Negative regulation of Lck by Cbl ubiquitin ligase

Rao, Navin; Miyake, Sachiko; Reddi, Alagarsamy Lakku; Douillard, Patrice; Ghosh, Amiya K.; Dodge, Ingrid; Zhou, Pengcheng; Fernandes, Norvin D.; Band, Hamid

doi:10.1073/pnas.062055999

Cited by 115 publications

(98 citation statements)

References 40 publications

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“…The interaction between SHP-2 and c-Cbl has been described earlier [44,45], but effects of this interaction for T cell function has not been shown to date. Potential target molecules of SHP-2/Cbl complex in T cells that are located upstream of PKC in the TCR signal transduction cascade are CD3-e, CD3-f, Lck, ZAP-70 or in particular PI3K [52][53][54][55][56][57]. By targeting one or more of these molecules for ubiquitin-mediated degradation, the increased complex formation between SHP-2 and c-Cbl could account for reduced effector function in full-length SHP-2-transduced Th cells.…”

Section: Discussionmentioning

confidence: 99%

The tyrosine phosphatase SHP‐2 regulates differentiation and apoptosis of individual primary T lymphocytes

Hoff

Brunner‐Weinzierl

2007

Eur J Immunol

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Although phosphatases are key players of intracellular processes, not much is known about the phosphatase SHP-2 during T cell differentiation. Here we show that ectopic over-expression of SHP-2 in primary T helper cells directly reduced the frequency of individual lymphocytes expressing pro-inflammatory cytokines after antigen-specific stimulation by a mechanism impairing activation of protein kinase C. In addition we demonstrate that SHP-2 mediates enhanced migration upon CXCR4 signaling in a G-protein-dependent manner. Most strikingly, SHP-2 mediated a dramatic increase in apoptosis by highly enhanced activation of caspases. Co-immunoprecipitations of SHP-2 and c-Cbl from primary T helper cells demonstrated that SHP-2 strongly interacts with the ubiquitin ligase c-Cbl, indicating that c-Cbl could mediate the negative signals of SHP-2. Our results show that SHP-2 signal transduction regulates central checkpoints of T cell differentiation by the activation of distinct signaling cascades.

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Section: Discussionmentioning

confidence: 99%

The tyrosine phosphatase SHP‐2 regulates differentiation and apoptosis of individual primary T lymphocytes

Hoff

Brunner‐Weinzierl

2007

Eur J Immunol

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“…These cells were grown in minimal essential-␣ medium with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 0.1 mM non-essential amino acids, and penicillin/streptomycin. The MEF cells used here are independent of another pair that we have reported previously (37,38).…”

Section: Methodsmentioning

confidence: 99%

Cbl-mediated Ubiquitinylation Is Required for Lysosomal Sorting of Epidermal Growth Factor Receptor but Is Dispensable for Endocytosis

Duan

Miura

Dimri

et al. 2003

Journal of Biological Chemistry

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184

176

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Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identifiedCbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl ؊/؊ mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization. Growth factor receptor tyrosine kinases (RTKs)1 play crucial roles in cellular proliferation, survival, migration, and differentiation. Epidermal growth factor receptor (EGFR/ErbB1) is a member of the ErbB family (ErbB1-4) of RTKs, which play crucial homeostatic roles and are implicated in oncogenesis. Ligand-induced activation of RTKs leads to the assembly of signaling protein complexes and subsequent activation of downstream signaling pathways. The ligand-activated RTKs also undergo rapid endocytosis (1). The endocytosed receptors then undergo a sorting process, which determines receptor fate and signal intensity. The receptors can be targeted to the lysosome for degradation, which terminates receptor signals. Alternatively, the internalized receptors can be recycled back to the cell surface for continued ligand binding and signaling (2-5). The relative efficiency of lysosomal sorting versus recycling is a key determinant of the signaling potency of RTKs (6). For example, EGFR is predominantly delivered to lysosomes when activated by EGF. In contrast, heregulin-activated ErbB2 is primarily recycled. The greater efficiency of the recycling process is thought to be a major determinant of the signaling superiority of ErbB2 over EGFR (7-9).Despite a critical role of endocytic sorting as a determinant of ErbB receptor down-regulation, the biochemical mechanisms that regulate this process have only recently begun to be elucidated. We, and others, have identified Cbl as one such regulator (10 -12). Cbl is recruited to the activated EGFR through both direct and indirect binding. Direct Cbl-EGFR interaction is mediated through the N-terminal tyrosine kinase-binding domain of Cbl, which binds to phosphorylated Tyr-1045 on EGFR (13). Indirect Cbl-E...

