hScrib interacts with ZO-2 at the cell-cell junctions of epithelial cells

In Drosophila, the tumor suppressor Scribble is localized at the septate junctions of epithelial cells. Its mammalian homologue, hScrib, is a basolateral protein likely associated to proteins of the cell-cell junctions. We report the direct interaction between hScrib and ZO-2, a junction-associated protein. This interaction relies on two PDZ domains of hScrib and on the C-terminal motif of ZO-2. Both proteins localise at cell-cell junctions of epithelial cells. A point mutation in the LRR of hScrib delocalises the protein from the plasma membrane and abrogates the interaction with ZO-2 but not with betaPIX. Tyrosine phosphorylation of hScrib does not impair the interaction with ZO-2. We show a direct link between two junctional proteins that are down-regulated during cancer progression.     

Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration

BACKGROUND: Mammalian Scribble (Scrib) plays a conserved role in polarization of epithelial and neuronal cells. Polarization is essential for migration of a variety of cell types; however, the function of Scrib in this context remains unclear. Scrib has been shown to interact with betaPIX, a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42. Cdc42 controls cell polarity from yeast to mammals during asymmetric cell division and epithelial cell polarization, as well as during cell migration. Cdc42 is, in particular, required for polarization and orientation of astrocytes in a scratch-induced polarized migration assay. Using this assay, we characterized Scrib function during polarized cell migration. RESULTS: Depletion of Scrib by siRNA or expression of dominant-negative constructs inhibits astrocyte polarization. Like Cdc42, Scrib controls protrusion formation, cytoskeleton polarization, and centrosome and Golgi reorientation. Scrib interacts and colocalizes with betaPIX at the front edge of polarizing astrocytes. Perturbation of Scrib localization or of Scrib-betaPIX interaction inhibits betaPIX polarized recruitment. We further show that betaPIX is required for astrocyte polarization and that both the Scrib-binding motif and the GEF activity of betaPIX are essential for its function. Scrib and betaPIX control Cdc42 activation and localization during astrocyte polarization. Thereby, Scrib regulates Cdc42-dependent APC and Dlg1 recruitment to the leading edge to promote cell orientation. CONCLUSION: We conclude that Scrib plays a key role in the establishment of cell polarity during migration. By interacting with betaPIX, Scrib controls localization and activation of the small GTPase Cdc42 and regulates Cdc42-dependent polarization pathways.     

Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase

The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.     

Thyrotropin receptor trafficking relies on the hScrib-betaPIX-GIT1-ARF6 pathway

G protein-coupled receptors are regulated by ligand stimulation, endocytosis, degradation of recycling to the cell surface. Little information is available on the molecular mechanisms underlying G protein-coupled receptors recycling. We have investigated recycling of the G protein-coupled thyroid stimulating hormone receptor (TSHR) and found that it relies on hScrib, a membrane-associated PDZ protein. hScrib directly binds to TSHR, inhibits basal receptor endocytosis and promotes recycling, and thus TSHR signalling, at the cell membrane. We previously demonstrated that hScrib is associated with a betaPIX-GIT1 complex comprised of a guanine nucleotide exchange factor and a GTPase-activating protein for ADP ribosylation factors that is involved in vesicle trafficking. We used dominant-negative constructs and small interfering RNA to show that TSHR recycling is regulated by the interaction between hScrib and betaPIX, and by the activity of GIT1. In addition, ARF6, a major target for GIT1, is activated during TSH stimulation of HEK293 and FRTL-5 thyroid cells, and plays a key role in TSHR recycling. Thus, we have uncovered an hScrib-betaPIX-GIT1-ARF6 pathway devoted to TSHR trafficking and function.     

Regulation of the hDlg/hScrib/Hugl-1 tumour suppressor complex

The proper function of the Scribble tumour suppressor complex is dependent upon the correct localisation of its components. Previously we observed dynamic relocalisation of the hDlg component under conditions of osmotic stress. We now show that the other two components of the complex, hScrib and Hugl-1 display similar patterns of expression. We demonstrate, by shRNA ablation of hScrib expression, that hDlg and Hugl-1 are in part dependent upon hScrib for their correct localization. However under conditions of osmotic stress this apparent dependency no longer exists: hDlg and Hugl-1 localise to cell membranes independently of hScrib. We also demonstrate an interaction between the three components of the hScrib complex and the tSNARE syntaxin 4, and show that correct localization of the Scrib complex is in part tSNARE dependent. This is the first detailed analysis of the co-localisation and function of the hScrib complex in mammalian cells and demonstrates a direct link between the control of the hScrib complex and vesicle transport pathways.     

Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin–catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin–catenin system and may control cadherin-mediated cell–cell adhesion.     

Phosphorylation of p85 beta PIX,a Rac/Cdc42-specific guanine nucleotide exchange factor,via the Ras/ERK/PAK2 pathway is required for basic fibroblast growth factor-induced neurite outgrowth

Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 betaPIX complex via the ERK cascade and subsequent translocation of this complex.     

Molecular and functional analysis of apical junction formation in the gut epithelium of Caenorhabditis elegans

The Caenorhabditis elegans intestine is a simple and accessible model system to analyze the mechanism of junction assembly. In comparison to Drosophila and vertebrates, the C. elegans apical junction is remarkable because a single electron-dense structure is implicated in complex processes such as epithelial tightness, vectorial transport and cell adhesion. Here we present evidence in support of a heterogeneous molecular assembly of junctional proteins found in Drosophila and vertebrate epithelia associated with different junctions or regions of the plasma membrane. In addition, we show that molecularly diverse complexes participate in different aspects of epithelial maturation in the C. elegans intestine. DLG-1 (Discs large) acts synergistically with the catenin-cadherin complex (HMP-1-HMP-2-HMR-1) and the Ezrin-Radixin-Moesin homolog (ERM-1) to ensure tissue integrity of the intestinal tube. The correct localization of DLG-1 itself depends on AJM-1, a coiled-coil protein. Double depletion of HMP-1 (alpha-catenin) and LET-413 (C. elegans homolog of Drosophila Scribble) suggests that the catenin-cadherin complex is epistatic to LET-413, while additional depletion of subapically expressed CRB-1 (Crumbs) emphasizes a role of CRB-1 concerning apical junction formation in the C. elegans intestine.     

Phosphorylation-dependent interaction between tumor suppressors Dlg and Lgl

The tumor suppressors Discs Large (Dlg), Lethal giant larvae (Lgl) and Scribble are essential for the establishment and maintenance of epithelial cell polarity in metazoan. Dlg, Lgl and Scribble are known to interact strongly with each other genetically and form the evolutionarily conserved Scribble complex. Despite more than a decade of extensive research, it has not been demonstrated whether Dlg, Lgl and Scribble physically interact with each other. Here, we show that Dlg directly interacts with Lgl in a phosphorylation-dependent manner. Phosphorylation of any one of the three conserved Ser residues situated in the central linker region of Lgl is sufficient for its binding to the Dlg guanylate kinase (GK) domain. The crystal structures of the Dlg4 GK domain in complex with two phosphor-Lgl2 peptides reveal the molecular mechanism underlying the specific and phosphorylation-dependent Dlg/Lgl complex formation. In addition to providing a mechanistic basis underlying the regulated formation of the Scribble complex, the structure of the Dlg/Lgl complex may also serve as a starting point for designing specific Dlg inhibitors for targeting the Scribble complex formation.     

The Scribble and Par complexes in polarity and migration: friends or foes?

The Par complex [consisting of Bazooka (also called Par3), Par6 and aPKC] is a well-described regulator of cell polarity whose role in many aspects of cell morphogenesis is under intense investigation. Recently, another set of proteins known as the Scribble complex (consisting of Scribble, Discs large and Lethal giant larvae) has also been shown to be important in polarity regulation in several settings. Here, we describe the current status of Scribble in polarity and review evidence from various model systems that indicates an essential but context-dependent role for the Scribble and Par complexes in directed cell migration. Based on the known interactions of Scribble and Par complexes with each other and with other signalling pathways, we propose models by which Par and Scribble might interact to regulate cell migration.     

Basolateral targeting by leucine-rich repeat domains in epithelial cells

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure–function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.     

