca2+ в процессе, обеспечиваемого посредством гетеротримерных G-белков и с участием многочисленных др. молеекул, включая phospholipase C (PLC), calcium-calmodulin-dependent kinase 2 (CamK2) и protein kinase C (PKC) [9,18]. Wnt-Ca2+ путь важен для клеточной слипчивости и клеточных движений во время гаструляции.


Signal specificity


С таким устрашающим количеством Wnt лигандов и Frizzled рецепторов, остаются нерешенными два вопроса, являются ли (и если да, то как) Wnt лиганды специфичными для определенных путей и как сигналы передаются (channeled) каждому из путей. Очевидно, что некоторые Wnt лиганды активируют и канонический и не-канонический пути, такие как Wnt3a, тогда как др., такие как Wnt5a, по-видимому, специфичны для неканонической передачи сигналов. Столь же важно понять, как сигнал передается от комплекса рецептор/корецептор к Dsh, хотя два недавних исследования продемонстрировали непосредственное взаимодействие между Dsh и Frizzled [19,20]. Наиболее важно, что способ, с помощью которого Dsh купирует и распределяет сигналы Wnt по трем сигнальным ветвям изучен хуже всего.
Dsh занимает ключевое положение на перекрестке всех ветвей сигнального каскада Wnt. Было предположено, что как субклеточная локализация Dsh, так и выбор эффекторных молекул, стоящих ниже Dsh, управляют избирательной активацией специфических путей. Исследования по локализации Dsh на Drosophila [21] и Caenorhabditis elegans [22] выявили корреляцию между локализацией Dsh на мембране и активацией пути PCP. В самом деле, мутации в DEP домене, который необходим для передачи сигналов PCP, демонстрируют нарушение мембранной локализации, что коррелирует с нарушением передачи сигналов PCP [16,21]. Эти исследования подтвердили гипотезу, что локализация на мембране Dsh необходима, по крайней мере, для осуществления одного исхода передачи сигналов Wnt. В не стимулированных клетках Dsh располагается в точечных везикулярных структурах в цитоплазме [23] с помощью процесса, который нуждается в DIX домене; в ответ на определенные Wnt лиганды, Dsh транслоцируется в плазматическую мембрану или в околоядерную/ядерную область, а локализация в мембране во всех изученных случаях нуждается в DEP домене [24,25]. Значение ядерной/околоядерной локализации остается неясным, но заслуживает внимания то, что ряд компонентов канонического сигнального пути, такие как APC, Axin, and GSK3β, по-видимому, переносятся между цитоплазмой и ядром вместе с β-catenin [26-28].
Эти исследования сформировали основу для работы Sokol и др. в Journal of Biology [10], которые идентифицировали два дополнительных домена в Dsh, которые модулируют как его субклеточное распределение, тае и его способность активировать канонический сигнальный Wnt путь. первый вновь идентифицированный домен расположен С-терминальнее относительно DEP домена, он модулирует локализацию Dsh благодаря своему действию в качестве nuclear export signal. Dsh белок, лишенный этого домена, или несущий мутацию в критическом лизине, строго накапливается в ядре у эмбрионов Xenopus и в культивируемых клетках млекопитающих. Неожиданно, однако, этот мутантный белок сохраняет свою способность обеспечивать передачу канонических сигналов столь же эффективно, что и дикого типа Dsh. Фармакологические агенты, затрудняющие экспорт в ядро и клеточные фракционные исследования предоставили дальнейшие доказательства, что эндогенный Dsh проникает в ядро, это подкрепляет мнение, что Dsh снует между цитоплазмой и ядром. Авт. затем идентифицировали второй домен, расположенный непосредственно С-терминальнее PDZ домена, который необходим для ядерной локализации; последовательности в этом домене атипичны для nuclear localization sequence (NLS). Мутации в этом втором домене устраняют накопление в ядре Dsh в присутствии ингибиторов ядерного экспорта и, что удивительно, нарушают способность Dsh индуцировать стабилизацию β-catenin и передачать канонические сигналы Wnt. Интересно, что замещение этого атипичного NLS на прототипический NLS из T антигена от simian virus SV40 перенаправляет Dsh в ядро и в основном восстанавливает передачу сигналов Wnt. Авт. еще больше подкрепили свои находки, продемонстрировав, что стимуляция культивируемых клеток млекопитающих с помощью Wnt3a ведет к накоплению части эндогенного Dsh (Dvl2 в данном случае) в или около ядра.


