James P. Fawcett

A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity

Cellular asymmetry is critical for the development of multicellular organisms. Here we show that homologues of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. We conclude that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signalling proteins that are implicated in mammalian cell polarity.

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

BACKGROUND 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

Par6–aPKC uncouples ErbB2 induced disruption of polarized epithelial organization from proliferation control

The polarized glandular organization of epithelial cells is frequently lost during development of carcinoma. However, the specific oncogene targets responsible for polarity disruption have not been identified. Here, we demonstrate that activation of ErbB2 disrupts apical–basal polarity by associating with Par6–aPKC, components of the Par polarity complex. Inhibition of interaction between Par6 and aPKC blocked the ability of ErbB2 to disrupt the acinar organization of breast epithelia and to protect cells from apoptosis but was not required for cell proliferation. Therefore, oncogenes target polarity proteins to disrupt glandular organization and protect cells from apoptotic death during development of carcinoma.

Par3 and Dynein Associate to Regulate Local Microtubule Dynamics and Centrosome Orientation during Migration

BACKGROUND Centrosome orientation toward the leading edge of migrating cells depends on dynein and microtubules (MTs), as well as a number of signaling factors at the leading edge. However, centrosomes are maintained at the cell center during orientation in fibroblasts, suggesting that factors working at sites other than the leading edge may also be involved. RESULTS In a search for factors that function with dynein in centrosome orientation, we found that the polarity protein Par3 associated with dynein and that knockdown of Par3 inhibited centrosome orientation by disrupting the position of the centrosome at the cell center; this disrupted centrosome positioning is the same phenotype as that observed with dynein inhibition. Par3 associated with dynein through its N-terminal dimerization and PDZ1 domains and interacted specifically with dynein light intermediate chain 2 (LIC2). siRNA knockdown of LIC2, but not LIC1, or overexpression of LIC2 or the N-terminal domain of Par3, also inhibited centrosome orientation by disrupting centrosome position. In wound-edge fibroblasts, Par3 specifically localized to cell-cell contacts where it overlapped with MT ends and dynein puncta in a LIC2-dependent fashion. Live imaging showed that MTs exhibited increased pausing at cell-cell contacts compared to the leading edge and that this elevated pausing was dependent on Par3 and LIC2. CONCLUSIONS Par3 associates with dynein and contributes to the local regulation of MT dynamics at cell-cell contacts and proper positioning of the centrosome at the cell center. We propose that Par3 acts as a cortical factor that tethers MTs through its association with LIC2 dynein.

Nck adaptor proteins control the organization of neuronal circuits important for walking

The intracellular signaling targets used by mammalian axon guidance receptors to organize the nervous system in vivo are unclear. The Nck1 and Nck2 SH2/SH3 adaptors (collectively Nck) can couple phosphotyrosine (pTyr) signals to reorganization of the actin cytoskeleton and are therefore candidates for linking guidance cues to the regulatory machinery of the cytoskeleton. We find that selective inactivation of Nck in the murine nervous system causes a hopping gait and a defect in the spinal central pattern generator, which is characterized by synchronous firing of bilateral ventral motor neurons. Nck-deficient mice also show abnormal projections of corticospinal tract axons and defective development of the posterior tract of the anterior commissure. These phenotypes are consistent with a role for Nck in signaling initiated by different classes of guidance receptors, including the EphA4 receptor tyrosine kinase. Our data indicate that Nck adaptors couple pTyr guidance signals to cytoskeletal events required for the ipsilateral projections of spinal cord neurons and thus for normal limb movement.

NOS1AP associates with Scribble and regulates dendritic spine development.

The formation and function of the neuronal synapse is dependent on the asymmetric distribution of proteins both presynaptically and postsynaptically. Recently, proteins important in establishing cellular polarity have been implicated in the synapse. We therefore performed a proteomic screen with known polarity proteins and identified novel complexes involved in synaptic function. Specifically, we show that the tumor suppressor protein, Scribble, associates with neuronal nitric oxide synthase (nNOS) adaptor protein (NOS1AP) [also known as C-terminal PDZ ligand of nNOS (CAPON)] and is found both presynaptically and postsynaptically. The Scribble–NOS1AP association is direct and is mediated through the phosphotyrosine-binding (PTB) domain of NOS1AP and the fourth PDZ domain of Scribble. Further, we show that Scribble bridges NOS1AP to a β-Pix [β-p21-activated kinase (PAK)-interacting exchange factor]/Git1 (G-protein-coupled receptor kinase-interacting protein)/PAK complex. The overexpression of NOS1AP leads to an increase in dendritic protrusions, in a fashion that depends on the NOS1AP PTB domain. Consistent with these observations, both full-length NOS1AP and the NOS1AP PTB domain influence Rac activity. Together these data suggest that NOS1AP plays an important role in the mammalian synapse.

Guidance of postural motoneurons requires MAPK/ERK signaling downstream of fibroblast growth factor receptor 1.

