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Biomedical subjects

Alan Hall

Publications and source records attributed to Alan Hall.

At least 19 recordsLinked to original sources
PubMed2006

Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3beta) independently of Akt/PKB serine phosphorylation.

An essential step during the development of hippocampal neurons is the polarised outgrowth of a single axon. Recently, it has been suggested that inhibition of glycogen synthase kinase-3beta (GSK-3beta) via Akt/PKB-dependent phosphorylation of Ser9, specifically at the tip of the presumptive axon, is required for selective axonal outgrowth. We now report that, by using neurons from double knock-in mice in which Ser9 and Ser21 of the two GSK-3beta isoforms have been replaced by Ala, polarity develops independently of phosphorylation at these sites. Nevertheless, global inhibition of GSK-3beta disturbs polarity development by leading to the formation of multiple axon-like processes in both control and knock-in neurons. This unpolarised outgrowth is accompanied by the symmetric delivery of membrane components to all neurites. Finally, the adenomatous polyposis coli (APC) protein accumulates at the tip of one neurite before and during axon elongation, but global inhibition of GSK-3beta leads to APC protein accumulation in all neurites. We conclude that GSK-3beta inhibition promotes the development of neuronal polarity, but that this is not mediated by Akt/PKB-dependent phosphorylation.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

Plexin-induced collapse assay in COS cells.

Semaphorins are a family of growth cone guidance molecules. When associated with their receptors and coreceptors, plexins and neuropilins, they act as chemorepellents for an extensive range of neuronal populations. The prototypic semaphorin, Sema3A, has a potent inhibitory effect on sensory axons emanating from dorsal root ganglia. This has formed the basis of the most famous assay for semaphorin activity, the chick dorsal root ganglia collapse assay. Recently, a heterologous, highly tractable assay has been used to investigate semaphorin signaling. In this system, the binding of recombinant semaphorins to COS cells expressing plexins and neuropilins induces a morphological collapse that may correlate with growth cone collapse. This chapter describes the optimization of this assay and outlines the subtle differences required to enable Sema3A-Fc and Sema4D-Fc to induce identical collapse phenotypes in COS cells expressing Plexin-A1 and neuropilin-1, or Plexin-B1, respectively.

Animals↗
PubMed2005

Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex.

Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.

Actins↗
PubMed2005

Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization.

Cell polarization is essential in a wide range of biological processes such as morphogenesis, asymmetric division, and directed migration. In this study, we show that two tumor suppressor proteins, adenomatous polyposis coli (APC) and Dlg1-SAP97, are required for the polarization of migrating astrocytes. Activation of the Par6-PKCzeta complex by Cdc42 at the leading edge of migrating cells promotes both the localized association of APC with microtubule plus ends and the assembly of Dlg-containing puncta in the plasma membrane. Biochemical analysis and total internal reflection fluorescence microscopy reveal that the subsequent physical interaction between APC and Dlg1 is required for polarization of the microtubule cytoskeleton.

Adaptor Proteins, Signal Transducing↗
PubMed2005

ALS2/Alsin regulates Rac-PAK signaling and neurite outgrowth.

Rac and its downstream effectors p21-activated kinase (PAK) family kinases regulate actin dynamics within growth cones to control neurite outgrowth during development. The activity of Rac is stimulated by guanine nucleotide exchange factors (GEFs) that promote GDP release and GTP binding. ALS2/Alsin is a recently described GEF that contains a central domain that is predicted to regulate the activities of Rac and/or Rho and Cdc42 activities. Mutations in ALS2 cause some recessive familial forms of amyotrophic lateral sclerosis (ALS) but the function of ALS2 is poorly understood. Here we demonstrate that ALS2 is present within growth cones of neurons, in which it co-localizes with Rac. Furthermore, ALS2 stimulates Rac but not Rho or Cdc42 activities, and this induces a corresponding increase in PAK1 activity. Finally, we demonstrate that ALS2 promotes neurite outgrowth. Defects in these functions may therefore contribute to motor neuron demise in ALS.

Amyotrophic Lateral Sclerosis↗
PubMed2005

Cdc42 controls the polarity of the actin and microtubule cytoskeletons through two distinct signal transduction pathways.