Making sense of nuclear localization


Какова же роль Dsh в ядре? Эти новые находки [10] выглядят неожиданными. Чтобы подтвердить свом заключения авт. показали, что Dsh находится в ядерных фракциях, но этот подход не является достаточно подходящим, т.к. может предположить, что Dsh имеет околоядерную локализацию и ко-фракционируется вместе с наружной ядерной оболочкой. Наиболее строгие доказательства ядерной роли Dsh получены в экспериментах, в которых меняется ядерный импорт и экспорт, показавших, что импорт является критическим для функции. Теперь, когда основное заключение о ядерной локализации и функции Dsh принято, остается несколько вопросов. Если функция Dsh необходима в ядре для канонической передачи сигналов Wnt, то почему не происходит гиперактивации пути, наблюдаемой при направлении Dsh в ядро? Авт. отмечают эту точку и постулируют, что 'steady state' скорее, чем собственно локализация необходимо для функционирования. Однако, необходимо исходить из того, что β-catenin, который д.б. стабилизирован с помощью такого Dsh-targeted подхода и д. увеличивать передачу сигналов, не делает этого.
Вообще-то наиболее важным вопросом является, почему многие исследования наблюдали транслокацию Dsh в плазматическую мембрану в ответ на Wnt стимуляцию или экспрессию Frizzled [8,12,17,29], но не обнаруживали Dsh в ядре. Возможно, что небольшой избирательный пул Dsh транслоцируется в ядро, чтобы обеспечить каноническую передачу сигналов, тогда как большая часть Dsh поступает в мембрану. Если это так, то релокализация в мембрану большей части Dsh кажется без причинным клеточным поведением без смысла? Наконец, какова функция Dsh в ядре? Возможно. что ядерный Dsh участвует в транскрипционной регуляции независимо от β-catenin, обеспечивая передачу сигналов Wnt, как Sokol и др. уже предполагали это ранее для Dsh-связывающего белка Frodo [30]. Perhaps the nuclear localization of Dsh will indeed provide clues to elucidate the final frontier of understanding the diverse mechanisms of Wnt regulation of gene transcription in the nucleus.

Dishevelled function in Wnt/β-catenin signaling
Keiji Itoh, Barbara K Brott, Gyu-Un Bae, Marianne J Ratcliffe and Sergei Y Sokol
Journal of Biology 2005, 4:3 doi:10.1186/jbiol22

Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined.
We report that Dsh protein accumulates in cell nuclei when Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein.
These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/β-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus.


Рис.1.  | Nuclear export of Dsh is not critical for its activity


Рис.2.  | Accumulation of Dsh in the nucleus in the absence of nuclear export


Рис.3.  | Endogenous Dsh shuttles between the cytoplasm and nucleus


Рис.4.  | Mapping nuclear localization signals in Dsh


Рис.5.  | Activation of the Wnt/β-catenin pathway requires nuclear localization of Dsh


Рис.6.  |  Dominant inhibition of Wnt-dependent transcription by Dsh mutants


Рис.7.  | Dsh mutants retain the ability to bind CK1ε and XARP


Рис.8.  |  DsNLSm, defective in the ?-catenin pathway, is active in noncanonical signaling


