102 research outputs found
CSN-mediated deneddylation differentially modulates Ci155 proteolysis to promote Hedgehog signalling responses
The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCFSlimb-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCFSlimb-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCFSlimb is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate–E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo
HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease
Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)–dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses
Inhibition of the Nuclear Import of Cubitus Interruptus by Roadkill in the Presence of Strong Hedgehog Signal
Hedgehog (Hh) signalling plays an important role in various developmental processes by activating the Cubitus interruptus (Ci)/Glioblastoma (Gli) family of transcription factors. In the process of proper pattern formation, Ci activity is regulated by multiple mechanisms, including processing, trafficking, and degradation. However, it remains elusive how Ci distinctly recognizes the strong and moderate Hh signals. Roadkill (Rdx) induces Ci degradation in the anterior region of the Drosophila wing disc. Here, we report that Rdx inhibited Ci activity by two different mechanisms. In the region abutting the anterior/posterior boundary, which receives strong Hh signal, Rdx inhibited the nuclear import of Ci by releasing importin α3 from Ci. In this region, Rdx negatively regulated the expression of transcription factor Knot/Collier. In farther anterior regions receiving moderate levels of Hh signal, Rdx induced Ci degradation, as reported previously. Thus, two different mechanisms by which Rdx negatively regulates Ci may play an important role in the fine-tuning of Hh responses
SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2
Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus
SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2
The Hedgehog Signaling Pathway Emerges as a Pathogenic Target
The Hedgehog (Hh) signaling pathway plays an essential role in the growth, development, and homeostatis of many tissues in vertebrates and invertebrates. Much of what is known about Hh signaling is in the context of embryonic development and tumor formation. However, a growing body of evidence is emerging indicating that Hh signaling is also involved in postnatal processes such as tissue repair and adult immune responses. To that extent, Hh signaling has also been shown to be a target for some pathogens that presumably utilize the pathway to control the local infected environment. In this review, we discuss what is currently known regarding pathogenic interactions with Hh signaling and speculate on the reasons for this pathway being a target. We also hope to shed light on the possibility of using small molecule modulators of Hh signaling as effective therapies for a wider range of human diseases beyond their current use in a limited number of cancers
Processing of the Drosophila Hedgehog Signaling Effector Ci-155 to the Repressor Ci-75 Is Mediated by Direct Binding to the SCF Component Slimb
SummarySignaling by extracellular Hedgehog (Hh) molecules is crucial for the correct allocation of cell fates and patterns of cell proliferation in humans and other organisms [1, 2]. Responses to Hh are universally mediated by regulating the activity and the proteolysis of the Gli family of transcriptional activators such that they induce target genes only in the presence of Hh [1, 3]. In the absence of Hh, the sole Drosophila Gli homolog, Cubitus interruptus (Ci), undergoes partial proteolysis to Ci-75, which represses key Hh target genes [4]. This processing requires phosphorylation of full-length Ci (Ci-155) by protein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), as well as the activity of Slimb [5–7]. Slimb is homologous to vertebrate β-TRCP1, which binds as part of an SCF (Skp1/Cullin1/F-box) complex to a defined phosphopeptide motif to target proteins for ubiquitination and subsequent proteolysis [8–10]. Here, we show that phosphorylation of Ci at the specific PKA, GSK-3, and CK1 sites required in vivo for partial proteolysis stimulates binding to Slimb in vitro. Furthermore, a consensus Slimb/β-TRCP1 binding site from another protein can substitute for phosphorylated residues of Ci-155 to direct conversion to Ci-75 in vivo. From this, we conclude that Slimb binds directly to phosphorylated Ci-155 to initiate processing to Ci-75. We also explore the phosphorylated motifs in Ci that are recognized by Slimb and provide some evidence that silencing of Ci-155 by phosphorylation may involve more than binding to Slimb
Lattice Dynamics in Covalent Solids: Sn in Sns2_Xsex (0≤X≤2)
Mössbauer effect studies using the 23.8 keV gamma transition in 119Sn have been carried out on the system SnS2-xSe x (O≤x≤2) on both microcrystalline powders and single crystal samples. The composition dependence of the isomer shift parameter for the single line resonance spectrum shows that the covalency increases as x increases; the 5s electron population of tin in SnSe2 is ∼0.13 greater than it is in SnS2. From the temperature dependence of the area under the resonance curve it is possible to extract a Mössbauer lattice temperature θM which is only weakly dependent on composition for samples prepared in an identical manner. Single crystal orientation experiments with SnS2 and SnSe2 have been carried out at 295 ± 1 K to investigate the anisotropy in the metal atom motion parallel and perpendicular to the crystallographic c axis (perpendicular and parallel to the easy cleavage plane). The vibrational anisotropy ratio, 〈x ∥〉/〈x⊥〉 is found to be 2.25 in SnS2 and 1.58 in SnSe2 at room temperature. The absolute values of the tin atom vibrational amplitudes have been calculated using the recoil-free fraction data for metallic (β) tin under identical conditions
Phosphoproteome Profiling of the Macrophage Response to Different Toll-Like Receptor Ligands Identifies Differences in Global Phosphorylation Dynamics
Toll-like
receptors (TLRs) are among the first sensors that detect
infection and drive immune response. Macrophages encountering a pathogen
are usually stimulated not by one TLR, but by a combination of TLRs
engaged by distinct microbe ligands. To understand the integrated
signaling under complex conditions, we investigated the differences
in the phosphoprotein signaling cascades triggered by TLR2, TLR4,
and TLR7 ligands using a single responding cell population. We performed
a global, quantitative, early poststimulation kinetic analysis of
the mouse macrophage phosphoproteome using stable isotope labeling
with amino acids coupled to phosphopeptide enrichment and high-resolution
mass spectrometry. For each TLR ligand, we found marked elevation
of phosphorylation of cytoskeleton components, GTPases of the Rho
family, and phospholipase C signaling pathway proteins. Phosphorylation
of proteins involved in phagocytosis was only seen in response to
TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation
of proteins involved in endocytosis were delayed in response to TLR2
as compared to TLR4 ligands. These findings reveal that the phosphoproteomic
response to stimulation of distinct TLRs varies both in the major
modification targets and the phosphorylation dynamics. These results
advance the understanding of how macrophages sense and respond to
a diverse set of TLR stimuli
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