scholarly journals Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiatao Zhou ◽  
Zhihui Xiao ◽  
Yanli Zhan ◽  
Xuemei Qu ◽  
Sisi Mou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Spectrometric Analysis ◽  
Jurkat T Cells ◽  
Origin And Evolution ◽  
Inhibitory Function ◽  
Mutant Sequence

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

Distinct Roles Of PD-1 Itsm and ITIM In Regulating Interactions With SHP-2, ZAP-70 and Lck, and PD-1-Mediated Inhibitory Function

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 191-191 ◽  
Author(s):  
Pranam Chatterjee ◽  
Nikolaos Patsoukis ◽  
Gordon J. Freeman ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Fusion Proteins ◽  
Cell Activation ◽  
Cytoplasmic Tail ◽  
Inhibitory Function ◽  
Gst Fusion Proteins

Abstract Programmed death (PD)-1 plays a prominent role in the induction and maintenance of peripheral tolerance. The biochemical mechanisms via which PD-1 mediates its inhibitory function remain poorly understood. The cytoplamsic tail of PD-1 contains two structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 tyrosine phosphatase constitutively interacts with PD-1 ITSM and is involved in PD-1-mediated inhibitory function. We sought to identify the nature of PD-1: SHP-2 interaction and to determine whether other TCR-proximal signaling molecules might interact with PD-1 cytoplasmic tail. SHP-2 has two SH2 domains (N-SH2 and C-SH2) and one PTP domain. To identify the region of SHP-2 that interacts with PD-1 we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Pull down assays with each GST-fusion protein using lysates from naive and activated primary human T cells revealed that PD-1 interacted with GST-SHP-2 fusion protein only after T cell activation along with simultaneous PD-1 ligation. This interaction was mediated selectively via the SH2 domains of SHP-2, indicating that PD-1 requires prior tyrosine phosphorylation in order to undergo interaction with SHP-2. To identify the mechanism of PD-1 tyrosine phosphorylation governing PD-1: SHP-2 interaction, we used COS cells to express PD-1 along with either empty vector, the TCR proximal tyrosine kinase Fyn, or a kinase inactive mutant of Fyn, followed by pull down with each SHP-2-GST fusion protein. No interaction between PD-1 and SHP-2-GST fusion proteins was detected in lysates from COS cells expressing empty vector or kinase inactive Fyn mutant. In contrast, in the presence of active Fyn, PD-1 underwent tyrosine phosphorylation and was able to interact with GST fusion proteins of SHP-2-FL, SHP-2-N-SH2, SHP-2-C-SH2 and SHP-2-ΔN-SH2 but not SHP-2-PTP, providing evidence that PD-1: SHP-2 interaction requires tyrosine phosphorylation of PD-1 by Src family kinases for subsequent SH2-mediated recruitment of SHP-2. To determine the structural and functional role of each individual tyrosine in the ITIM and the ITSM of PD-1 cytoplasmic tail in PD-1: SHP-2 interaction in vivo, we used Jurkat T cells to express cDNA of either PD-1 wild type, PD-1 with the ITIM tyrosine mutated to phenylalanine (PD-1.Y223F), PD-1 with the ITSM tyrosine mutated to phenylalanine (PD-1.Y248F) or PD-1 with both ITIM and ITSM tyrosines mutated to phenylalanine (PD-1.Y223F/Y248F). After activation, PD-1 wild type underwent tyrosine phosphorylation and developed a robust interaction with SHP-2. PD-1.Y223F retained the ability to undergo interaction with SHP-2 after activation, whereas PD-1.Y248F and PD-1.Y223F/Y248F were unable to interact with SHP-2. We examined whether the PD-1 cytopasmic phosphotyrosines might interact with other SH2 domain containing proteins with critical role in T cell activation. We determined that after T cell activation, PD-1 displayed interaction with ZAP-70 and with activated Lck as determined by PD-1 immunoprecipitation followed by immunoblot with antibodies specific for ZAP-70 and for the activation-specific phospho-LckY394. These interactions remained unaffected in T cells expressing PD-1.Y223F but were abrogated in T cells expressing PD-1.Y248F or PD-1.Y223F/Y248F indicating a mandatory role of phosphorylated ITSM but not ITIM for these associations. However, despite their distinct ability to mediate interactions of PD-1 with SHP-2, Lck and ZAP-70, both phosphorylated ITSM and ITIM had a mandatory role in the inhibitory effect of PD-1 on T cell activation. In T cells expressing either PD-1.Y223F or PD-1.Y248F, PD-1-mediated inhibition of IL-2 production was diminished by 50%, but was almost abrogated in T cells expressing the double mutant PD-1.Y223F/Y248F. Our results indicate that the cytoplasmic tail of PD-1 requires tyrosine phosphorylation in order to mediate phosphorylation-dependent interactions and inhibition on T cell activation. Although phosphorylation-dependent interactions of PD-1 with SHP-2, ZAP-70 and Lck involve Y248 in the ITSM, yet unidentified interactions of Y223 in the ITIM are mandatory for PD-1-mediated inhibitory function on T cell activation. Disclosures: Freeman: Boehringer-Ingelheim: Patents & Royalties; Bristol-Myers-Squibb/Medarex: Patents & Royalties; Roche/Genentech: Patents & Royalties; Merck: Patents & Royalties; EMD-Serrono: Patents & Royalties; Amplimmune: Patents & Royalties; CoStim Pharmaceuticals: Patents & Royalties; Costim Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