Рис.9.  |  Nuclear translocation of Dvl2 upon Wnt3a treatment

Табл. 1.  |  Axis induction by Dsh constructs

The specification of cell fates during embryonic development frequently depends on inductive interactions, which involve transmission of extracellular signals from the cell surface to the nucleus. In the transforming growth factor ? (TGF?) signal transduction pathway, Smad proteins that are initially associated with TGF? receptors move to the nucleus to regulate target genes [1]. Another example of a direct link between the cell surface and the nucleus during embryonic development is the proteolytic cleavage and nuclear translocation of the cytoplasmic fragment of the Notch receptor [2]. In contrast, multiple steps appear to be required for a Wnt signal to reach the nucleus. In this molecular pathway, signals from Frizzled receptors are transduced to Dishevelled (Dsh), followed by inactivation of the β-catenin degradation complex that includes the adenomatous polyposis coli protein (APC), Axin and glycogen synthase kinase 3 (GSK3) [3,4]. Stabilization of β-catenin is thought to promote its association with members of the T-cell factor (Tcf) transcription factor family in the nucleus, resulting in the activation of target genes [5,6]. As well as the canonical β-catenin-dependent pathway, Frizzled receptors also activate small GTPases of the Rho family, protein kinase C and Jun-N-terminal kinases (JNKs) to regulate planar cell polarity in Drosophila and convergent extension cell movements and tissue separation in Xenopus [7-12]. Thus, the Wnt/Frizzled pathway serves as a model for molecular target selection during signal transduction.
Dsh is a common intracellular mediator of several pathways activated by Frizzled receptors and is composed of three conserved regions that are known as the DIX, PDZ and DEP domains [13]. Different domains of Dsh are engaged in specific interactions with different proteins, thereby leading to distinct signaling outcomes [13]. Daam, a formin-related protein, promotes RhoA activation by Dsh [9], whereas Frodo, another Dsh-binding protein, is required for Wnt/β-catenin signaling in the nucleus [14]. These interactions may take place in various cellular compartments, linking specific activities of Dsh to its distribution inside the cell. Dsh is found in a complex with microtubules and with the actin cytoskeleton [15-17]. Dsh is also associated with cytoplasmic lipid vesicles, and this localization was shown to require the DIX domain [7,16,18]. Overexpressed Frizzled receptors can recruit Dsh to the cell membrane in Xenopus ectoderm, and this redistribution requires the DEP domain [7,18,19]. The DIX domain is essential for the Wnt/β-catenin pathway, whereas the DEP domain plays a role in the planar cell polarity pathway [7,8,16,18,20,21]. Thus, the specific subcellular localization of Dsh may be crucial for local signaling events.
The current study was based on our initial observation that a Dsh construct lacking the carboxy-terminal DEP domain was found in cell nuclei. We have now identified a nuclear export signal in the deleted region and also discovered that Dsh proteins accumulate in the nuclei of Xenopus ectodermal cells and mammalian cells upon inhibition of nuclear export. Dsh also accumulated in the nuclei after stimulation of mammalian cells with Wnt3a-containing culture medium. By analyzing various mutant Dsh constructs in Xenopus ectoderm, we show that the signals responsible for Dsh nuclear localization reside in a novel domain and that the nuclear translocation of Dsh is essential for its ability to activate Wnt/β-catenin signaling.


Results and discussion


A nuclear export signal in Dsh is responsible for the cytoplasmic localization of Dsh


We studied the subcellular distribution of fusions of Dsh with green fluorescent protein (GFP) in Xenopus ectodermal cells. In contrast to Dsh-GFP, which is localized in punctate structures within the cytoplasm [7,18], the Ds2 construct, lacking the carboxy-terminal region, accumulates in the nucleus (Figure 1a–c), indicating that the carboxyl terminus contains sequences for nuclear export. Indeed, we found a potential leucine-rich nuclear export signal (NES) in Dsh at positions 510–515, corresponding to the conserved consensus M/LxxLxL (single letter amino-acid code, where x is any amino acid) [22,23]. When leucines 513 and 515 in this putative NES were substituted with alanines, the mutated Dsh fusion construct, DsNESm, was localized predominantly in the nucleus (Figure 1a,d), demonstrating that the sequence is a functional nuclear export signal.
To examine whether inhibition of nuclear export abrogates Dsh activity, we compared the abilities of DsNESm and wild-type Dsh-GFP to induce secondary axes in frog embryos. Although the molecular mechanism operating during axis induction remains to be elucidated, this assay faithfully reflects the biological activity of Dsh in the canonical Wnt/β-catenin pathway [14,16,18,24]. DsNESm, which was expressed at similar levels to the wild-type Dsh-GFP (data not shown), induced secondary axes at least as efficiently as Dsh-GFP (Table 1). Induced axes contained pronounced head structures with eyes and cement glands (Figure 1e–g). These results suggest that Dsh may function in the nucleus to trigger dorsal axial development.