Identification of intracellular signaling pathways necessary for appropriate axon guidance is challenging because many CNS populations used to study these events contain multiple cell types. Here, we resolve this issue by using mouse embryonic stem (ES) cells that were directed to differentiate into a population of motoneurons that exclusively innervate epaxial muscles [medial median motor column (MMCm) motoneurons]. These ES cell-derived MMCm motoneurons, like their endogenous counterparts, express fibroblast growth factor receptor 1 (FGFR1) and selectively extend axons toward the epaxial trophin FGF8. Unlike wild-type MMCm motoneurons, FGFR1−/− MMCm motoneurons show guidance defects when transplanted into the neural tube of chick embryos. Furthermore, activation of FGFR1 selectively signals through mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) for appropriate guidance in vitro, whereas overexpression of constitutively active MAPK/ERK in transplanted, or endogenous chick, MMCm cells causes guidance defects in vivo. These results indicate that MAPK/ERK activation downstream of FGFR1 is necessary for MMCm motor axon guidance and that ES cell-derived neurons provide an important tool for dissecting intracellular pathways required for axon guidance.

Multiple KCNQ Potassium Channel Subtypes Mediate Basal Anion Secretion from the Human Airway Epithelial Cell Line Calu-3

Potassium channels play an important role in providing a driving force for anion secretion from secretory epithelia. To investigate the role of KCNQ K+ channels in mediating rates of basal anion secretion across the human airway submucosal gland serous cell model, the Calu-3 cell, we examined the expression, localization and function of these channels. In addition to our previous knowledge that Calu-3 cells express KCNQ1, using reverse transcriptase polymerase chain reaction we determined expression of KCNQ3, KCNQ4 and KCNQ5 mRNA transcripts. Immunoblotting detected KCNQ1, KCNQ3 and KCNQ5 proteins, while KCNQ4 protein was not found. Immunolocalization using polarized Calu-3 cell monolayers revealed that KCNQ1 and KCNQ3 were located in or toward the apical membrane of the cells, while KCNQ5 was detected in the apical and lateral membranes. Transepithelial transport studies revealed a small chromanol 293B-sensitive current at the apical membrane, likely KCNQ1. Application of XE991, an inhibitor of all members of the KCNQ channel family, inhibited the basal short-circuit current when applied to both sides of the cells to a greater extent than 293B, with the largest inhibition seen upon apical application. This result was confirmed using linopiridine, a less potent analogue of XE991, and suggests that functional KCNQ3 and KCNQ5, in addition to KCNQ1, are present at the apical aspect of these cells. These results demonstrate the role of a number of KCNQ channel members in controlling basal anion secretion across Calu-3 cells, while also demonstrating the importance of apically located K+ channels in mediating anion secretion in the airway epithelium.

Motoneurons derived from induced pluripotent stem cells develop mature phenotypes typical of endogenous spinal motoneurons.

Induced pluripotent cell-derived motoneurons (iPSCMNs) are sought for use in cell replacement therapies and treatment strategies for motoneuron diseases such as amyotrophic lateral sclerosis (ALS). However, much remains unknown about the physiological properties of iPSCMNs and how they compare with endogenous spinal motoneurons or embryonic stem cell-derived motoneurons (ESCMNs). In the present study, we first used a proteomic approach and compared protein expression profiles between iPSCMNs and ESCMNs to show that <4% of the proteins identified were differentially regulated. Like ESCs, we found that mouse iPSCs treated with retinoic acid and a smoothened agonist differentiated into motoneurons expressing the LIM homeodomain protein Lhx3. When transplanted into the neural tube of developing chick embryos, iPSCMNs selectively targeted muscles normally innervated by Lhx3 motoneurons. In vitro studies showed that iPSCMNs form anatomically mature and functional neuromuscular junctions (NMJs) when cocultured with chick myofibers for several weeks. Electrophysiologically, iPSCMNs developed passive membrane and firing characteristic typical of postnatal motoneurons after several weeks in culture. Finally, iPSCMNs grafted into transected mouse tibial nerve projected axons to denervated gastrocnemius muscle fibers, where they formed functional NMJs, restored contractile force. and attenuated denervation atrophy. Together, iPSCMNs possess many of the same cellular and physiological characteristics as ESCMNs and endogenous spinal motoneurons. These results further justify using iPSCMNs as a source of motoneurons for cell replacement therapies and to study motoneuron diseases such as ALS.

NOS1AP Functionally Associates with YAP To Regulate Hippo Signaling

ABSTRACT Deregulation of cellular polarity proteins and their associated complexes leads to changes in cell migration and proliferation. The nitric oxide synthase 1 adaptor protein (NOS1AP) associates with the tumor suppressor protein Scribble to control cell migration and oncogenic transformation. However, how NOS1AP is linked to the cell signaling events that curb oncogenic progression has remained elusive. Here we identify several novel NOS1AP isoforms, NOS1APd, NOS1APe, and NOS1APf, with distinct cellular localizations. We show that isoforms with a membrane-interacting phosphotyrosine binding (PTB) domain can associate with Scribble and recognize acidic phospholipids. In a screen to identify novel binding proteins, we have discovered a complex consisting of NOS1AP and the transcriptional coactivator YAP linking NOS1AP to the Hippo signaling pathway. Silencing of NOS1AP reduces the phosphorylation of YAP and of the upstream kinase Lats1. Conversely, expression of NOS1AP promotes YAP and Lats1 phosphorylation, which correlates with reduced TEAD activity and restricted cell proliferation. Together, these data implicate a role for NOS1AP in the regulation of core Hippo signaling and are consistent with the idea that NOS1AP functions as a tumor suppressor.