T cells: a dedicated effector kinase pathways for every trait?

2021 ◽  
Vol 478 (6) ◽  
pp. 1303-1307
Author(s):  
Kriti Bahl ◽  
Jeroen P. Roose
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Spectrometric Analysis ◽  
Activated T Cells ◽  
Biological Responses ◽  
Extracellular Signal ◽  
Differentiation And Proliferation

Signaling pathways play critical roles in regulating the activation of T cells. Recognition of foreign peptide presented by MHC to the T cell receptor (TCR) triggers a signaling cascade of proximal kinases and adapter molecules that lead to the activation of Effector kinase pathways. These effector kinase pathways play pivotal roles in T cell activation, differentiation, and proliferation. RNA sequencing-based methods have provided insights into the gene expression programs that support the above-mentioned cell biological responses. The proteome is often overlooked. A recent study by Damasio et al. [Biochem. J. (2021) 478, 79–98. doi:10.1042/BCJ20200661] focuses on characterizing the effect of extracellular signal-regulated kinase (ERK) on the remodeling of the proteome of activated CD8+ T cells using Mass spectrometric analysis. Surprisingly, the Effector kinase ERK pathway is responsible for only a select proportion of the proteome that restructures during T cell activation. The primary targets of ERK signaling are transcription factors, cytokines, and cytokine receptors. In this commentary, we discuss the recent findings by Damasio et al. [Biochem. J. (2021) 478, 79–98. doi:10.1042/BCJ20200661] in the context of different Effector kinase pathways in activated T cells.

scholarly journals Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Matthias Kästle ◽  
Camilla Merten ◽  
Roland Hartig ◽  
Thilo Kaehne ◽  
Ardiyanto Liaunardy-Jopeace ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Enzymatic Activity ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Jurkat T Cells

Abstract Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

18β-Glycyrrhetinic acid potently inhibits Kv1.3 potassium channels and T cell activation in human Jurkat T cells

2013 ◽  
Vol 148 (2) ◽  
pp. 647-654 ◽  
Author(s):  
Xiao-Xing Fu ◽  
Li-Li Du ◽  
Ning Zhao ◽  
Qian Dong ◽  
Yu-Hua Liao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Potassium Channels ◽  
T Cell Activation ◽  
Cell Activation ◽  
Glycyrrhetinic Acid ◽  
Jurkat T Cells

scholarly journals PAG-Associated FynT Regulates Calcium Signaling and Promotes Anergy in T Lymphocytes

2007 ◽  
Vol 27 (5) ◽  
pp. 1960-1973 ◽  
Author(s):  
Dominique Davidson ◽  
Burkhart Schraven ◽  
André Veillette
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Anergic T Cells ◽  
Selective Enhancement

ABSTRACT Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.

Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3ζ expression

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3851-3859 ◽  
Author(s):  
Sandeep Krishnan ◽  
Vishal G. Warke ◽  
Madhusoodana P. Nambiar ◽  
Henry K. Wong ◽  
George C. Tsokos ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Effector T Cells ◽  
T Cell Differentiation ◽  
Effector T Cell

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon γ (IFN-γ) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3ζ and CD3ε signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3ζ has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3ζ down-regulation were examined. The loss of CD3ζ expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3ζ expression may be a feature of chronic T-cell activation and effector generation in vivo.

scholarly journals The Role of E3 Ubiquitin Ligase Cbl Proteins in Interleukin-2-Induced Jurkat T-Cell Activation

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Ming-Fang Zhao ◽  
Xiu-Juan Qu ◽  
Jing-Lei Qu ◽  
You-Hong Jiang ◽  
Ye Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathway ◽  
T Cell Activation ◽  
Ubiquitin Ligase ◽  
Cell Activation ◽  
Erk Signaling ◽  
Jurkat T Cells ◽  
Jurkat T Cell ◽  
Cbl Proteins

Interleukin- (IL-) 2 is the major growth factor for T-cell activation and proliferation. IL-2 has multiple functions in the regulation of immunological processes. Although most studies focus on T-cell immunomodulation, T-cell activation by IL-2 is the foundation of priming the feedback loop. Here, we investigated the effect of MAPK/ERK and PI3K/Akt signaling pathways on IL-2-induced cell activation and the regulatory mechanisms of upstream ubiquitin ligase Cbl-b and c-Cbl. Morphological analysis of Jurkat T cells was performed by cytospin preparations with Wright-Giemsa stain. CD25 expression on Jurkat T cells was determined by flow cytometry. Changes in cell activation proteins such as p-ERK, ERK, p-Akt, Akt, and ubiquitin ligase Casitas B-cell Lymphoma (Cbl) proteins were analyzed by western blot. Following IL-2-induced activation of Jurkat T cells, p-ERK expression was upregulated, while there was no change in p-Akt, ERK, or Akt expression. Thus, the MAPK/ERK signaling pathway, but not PI3K/Akt, was involved in IL-2-induced T-cell activation. Either using PD98059 (a specific inhibitor for p-ERK) or depletion of ERK with small interfering RNA (siRNA) reduced the expression of CD25. This study also showed that ubiquitin ligase proteins Cbl-b and c-Cbl might be involved in IL-2-induced Jurkat T-cell activation by negatively regulating the MAPK/ERK signaling pathway.

scholarly journals Partially Phosphorylated T Cell Receptor ζ Molecules Can Inhibit T Cell Activation

1999 ◽  
Vol 190 (11) ◽  
pp. 1627-1636 ◽  
Author(s):  
Ellen N. Kersh ◽  
Gilbert J. Kersh ◽  
Paul M. Allen
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Inhibitory Function ◽  
Cell Clones ◽  
Agonist And Antagonist ◽  
Inhibitory Signaling

The T cell receptor complex (TCR) ζ chain is constitutively tyrosine phosphorylated specifically at two of the six ζ immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues in resting peripheral T cells. Further phosphorylation of ζ is induced by both agonist and antagonist ligands of the TCR, with agonists inducing complete phosphorylation of the ζ ITAM tyrosines. After antagonist stimulation, ζ phosphorylation is incomplete and generates discrete forms of partially phosphorylated ITAMs. Here, we mutate specific tyrosines in chimeric human CD8-ζ molecules to reflect phosphorylation in resting T cells as well as phosphorylation induced by agonist and antagonist ligands. We demonstrate that such partially phosphorylated TCR-ζ species can inhibit IL-2 production in T cell hybridomas and proliferation in T cell clones. This reveals a previously unrecognized, inhibitory function of partially phosphorylated ITAMs. These findings support the concept that TCR antagonism can arise through the generation of an inhibitory signal within the TCR complex and that constitutive ζ phosphorylation in resting T cells is an inhibitory signaling environment.