The apical/basal-polarity determinant Scribble cooperates with the PCP core factor Stbm/Vang and functions as one of its effectors

Most tissues display several features of cellular polarization. Besides the ubiquitous epithelial polarization in the Apical–Basal (A/B) axis, many epithelia (and associated organs) display a Planar Cell Polarization (PCP). Recently, a crosstalk between the PCP and A/B polarity determinants has been suggested, i.e. the activity or stability of the PCP factor Frizzled is regulated by the A/B determinants aPKC and Bazooka in the Drosophila eye. We have systematically investigated genetic and physical interactions between the Drosophila A/B factors and the core PCP component Strabismus (Stbm)/Van Gogh (Vang). The A/B determinant Scribble was found to interact both genetically and physically with Stbm/Vang. We demonstrate that Scribble binds Stbm/Vang through its PDZ domain 3 and that it cooperates with Stbm/Vang in PCP establishment. Our data indicate that Scribble, in addition to its role in A/B polarity, has a distinct requirement in PCP establishment in the Drosophila eye and wing. We define a scribble allele that is largely PCP specific. Our data show that Scribble is part of the Stbm/Vang PCP complex and further suggest that it might act as an effector of Stbm/Vang during PCP establishment.     

Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/betaPIX signaling complex

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. We have delineated a Ca(2+)-signaling pathway downstream of the NMDA receptor that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) betaPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in betaPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the betaPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or betaPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity.     

Role of the polarity protein Scribble for podocyte differentiation and maintenance

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribble(flox/flox) mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.     

Novel putative targets of N-ethylmaleimide sensitive fusion protein (NSF) and alpha/beta soluble NSF attachment proteins (SNAPs) include the Pak-binding nucleotide exchange factor betaPIX

N-ethylmaleimide sensitive fusion protein (NSF) is a chaperone that plays a crucial role in the fusion of vesicles with target membranes. NSF mediates the ATP-consuming dissociation of a core protein complex that assembles during vesicle fusion and it thereby recharges the fusion machinery to perform multiple rounds of fusion. The binding of NSF to the core complex is mediated by co-chaperones named soluble NSF attachment proteins (SNAPs), for which three isoforms (alpha, beta and gamma) are known. Here, we sought to identify novel targets of the NSF-SNAP complex. A yeast two-hybrid screen using the brain specific betaSNAP isoform as bait revealed, as expected, NSF and several isoforms of the SNARE protein syntaxin as interactors. In addition, three isoforms of the reticulon protein family and two isoforms of BNIP3 interacted with betaSNAP. A yeast two-hybrid screen using NSF as bait identified Rab11-FIP3 and the Pak-binding nucleotide exchange factor betaPIX as putative binding partners. betaPIX interacts with recombinant NSF in co-sedimentation assays and the two proteins may be co-immunoprecipitated. A leucine zipper (LZ) motif within the C-terminus of betaPIX mediates binding to NSF; however, this fragment of betaPIX does not exhibit dominant negative effects in a cellular assay. In summary, our results support the evolving view that NSF has numerous targets in addition to conventional SNARE complexes.     

The PDZ domain-binding motif of the human T cell leukemia virus type 1 tax protein induces mislocalization of the tumor suppressor hScrib in T cells

Interactions with cellular PDZ domain-containing proteins obviously contribute to the tumorigenic potential of several viral oncoproteins. In this regard, the oncogenic potential of the human T cell leukemia virus type 1 Tax protein correlates with its binding capacity to the tumor suppressor hDlg. Recent results show that hDlg in T cells is associated to a network of scaffolding proteins including another PDZ domain-containing protein termed hScrib. Interestingly, previous studies have revealed complementary activities of both proteins in the control of epithelial cell polarity. Here, we demonstrate that Tax can bind to hScrib and that the resulting Tax/hScrib complex is present in human T cell leukemia virus type 1-infected T cells. By confocal microscopy, we show that Tax modifies the localization of hScrib in transfected COS cells as well as in infected T cell lines and targets hScrib to particular spots exhibiting a granular distribution, mainly distributed in the cytoplasm. Given that Tax sequesters hScrib to these particular structures, we postulate that Tax might inhibit hScrib activity. Providing further support to this idea, we find that transient overexpression of hScrib attenuates T cell receptor-induced NFAT activity but that the presence of Tax counteracts this negative effect on the NFAT pathway. The fact that hDlg and hScrib are both targeted by Tax underlies their importance in T cell function.     

