Kevin S. Dingwell

The Extracellular Domain of Herpes Simplex Virus gE Is Sufficient for Accumulation at Cell Junctions but Not for Cell-to-Cell Spread

ABSTRACT Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or cell-to-cell spread.

The multiple decisions made by growth cones of RGCs as they navigate from the retina to the tectum in Xenopus embryos.

Retinal ganglion cells (RGCs) of Xenopus laevis send axons along a stereospecific pathway from the retina to their target the optic tectum. Viewed from the point of the growth cone, this journey is reflected by discrete processes of axon initiation, axon outgrowth, navigation, target recognition, and innervation. These processes are characterised by distinct signalling mechanisms that trigger dynamic changes in growth cone morphology and behavior. Here we review work primarily from our laboratory, examining these events from a cellular and molecular perspective, focusing on the roles of FGFs, netrins, receptors, and intracellular effectors.

Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

Intramembrane proteolytic cleavage by signal peptide peptidase is required for the turnover of some ER-resident, tail-anchored membrane proteins.

USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis.

Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling

Bone morphogenetic proteins (BMPs) control multiple cellular processes in embryos and adult tissues. BMPs signal through the activation of type I BMP receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical pathway, this triggers the association of these SMADs with SMAD4 and their translocation to the nucleus, where they regulate gene expression. BMPs can also signal independently of SMAD4, but this pathway is poorly understood. Here, we report the discovery and characterization of PAWS1/FAM83G as a novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4-independent manner, and BMP signalling induces the phosphorylation of PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP is essential for activation of the SMAD4-independent BMP target genes NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD substrate for type I BMP receptor kinases and as a novel player in the BMP pathway. We also demonstrate that PAWS1 regulates the expression of several non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway.

Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk-1 and Egr-1.

Signalling by members of the FGF family is required for induction and maintenance of the mesoderm during amphibian development. One of the downstream effectors of FGF is the SRF-interacting Ets family member Elk-1, which, after phosphorylation by MAP kinase, activates the expression of immediate-early genes. Here, we show that Xenopus Elk-1 is phosphorylated in response to FGF signalling in a dynamic pattern throughout the embryo. Loss of XElk-1 function causes reduced expression of Xbra at neurula stages, followed by a failure to form notochord and muscle and then the partial loss of trunk structures. One of the genes regulated by XElk-1 is XEgr-1, which encodes a zinc finger transcription factor: we show that phosphorylated XElk-1 forms a complex with XSRF that binds to the XEgr-1 promoter. Superficially, Xenopus tropicalis embryos with reduced levels of XEgr-1 resemble those lacking XElk-1, but to our surprise, levels of Xbra are elevated at late gastrula stages in such embryos, and over-expression of XEgr-1 causes the down-regulation of Xbra both in whole embryos and in animal pole regions treated with activin or FGF. In contrast, the myogenic regulatory factor XMyoD is activated by XEgr-1 in a direct manner. We discuss these counterintuitive results in terms of the genetic regulatory network to which XEgr-1 contributes.

PAWS1 controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP

ABSTRACT Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells. This article has an associated First Person interview with the first authors of the paper. Summary: PAWS1 (also known as FAM83G) controls cell migration by influencing the organization of F-actin and focal adhesions and the distribution of the actin stress fibre network through its association with CD2AP.

PAWS1 controls Wnt signalling through association with casein kinase 1α

The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co‐localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.

The aryl hydrocarbon receptor controls cyclin O to promote epithelial multiciliogenesis.

Epithelia function as barriers against environmental insults and express the transcription factor aryl hydrocarbon receptor (AhR). However, AhR function in these tissues is unknown. Here we show that AhR regulates multiciliogenesis in both murine airway epithelia and in Xenopus laevis epidermis. In air-exposed airway epithelia, induction of factors required for multiciliogenesis, including cyclin O (Ccno) and Multicilin (Mcidas), is AhR dependent, and air exposure induces AhR binding to the Ccno promoter. Submersion and hypoxic conditions impede AhR-dependent Ccno induction. This is mediated by the persistence of Notch signalling, as Notch blockade renders multiciliogenesis and Ccno induction by AhR independent from air exposure. In contrast to Ccno induction, air exposure does not induce the canonical AhR target cytochrome P450 1a1 (Cyp1a1). Inversely, exposure to AhR ligands induces Cyp1a1 but not Ccno and impeded ciliogenesis. These data indicate that AhR involvement in detoxification of environmental pollutants may impede its physiological role, resulting in respiratory pathology.

The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms

The FAM83 proteins anchor various isoforms of the constitutively active kinase CK1 to specific subcellular locations. Subcellular targeting of CK1 FAM83 proteins participate in various cellular processes and are characterized by an N-terminal “domain of unknown function” called DUF1669. Fulcher et al. found that FAM83 family members interacted with a specific subset of casein kinase 1 (CK1) isoforms in vitro through the DUF1669 domain. Each of the eight FAM83 family members exhibited a distinct pattern of subcellular localization and colocalized with specific CK1 isoforms in cultured cells. Experiments in which DUF1669 domains were swapped among FAM83 family members suggested that DUF1669 determines the specificity of the FAM83 protein for particular CK1 isoforms. Because CK1 isoforms are thought to be constitutively active protein kinases, the ability of FAM83 proteins to anchor CK1 isoforms may be an important mechanism for targeting CK1 activity to specific subcellular locations and substrates. Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.