Cdc42, a Rho family GTPase, is a key regulator of cell polarity. In Saccharomyces cerevisiae, it is required for polarized bud formation and pheromone gradient sensing, while in higher eukaryotes, it participates in asymmetric cell division, directional sensing during migration, and morphogenesis. Using a scratch-induced fibroblast migration assay, we previously showed that Cdc42 controls the polarization of both membrane protrusions and the Golgi/centrosome. We now find that Golgi/centrosome polarity is mediated through activation of the Par6/aPKC complex, as previously described in astrocytes. However, this complex is not involved in Cdc42-dependent polarization of protrusions, which instead is mediated by Pak acting through the Rac guanine nucleotide exchange factor, betaPIX. Pak kinase activity is essential for spatially restricting Rac-dependent actin polymerization to the leading edge of the migrating cells, though it is not required for actin polymerization per se. We conclude that in migrating cells, Cdc42 co-ordinately regulates the polarity of the microtubule and actin cytoskeletons through two distinct pathways.

Actins↗
PubMed2005

Association of CNK1 with Rho guanine nucleotide exchange factors controls signaling specificity downstream of Rho.

BACKGROUND: Rho is a small GTPase that controls signal transduction pathways in response to a large number of extracellular stimuli. With over 15 potential Rho target proteins identified to date, however, it is not clear how distinct signaling outputs can be generated downstream of a particular stimulus. RESULTS: Several of the known Rho targets are structurally reminiscent of scaffold proteins, which are generally thought to play an important role in controlling signaling specificity. Here, we show that the Rho target CNK1 is a scaffold protein that interacts with Net1 or p115RhoGEF, two Rho-specific guanine nucleotide exchange factors (GEFs), as well with MLK2 and MKK7, two of the kinase components in the JNK MAP kinase cascade. CNK1 acts cooperatively with the two GEFs to activate JNK MAP kinase, but not other Rho-mediated pathways. In HeLa cells, serum or sphingosine-1-phosphate stimulate Rho-dependent activation of the JNK MAP kinase cascade, and this requires endogenous CNK1. CONCLUSIONS: We conclude that CNK1 couples a subset of Rho exchange factors to activation of the JNK MAP kinase pathway and that signaling specificity is achieved through complexes containing both upstream activators and downstream targets of Rho.

Blotting, Western↗
PubMed2005

Rho GTPases: biochemistry and biology.

Approximately one percent of the human genome encodes proteins that either regulate or are regulated by direct interaction with members of the Rho family of small GTPases. Through a series of complex biochemical networks, these highly conserved molecular switches control some of the most fundamental processes of cell biology common to all eukaryotes, including morphogenesis, polarity, movement, and cell division. In the first part of this review, we present the best characterized of these biochemical pathways; in the second part, we attempt to integrate these molecular details into a biological context.

Animals↗
PubMed2004

Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists. mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.

Actins↗
PubMed2004

Galphaz inhibits serum response factor-dependent transcription by inhibiting Rho signaling.

Galpha12/13 or Galphaq signals induce activation of Rho GTPase, leading to serum response factor (SRF)-mediated gene transcription and actin cytoskeletal organization; however, less is known regarding how Rho pathway signals are down-regulated. Here we report that Galphaz signals inhibit serum response factor (SRF)-dependent transcription. Galphaz expression inhibits Galpha12/13-, Galphaq-, and Rho guanine nucleotide exchange factor (GEF)-induced serum response element (SRE) reporter activation in human embryonic kidney 293T and PC-12 cells. Expression of Galphaz mutants with defective fatty acylation has no inhibitory effect. Expression of Galphaz, but not Galphai, attenuates serum-induced SRE reporter activation, suggesting that Galphaz can down-regulate endogenous signals leading to SRF. Whereas Galphaz also blocks SRE reporter induction by the activated mutant RhoAL63, it does not affect Galpha12- or Rho GEF-induced RhoA activation or RhoAL63-GTP binding in vivo. Moreover, Galphaz does not inhibit SRE reporter induction by an activated form of Rho kinase. Because Galphaz inhibits RhoAL63/A188-induced reporter activation, phosphorylation of RhoA on serine 188 does not seem to be involved; furthermore, RhoA subcellular localization was not affected. Use of pharmacologic inhibitors implies that Galphaz-induced reduction of SRE reporter activation occurs via a mechanism other than adenylate cyclase modulation. These findings suggest that Galphaz signals may attenuate Rho-induced stimulation of SRF-mediated transcription.