Scrib, a tumor suppressor gene involved in cell polarity

Polarity is a fundamental feature of all organisms both during development and in the adult. This reflects the key role of cell polarity during basic fundamental processes such as cell division, cell differentiation and cell migration. The control of cell polarity relies on functionally conserved proteins. Among these, Scribble, initially identified as a tumor suppressor gene in Drosophila, has been first involved in epithelial polarity. More recently Scribble function has been implicated in neuronal polarity and polarized cell migration. Scribble joins the growing family of tumor suppressors that play a key and conserved function in cell polarity. Scribble illustrates the more and more obvious link between regulation of cell polarity, cell transformation and tumor formation.     

Basic fibroblast growth factor stimulates activation of Rac1 through a p85 betaPIX phosphorylation-dependent pathway

In a previous study (Shin, E. Y., Shin, K. S., Lee, C. S., Woo, K. N., Quan, S. H., Soung, N. K., Kim, Y. G., Cha, C. I., Kim, S. R., Park, D., Bokoch, G. M., and Kim, E. G. (2002) J. Biol. Chem. 277, 44417-44430) we reported that phosphorylation of p85 betaPIX, a guanine nucleotide exchange factor (GEF) for Rac1/Cdc42, is a signal for translocation of the PIX complex to neuronal growth cones and is associated with basic fibroblast growth factor (bFGF)-induced neurite outgrowth. However, the issue of whether p85 betaPIX phosphorylation affects GEF activity on Rac1/Cdc42 is yet to be explored. Here we show that Rac1 activation occurs in a p85 betaPIX phosphorylation-dependent manner. A GST-PBD binding assay reveals that Rac1 is activated in a dose- and time-dependent manner in PC12 cells in response to bFGF. Inhibition of ERK or PAK2, the kinases upstream of p85 betaPIX in the bFGF signaling, prevents Rac1 activation, suggesting that phosphorylation of p85 betaPIX functions upstream of Rac1 activation. To directly address this issue, transfection studies with wild-type and mutant p85 betaPIX (S525A/T526A, a non-phosphorylatable form) were performed. Expression of mutant PIX markedly inhibits both bFGF- and nerve growth factor (NGF)-induced activation of Rac1, indicating that phosphorylation of p85 betaPIX is responsible for activation of this G protein. Both wild-type and mutant p85 betaPIX displaying negative GEF activity (L238R/L239S) are similarly recruited to growth cones, suggesting that Rac1 activation is not essential for translocation of the PIX complex (PAK2-p85 betaPIX-Rac1). However, expression of mutant p85 betaPIX (L238R/L239S) results in retraction of the pre-existing neurites. Our results provide evidence that bFGF- and NGF-induced phosphorylation of p85 betaPIX mediates Rac1 activation, which in turn regulates cytoskeletal reorganization at growth cones, but not translocation of the PIX complex.     

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth,viability, and directional outgrowth of epithelial seam cells

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical–basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.     

Scribble controls NGF-mediated neurite outgrowth in PC12 cells

Neurite outgrowth is mediated by dynamic changes of the cytoskeleton and is largely controlled by Rho GTPases and their regulators. Here, we show that the polarity protein Scribble controls PC12 cell neurite outgrowth in response to nerve growth factor. Scribble knockdown decreases neurite numbers and increases neurite length. This effect is linked to TrkA the cognate receptor for NGF as pharmacological inhibition of phosphorylated TrkA (pTrkA) reduces Scribble expression. Moreover, Scribble forms a complex with the MAPK components ERK1/2 in a growth factor dependent manner. In RNAi experiments where Scribble expression is efficiently depleted sustained ERK1/2 phosphorylation is reduced. Conversely, siRNA with intermediate Scribble silencing efficiency fails to match this effect indicating that ERK1/2 activation depends on basic Scribble protein levels. Finally, Scribble translocates to the plasma membrane in response to growth factor where it complexes with HRas and Rac1 suggesting that the phenotype activated by loss of Scribble may be a result of altered GTPase activity. Together, these results demonstrate a novel role for Scribble in neurite outgrowth of PC12 cells.