Nuclear localization of Dsh in cells treated with nuclear export inhibitors


Accumulation of DsNESm in the nucleus implies that the wild-type Dsh shuttles between the nucleus and the cytoplasm. We therefore studied the subcellular distribution of Dsh in Xenopus embryonic cells under conditions in which nuclear export is blocked. When ectodermal cells expressing Dsh-GFP were incubated with N-ethylmaleimide (NEM), an inhibitor of the nuclear export receptor CRM1/exportin [25,26], Dsh-GFP was detected predominantly in the nucleus, compared to the punctate cytoplasmic pattern of Dsh-GFP in untreated cells (Figure 2a,b). This effect was specific to full-length Dsh-GFP, as Ds3, a Dsh construct that lacks 48 amino acids adjacent to the PDZ domain (Figure 1a), did not accumulate in the nucleus after NEM treatment (Figure 2e,f). The nuclear retention of Dsh-GFP was also observed using leptomycin B (LMB), another inhibitor of CRM1-dependent nuclear export [22,23] (Figure 2c,d). These results indicate that Dsh shuttles between the cytoplasm and the nucleus, and that its abundance in the cytoplasm is due to highly efficient nuclear export.
To ensure that the Dsh-GFP fusion behaves similarly to the endogenous Dsh protein, we examined the localization of endogenous Dvl2, a mammalian homolog of Dsh, in human and rat tissue culture cells. Human embryonic kidney (HEK) 293 cells treated with LMB accumulated Dvl2 in the nucleus, contrasting with the cytoplasmic localization of Dvl2 in untreated cells (Figure 3a–c). We also evaluated the subcellular localization of endogenous Dvl2 in Rat-1 fibroblasts, which are known to respond to Wnt signaling. Fractionation of cells into nuclear and cytoplasmic protein pools revealed only a small amount of endogenous Dvl2 in intact nuclei, whereas after NEM treatment, Dvl2 was localized predominantly in the nuclear fraction (Figure 3d). The efficiency of subcellular fractionation was controlled for by staining with antibodies to glyceraldehyde phosphate dehydrogenase (GAPDH) and nuclear lamins. These proteins remained exclusively cytoplasmic or nuclear, respectively, in both untreated and NEM-treated cells (Figure 3d). Thus, our data show that Dsh translocates into the nucleus and is actively exported into the cytoplasm of both Xenopus ectodermal cells and mammalian fibroblasts.


Identification of sequences responsible for Dsh nuclear localization


To identify specific amino-acid sequences that direct the transport of Dsh to the nucleus, we studied the subcellular distribution of mutated Dsh-GFP fusion constructs (Figure 4a). The removal of the DIX and PDZ domains (Ds1) did not eliminate nuclear translocation in response to NEM or LMB (Figure 4a–d), indicating that these two domains are not required for the nuclear import. Similarly, the DEP domain is not required for Dsh nuclear localization (Ds2; Figure 1a,c). Comparison of Ds1 and Ds2 (see Figure 4a), both capable of nuclear localization, reveals a short stretch of shared amino acids located between the PDZ and DEP domains. Strikingly, the removal of just this 48 amino-acid region abrogated nuclear import of Dsh in the presence of NEM or LMB (Ds3; Figures 2e,f and 4a). Together these experiments identify amino acids 333–381 as the region required for nuclear localization of Dsh.
Although this short sequence is highly conserved in all Dsh homologs from Hydra to humans (Figure 4j), it does not bear detectable similarity to nuclear localization signals characterized in other proteins [27]. This sequence may interact directly with components of the nuclear import machinery or bind to a protein that itself binds a karyopherin/importin and mediates the nuclear import of Dsh by a piggyback mechanism. Interestingly, this region overlaps a novel proline-rich domain identified by mutational analysis of Dsh in Drosophila [28]. To define further the specific amino acids necessary for nuclear localization, a panel of Dsh constructs with point mutations spanning the conserved region was examined (data not shown). Nuclear import was eliminated with the substitution of three amino acids, converting IVLT into AVGA (DsNLSm; Figure 4a,e–g,j), indicating that these three amino acids are critical.