The E3 Ubiquitin Ligase TRIM36, a Transcriptional Target of Tob, Is Expressed in Anergic T Cells and Mediates Unresponsiveness through Proteolysis of Signaling Proteins PLC- γ1 and PKC-𝛉.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 113-113
Author(s):  
Dimitrios Tzachanis ◽  
Alla Berezovskaya ◽  
Esther M. Lafuente ◽  
Lequn Li ◽  
Gordon J. Freeman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Ubiquitin Ligase ◽  
Cell Activation ◽  
E3 Ubiquitin Ligase ◽  
Signaling Proteins ◽  
Jurkat T Cells ◽  
Empty Vector ◽  
Anergic T Cells

Abstract Ligation of the T cell receptor (TCR) and costimulatory receptors leads to cytokine secretion and clonal expansion, whereas ligation of TCR alone leads to anergy. We have previously determined that anergic cells express Tob, a member of the novel APRO gene family, which inhibits T cell activation. The precise molecular mechanisms via which Tob mediates its effects in T cells are not fully understood. Tob functions as transcriptional coactivator and enhances DNA binding of Smads. Therefore, Tob may regulate de novo mRNA synthesis or gene transcription. To identify genes that are induced by Tob, Jurkat T cells that lack endogenous Tob, were transfected with Tob cDNA or empty vector and differential gene expression was determined by suppression subtractive hybridization. TRIM36 was one of the genes induced by Tob. TRIM36 is a RING finger E3 ubiquitin ligase. It belongs to a recently identified tripartite motif (TRIM) gene family which also includes Pyrin/Marenosrtin, MID1, MUL, PML, RFP and TIF1, proteins implicated in familial human diseases and cancer. E3 proteins confer substrate specificity to the ubiquitin system. Previous studies have shown that the trancriptional profile of anergic cells includes the E3 ubiquitin ligases Cbl-b, GRAIL and Itch. Therefore, the finding that Tob, a transcriptional regulator expressed in anergic cells, induces expression of TRIM36 E3 ubiquitin ligase is very intriguing. Northern blot analysis confirmed that TRIM36 mRNA was selectively upregulated in anergic T cells. To determine the role of TRIM36 on IL-2 gene transcription, Jurkat T cells were transfected with full-length TRIM36 cDNA along with the IL-2 promoter/enhancer cDNA (2kb) linked to the luciferase gene. TRIM36 inhibited CD3+CD28-mediated IL-2 transcription by 90%. Interestingly, when cells were stimulated with PMA+Ionomycin, which bypass the TCR proximal signals, IL-2 transcription was almost unaffected. These results prompted us to search for candidate ubiquitination substrates among signaling molecules that have a critical role on TCR-mediated T cell activation and IL-2 transcription. Previous studies have shown that among T cell signaling molecules, TCRζ, ZAP70, PLC-γ1 and PKC-𝛉 undergo ubiquitin-targeted degradation. For this reason, we investigated whether any of these proteins might be substrates for TRIM36-mediated ubiquitination. V5-tagged TRIM36 or empty vector was expressed in Jurkat T cells followed by stimulation with anti-CD3+anti-CD28 mAbs in the presence of ubiquitin aldehyde that prevents substrate deubiquitination. Immunoblot with antibodies specific for TCR ζ, ZAP70, PLC-γ1 and PKC-𝛉 showed that expression of PLC-γ1 and PKC-𝛉 was selectively reduced in the presence of TRIM36. Immunoprecipitation with V5 mAb followed by immunoblot with substrate-specific antibodies revealed that PLC- γ1 and PKC-𝛉 coprecipitated with TRIM36. Immunoblot with ubiquitin-specific antibody revealed that PLC-γ1 and PKC- 𝛉 were substrates for ubiquitination by TRIM36. Our results show that at least one molecular mechanism via which Tob mediates its inhibitory effect on T cell activation involves the induction of TRIM36 ubiquitin ligase, which mediates degradation of two key signaling proteins, PLC- γ1 and PKC-𝛉. Moreover, these results suggest that TRIM36 may represent a novel target of molecular intervention for induction of transplantation tolerance.

Sign in / Sign up

Export Citation Format

Share Document