scholarly journals SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding

2022 ◽  
Author(s):  
Ben F Brian ◽  
Frances V Sjaastad ◽  
Tanya S Freedman
Keyword(s):  
Ubiquitin Ligase ◽  
Kinase Activity ◽  
Immune Cell ◽  
Sh3 Domain ◽  
Src Family Kinases ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Membrane Microdomains ◽  
Activation Loop ◽  
Human Polymorphism

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, i.e., Lck, Fyn, Lyn, Hck, Fgr, Blk, Src, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that nucleates an autoinhibitory complex. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (Pep/LYP), which dephosphorylate the SFK activation loop to promote autoinhibition. High local concentrations of Csk are required to promote its negative-regulatory function, and Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains. Purified Csk also homodimerizes in solution through an interface that overlaps the phosphatase binding site. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Csk W47A, an SH3-domain mutant commonly used to block PTPN22 binding, also impairs homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with both Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 596-625 ◽  
Author(s):  
Michal Smida ◽  
Anita Posevitz-Fejfar ◽  
Vaclav Horejsi ◽  
Burkhart Schraven ◽  
Jonathan A. Lindquist
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Kinase Activity ◽  
Src Kinase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Ras Activation ◽  
Anergic T Cells

Abstract In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Src-kinase activity by recruiting Csk (the C-terminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to enhanced Csk recruitment and inhibitory tyrosine phosphorylation within Fyn. This together with enhanced phosphorylation of a tyrosine within the SH2 domain of Fyn leads to the formation of a hyperactive conformation, thus explaining the enhanced Fyn kinase activity. In addition, we have also identified the formation of a multiprotein complex containing PAG, Fyn, Sam68, and RasGAP in stimulated T cells. We demonstrate that PAG-Fyn overexpression is sufficient to suppress Ras activation in Jurkat T cells and show that this activity is independent of Csk binding. Thus, in addition to negatively regulating Src family kinases by recruiting Csk, PAG also negatively regulates Ras by recruiting RasGAP to the membrane. Finally, by knocking down PAG, we demonstrate both enhanced Src kinase activity and Ras activation, thereby establishing PAG as an important negative regulator of T-cell activation.

scholarly journals HIV-1 Nef Selectively Activates Src Family Kinases Hck, Lyn, and c-Src through Direct SH3 Domain Interaction

2006 ◽  
Vol 281 (37) ◽  
pp. 27029-27038 ◽  
Author(s):  
Ronald P. Trible ◽  
Lori Emert-Sedlak ◽  
Thomas E. Smithgall
Keyword(s):  
Sh3 Domain ◽  
Src Family Kinases ◽  
Domain Interaction ◽  
Hiv 1

scholarly journals Staphylococcal Enterotoxin B-Induced MicroRNA-155 Targets SOCS1 To Promote Acute Inflammatory Lung Injury

2014 ◽  
Vol 82 (7) ◽  
pp. 2971-2979 ◽  
Author(s):  
Roshni Rao ◽  
Prakash Nagarkatti ◽  
Mitzi Nagarkatti
Keyword(s):  
Lung Injury ◽  
Immune Cell ◽  
Tissue Injury ◽  
Staphylococcal Enterotoxin ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Staphylococcal Enterotoxin B ◽  
Functional Link ◽  
Ifn Γ ◽  
Enterotoxin B

ABSTRACTStaphylococcal enterotoxin B (SEB) causes food poisoning in humans. It is considered a biological weapon, and inhalation can trigger lung injury and sometimes respiratory failure. Being a superantigen, SEB initiates an exaggerated inflammatory response. While the role of microRNAs (miRNAs) in immune cell activation is getting increasing recognition, their role in the regulation of inflammatory disease induced by SEB has not been studied. In this investigation, we demonstrate that exposure to SEB by inhalation results in acute inflammatory lung injury accompanied by an altered miRNA expression profile in lung-infiltrating cells. Among the miRNAs that were significantly elevated, miR-155 was the most overexpressed. Interestingly, miR-155−/−mice were protected from SEB-mediated inflammation and lung injury. Further studies revealed a functional link between SEB-induced miR-155 and proinflammatory cytokine gamma interferon (IFN-γ). Through the use of bioinformatics tools, suppressor of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ, was identified as a potential target of miR-155. While miR-155−/−mice displayed increased expression ofSocs1, the overexpression of miR-155 led to its suppression, thereby enhancing IFN-γ levels. Additionally, the inhibition of miR-155 resulted in restoredSocs1expression. Together, our data demonstrate an important role for miR-155 in promoting SEB-mediated inflammation in the lungs throughSocs1suppression and suggest that miR-155 may be an important target in preventing SEB-mediated inflammation and tissue injury.

scholarly journals SLAP2 adaptor binding disrupts c-CBL autoinhibition to activate ubiquitin ligase function

2020 ◽  
Author(s):  
Leanne E. Wybenga-Groot ◽  
Andrea J. Tench ◽  
Craig D. Simpson ◽  
Jonathan St. Germain ◽  
Brian Raught ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Binding Site ◽  
Ubiquitin Ligase ◽  
Adaptor Proteins ◽  
Negative Regulator ◽  
Alternative Mechanism ◽  
Kinase Signaling ◽  
Tyrosine Kinase Signaling ◽  
Ligase Function

AbstractCBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

scholarly journals Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alan Sulpizio ◽  
Marena E Minelli ◽  
Min Wan ◽  
Paul D Burrowes ◽  
Xiaochun Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Ubiquitin Ligase ◽  
Legionella Pneumophila ◽  
Kinase Activity ◽  
Effector Protein ◽  
Dependent Manner ◽  
Biochemical Analyses ◽  
Human And Mouse

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

scholarly journals Differential Sensitivity of Src-Family Kinases to Activation by SH3 Domain Displacement

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e105629 ◽  
Author(s):  
Jamie A. Moroco ◽  
Jodi K. Craigo ◽  
Roxana E. Iacob ◽  
Thomas E. Wales ◽  
John R. Engen ◽  
...  
Keyword(s):  
Sh3 Domain ◽  
Src Family Kinases ◽  
Differential Sensitivity

scholarly journals Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance

2015 ◽  
Vol 112 (5) ◽  
pp. 1577-1582 ◽  
Author(s):  
Lars Ketscher ◽  
Ronny Hannß ◽  
David J. Morales ◽  
Anja Basters ◽  
Susana Guerra ◽  
...  
Keyword(s):  
Protease Activity ◽  
Protein Modification ◽  
Negative Regulator ◽  
Viral Resistance ◽  
Regulatory Function ◽  
Influenza B Virus ◽  
Influenza B ◽  
Minimal Risk ◽  
Independent Manner

Protein modification by the ubiquitin-like protein ISG15 is an interferon (IFN) effector system, which plays a major role in antiviral defense. ISG15 modification is counteracted by the isopeptidase USP18, a major negative regulator of IFN signaling, which was also shown to exert its regulatory function in an isopeptidase-independent manner. To dissect enzymatic and nonenzymatic functions of USP18 in vivo, we generated knock-in mice (USP18C61A/C61A) expressing enzymatically inactive USP18. USP18C61A/C61A mice displayed increased levels of ISG15 conjugates, validating that USP18 is a major ISG15 isopeptidase in vivo. Unlike USP18−/− mice, USP18C61A/C61A animals did not exhibit morphological abnormalities, fatal IFN hypersensitivity, or increased lethality, clearly showing that major USP18 functions are unrelated to its protease activity. Strikingly, elevated ISGylation in USP18C61A/C61A mice was accompanied by increased viral resistance against vaccinia virus and influenza B virus infections. Enhanced resistance upon influenza B infection in USP18C61A/C61A mice was completely reversed in USP18C61A/C61A mice, which additionally lack ISG15, providing evidence that the observed reduction in viral titers is ISG15 dependent. These results suggest that increasing ISGylation by specific inhibition of USP18 protease activity could constitute a promising antiviral strategy with only a minimal risk of severe adverse effects.

scholarly journals Evolutionary Plasticity of AmrZ Regulation in Pseudomonas

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. e00132-18 ◽  
Author(s):  
David A. Baltrus ◽  
Kevin Dougherty ◽  
Beatriz Diaz ◽  
Rachel Murillo
Keyword(s):  
Pseudomonas Syringae ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Dual Function ◽  
Genomic Context ◽  
Master Regulator ◽  
Content Type ◽  
Swimming Motility ◽  
Positive Regulator ◽  
Mode Of Regulation

ABSTRACT amrZ encodes a master regulator protein conserved across pseudomonads, which can be either a positive or negative regulator of swimming motility depending on the species examined. To better understand plasticity in the regulatory function of AmrZ, we characterized the mode of regulation for this protein for two different motility-related phenotypes in Pseudomonas stutzeri. As in Pseudomonas syringae, AmrZ functions as a positive regulator of swimming motility within P. stutzeri, which suggests that the functions of this protein with regard to swimming motility have switched at least twice across pseudomonads. Shifts in mode of regulation cannot be explained by changes in AmrZ sequence alone. We further show that AmrZ acts as a positive regulator of colony spreading within this strain and that this regulation is at least partially independent of swimming motility. Closer investigation of mechanistic shifts in dual-function regulators like AmrZ could provide unique insights into how transcriptional pathways are rewired between closely related species. IMPORTANCE Microbes often display finely tuned patterns of gene regulation across different environments, with major regulatory changes controlled by a small group of “master” regulators within each cell. AmrZ is a master regulator of gene expression across pseudomonads and can be either a positive or negative regulator for a variety of pathways depending on the strain and genomic context. Here, we demonstrate that the phenotypic outcomes of regulation of swimming motility by AmrZ have switched at least twice independently in pseudomonads, so that AmrZ promotes increased swimming motility in P. stutzeri and P. syringae but represses this phenotype in Pseudomonas fluorescens and Pseudomonas aeruginosa. Since examples of switches in regulatory mode are relatively rare, further investigation into the mechanisms underlying shifts in regulator function for AmrZ could provide unique insights into the evolution of bacterial regulatory proteins.

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