Dsh nuclear translocation is crucial for its function in the β-catenin pathway


To determine whether nuclear localization of Dsh is required for its activity, we compared the abilities of DsNLSm and wild-type Dsh to induce secondary axes in frog embryos. We also assessed activation of a luciferase reporter construct for Siamois [29], an immediate target of Wnt/β-catenin signaling. DsNLSm had impaired ability to induce secondary axes and to activate the Siamois reporter when compared with wild-type Dsh (Figure 5a,b; Table 1). Furthermore, DsNLSm failed to stabilize β-catenin (Figure 5c). This difference was not due to differences in protein expression, as both constructs were present in embryo lysates at similar levels (Figure 5c). Thus, these findings indicate that the nuclear localization of Dsh is critical for its functional activity in the β-catenin pathway.
Not only was the function of DsNLSm in the β-catenin pathway impaired, but we found that this construct behaved as a dominant inhibitor of Wnt signaling and prevented the activation of the Siamois reporter by Xwnt3a and Xwnt8 RNAs (Figure 6a,b). Consistent with these observations, another construct lacking the region responsible for the nuclear localization (Ds3; see Figure 4a) also suppressed Wnt signaling (Figure 6b). Despite these inhibitory properties, dorsally injected DsNLSm RNA, like Xdd1, a dominant negative deletion mutant [24], did not suppress primary axis formation (data not shown).
Impaired activity of the DsNLSm construct may be due to its inability to translocate to the nucleus, or due to a coincidental elimination of a binding site for an essential cofactor that functions together with Dsh in the cytoplasm. To exclude the latter possibility, the IVLT sequence of Dsh NLS was replaced with KKKRK, an unrelated NLS from SV40 T antigen [27]. This construct, DsSNLS, relocated to the nucleus even in the absence of nuclear export inhibitors (Figure 4a,i). Notably, all activities of wild-type Dsh, including induction of complete secondary axes, activation of the Siamois promoter and β-catenin stabilization were significantly restored in DsSNLS (Figure 5a–c; Table 1). In contrast to DsNLSm, DsSNLS did not inhibit the ability of Wnt ligands to activate pSia-Luc (Figure 6b), consistent with its being a positive regulator of the Wnt pathway. We note that the signaling activity of DsSNLS was not enhanced compared to wild-type Dsh, suggesting that the rate of the nuclear translocation of Dsh rather than its steady state levels in the nucleus is critical for target gene activation. It is also possible that other nuclear components, rather than Dsh, become rate-limiting for signaling. Overall, the simplest interpretation of our data is that the nuclear import of Dsh is essential for its activity.
We next examined the ability of DsNLSm to bind critical Wnt signaling components, such as casein kinase 1? (CK1?), a positive regulator of the β-catenin pathway [30,31], or Axin, a negative regulator [20,32-36], both of which are known to bind Dsh. Both DsSNLS, enriched in the nucleus, and DsNLSm and Ds3, which do not enter the nucleus, bound CK1? and XARP, a Xenopus Axin-related protein [20] (Figure 7). Thus, these mutated Dsh constructs retain the ability to associate with critical components of the Wnt/β-catenin pathway, arguing that defective nuclear translocation of DsNLSm is likely to be responsible for its inability to activate β-catenin signaling.