Amino Acid Substitution↗
PubMed2004

The activity of the plexin-A1 receptor is regulated by Rac.

Plexins constitute a large family of transmembrane proteins that act as receptors for the semaphorin family of ligands. They are best known for their role in growth cone guidance, although they also are widely expressed outside the nervous system. Plexins are thought to control axon guidance by modifying the growth cone cytoskeleton, and Rho GTPases have been strongly implicated in this response. However, the exact contribution of Rho proteins is unclear. Sema3A/Plexin-A1-induced growth cone collapse, for example, requires Rac activity, which is a surprising result given that this GTPase is usually associated with membrane protrusions. We show here that Sema3A-induced collapse of COS-7 cells expressing Plexin-A1 also requires Rac but not Rho activity and that the cytoplasmic tail of Plexin-A1 interacts directly with activated Rac. However, collapse induced by a constitutively activated version of Plexin-A1 does not require Rac. We propose a novel function for Rac, namely that it acts upstream of Plexin-A1 during semaphoring-induced collapse, to regulate the activity of the receptor.

Animals↗
PubMed2004

p27Kip1 modulates cell migration through the regulation of RhoA activation.

The tumor suppressor p27(Kip1) is an inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes and plays a crucial role in cell cycle regulation. However, p27(Kip1) also has cell cycle-independent functions. Indeed, we find that p27(Kip1) regulates cell migration, as p27(Kip1)-null fibroblasts exhibit a dramatic decrease in motility compared with wild-type cells. The regulation of motility by p27(Kip1) is independent of its cell-cycle regulatory functions, as re-expression of both wild-type p27(Kip1) and a mutant p27(Kip1) (p27CK(-)) that cannot bind to cyclins and CDKs rescues migration of p27(-/-) cells. p27(-/-) cells have increased numbers of actin stress fibers and focal adhesions. This is reminiscent of cells in which the Rho pathway is activated. Indeed, active RhoA levels were increased in cells lacking p27(Kip1). Moreover, inhibition of ROCK, a downstream effector of Rho, was able to rescue the migration defect of p27(-/-) cells in response to growth factors. Finally, we found that p27(Kip1) binds to RhoA, thereby inhibiting RhoA activation by interfering with the interaction between RhoA and its activators, the guanine-nucleotide exchange factors (GEFs). Together, the data suggest a novel role for p27(Kip1) in regulating cell migration via modulation of the Rho pathway.

Actins↗
PubMed2004

Regulation of cell migration by the C2 domain of the tumor suppressor PTEN.

PTEN is a tumor suppressor protein that dephosphorylates phosphatidylinositol 3,4,5 trisphosphate and antagonizes the phosphatidylinositol-3 kinase signaling pathway. We show here that PTEN can also inhibit cell migration through its C2 domain, independent of its lipid phosphatase activity. This activity depends on the protein phosphatase activity of PTEN and on dephosphorylation at a single residue, threonine(383). The ability of PTEN to control cell migration through its C2 domain is likely to be an important feature of its tumor suppressor activity.

Animals↗
PubMed2004

Cell migration: Rho GTPases lead the way.

Rho GTPases control signal transduction pathways that link cell surface receptors to a variety of intracellular responses. They are best known as regulators of the actin cytoskeleton, but in addition they control cell polarity, gene expression, microtubule dynamics and vesicular trafficking. Through these diverse functions, Rho GTPases influence many aspects of cell behavior. This review will focus specifically on their role in cell migration.

Actins↗
PubMed2004

Human CNK1 acts as a scaffold protein, linking Rho and Ras signal transduction pathways.

Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. Here, we describe the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways.

Adaptor Proteins, Signal Transducing↗