Suppression of Dsh nuclear import does not affect noncanonical signaling


Besides the β-catenin pathway, Dsh also functions in a planar cell polarity (PCP) pathway, which involves Rho GTPase and JNK and controls morphogenetic movements in early embryos [8,9,37-39]. We asked whether mutations in DsNLSm influence the β-catenin pathway exclusively or affect the PCP pathway as well. First, we observed that both Dsh-GFP and DsNLSm-GFP were efficiently recruited to the cell membrane by overexpressed Xfz8, a Frizzled family member [40] (Figure 8a). As Dsh relocalization to the cell membrane in response to Frizzled is associated with its ability to signal in the PCP pathway [7,8], this observation suggests that DsNLSm can respond to Frizzled signaling independent of β-catenin.
In Xenopus, the PCP pathway involving Dsh is implicated in the control of convergent extension movements [24,41,42]. Overexpression of the Xdd1 deletion mutant leads to the development of short embryos when expressed in dorsal marginal cells ([24]; Figure 8b). Severe convergent extension defects (Figure 8b) were observed in 22%, and mild defects were observed in 28% of the embryos injected with Xdd1 RNA (N = 35). In contrast, only mild morphogenetic defects were observed in embryos coinjected with Dsh (15%; N = 40) or DsNLSm RNA (18%; N = 39), indicating that both Dsh and DsNLSm partially rescued the effect of Xdd1. This indicates that DsNLSm is active in noncanonical PCP-like signaling. We also examined whether DsNLSm activates c-Jun N-terminal kinase (JNK), which is thought to function downstream of Dsh in the PCP pathway [8,37-39]. Both DsNLSm and Dsh activated JNK at equivalent levels (Figure 8c), suggesting that nuclear localization of Dsh is not required for its function in noncanonical signaling.


Nuclear accumulation of Dsh following Wnt3a stimulation


Our findings are consistent with a scenario in which Wnt signaling may cause nuclear translocation of Dsh followed by formation of a stable β-catenin/Tcf3 complex and transcriptional activation of target genes. In support of this hypothesis, Dsh was reported to move to the nucleus in response to Wnt signaling in primary embryonic kidney cells [17]. In Rat-1 cells, we did not detect a significant change in Dsh distribution in response to Wnt signals (data not shown), possibly due to highly efficient nuclear export of Dsh in these cells. But immunofluorescence staining for Dvl2 revealed the nuclear accumulation of the protein in HEK293 and MCF7 cells after 3–6 h stimulation with Wnt3a-containing medium (Figure 9a, and data not shown). The effect was quantified by measuring nuclear to cytoplasmic (N/C) ratios of fluorescence intensity. The N/C ratio averaged 28% after 6 h treatment with the control medium, but increased to 91% after stimulation with Wnt3a-conditioned medium (Figure 9b). These observations are consistent with the view that Dsh regulates Wnt-dependent gene targets in the nucleus.


A role for Dsh in the nucleus


In the current view, Wnt signaling causes inactivation of the β-catenin degradation complex, leading to stabilization and nuclear translocation of β-catenin [3]. Given that Dsh is genetically upstream of the β-catenin degradation complex [3,4] and that β-catenin degradation is thought to occur in the cytoplasm [43], Dsh nuclear import is unexpected. Nevertheless, our data demonstrate that Dsh shuttles between the cytoplasm and the nucleus and that its presence in the nucleus is critical for signaling. One explanation of these results is that β-catenin degradation may occur in the nucleus. Consistent with this possibility, APC, Axin and GSK3, components of the β-catenin degradation complex, have also recently been found to shuttle between the cytoplasm and the nucleus [22,23,44-47]. Moreover, Frat/GBP, a positive regulator of β-catenin, has been reported to expel GSK3 from the nucleus [47]. We show that the ability of Dsh constructs to enter the nucleus correlates with their ability to stabilize β-catenin (Figure 5c). These observations indicate that Wnt/β-catenin signaling may depend on the nuclear localization of pathway components.
Alternatively, nuclear localization of Dsh may affect β-catenin stability indirectly, by regulating protein interactions that sequester β-catenin in the nucleus, thereby preventing its cytoplasmic degradation [48]. Although we did not detect a significant change in nuclear import of β-catenin-GFP in Xenopus ectoderm cells overexpressing Dsh (data not shown), this process may be cell-context-dependent. On the other hand, we recently showed that Frodo, a nuclear Dsh-interacting protein, associates with Tcf3 and influences Tcf3-dependent transcription [49]. It is thus possible that Frodo links Tcf3 and Dsh to regulate Wnt target genes. Future studies should examine molecular components critical for the nuclear function of Dsh.
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