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“…The E3 ubiquitin ligase c-Cbl functions as a negative regulator of many signaling pathways and ubiquitination marks active enzymes and receptors for degradation [reviewed in 7 and 8]. Notable targets for c-Cbl-mediated ubiquitination are Lck [9], Vav [10], Fyn [11][12][13], , as well as the already mentioned TCR/ CD3-complex. c-Cbl itself is activated by tyrosine phosphorylation on several residues, most importantly Y700, Y731 and Y774 [13,[15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Reduced Cbl phosphorylation and degradation of the ζ‐chain of the T‐cell receptor/CD3 complex in T cells with low Lck levels

et al. 2008

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T cells with short interfering RNA-mediated Lck-knockdown (kd) display paradoxical hyper-responsiveness upon TCR ligation. We have previously reported a possible mechanism for T-cell activation in cells with low levels of Lck depending on Grb2-SOS1 recruitment to the zeta-chain of TCR/CD3 (Methi et al., Eur. J. Immunol. 2007, 37: 2539-2548. Here, we show that short interfering RNA-mediated targeting of Lck caused a dramatic reduction in c-Cbl phosphorylation and a general reduction in protein ubiquitination after TCR stimulation. Specifically, this resulted in reduced ubiquitination of the zeta-chain, yet internalization of TCR/CD3 appeared to be normal after receptor engagement. However, zeta-chain levels were elevated in Lck-kd cells, and confocal microscopy revealed reduced colocalization of CD3-containing vesicles with endosomal and lysosomal compartments.We hypothesize that prolonged stability of internalized T-cell receptor complex may result in extended signaling in T cells with low Lck levels. Key words: Cbl Á CD3 Á Lck Á T cells Á T-cell receptors IntroductionEngagement of the TCR leads not only to activation of T cells, but also to internalization and lysosomal degradation of the receptor complex [1]. The latter process serves to terminate signaling, and has been shown to be dependent on the tyrosine kinase activity of Lck [2,3] and ubiquitination by c-Cbl [4][5][6]. The E3 ubiquitin ligase c-Cbl functions as a negative regulator of many signaling pathways and ubiquitination marks active enzymes and receptors for degradation [reviewed in 7 and 8]. Notable targets for c-Cbl-mediated ubiquitination are Lck [9], Vav [10], Fyn [11][12][13], , as well as the already mentioned TCR/ CD3-complex. c-Cbl itself is activated by tyrosine phosphorylation on several residues, most importantly Y700, Y731 and Y774 [13,[15][16][17]. These residues are not, however, substrates of Lck or but of Fyn and Syk [13,16,18,19], which are activated directly or indirectly by Lck [20].Cbl exists in two main isoforms, c-Cbl and Cbl-b, with a high level of sequence conservation. Consistent with the negative regulation assigned to the Cbl family of proteins, T cells from c-Cbl À/À and Cbl-b À/À mice were hyperactive upon TCR engagement, although some biochemical distinctions between the phenotypes exist [21][22][23][24][25]. T cells from double-knockout mice lacking both c-Cbl and Cbl-b failed to modulate surface TCR after ligand engagement, resulting in sustained TCR signaling and ERK1/2 phosphorylation. However, signaling through the major TCR pathways were not increased [26]. These observations are comparable to what we found in T cells with low levels of Lck, which also display prolonged ERK1/2 activation while expression levels of other proteins (Fyn, Csk, PKCa, PLCg1, LAT, FAK, and Pyk2) remained unaffected [27,28]. We therefore investigated the impact of short interfering RNA (siRNA)-mediated Lck-knockdown (kd) on c-Cbl activity and TCR/CD3 turnover in T cells. As expected, c-Cbl phosphorylation and ubiquitination of the z-c...

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Negative regulation of Lck by Cbl ubiquitin ligase

Cited by 115 publications

References 40 publications

The tyrosine phosphatase SHP‐2 regulates differentiation and apoptosis of individual primary T lymphocytes

The tyrosine phosphatase SHP‐2 regulates differentiation and apoptosis of individual primary T lymphocytes

Cbl-mediated Ubiquitinylation Is Required for Lysosomal Sorting of Epidermal Growth Factor Receptor but Is Dispensable for Endocytosis

Reduced Cbl phosphorylation and degradation of the ζ‐chain of the T‐cell receptor/CD3 complex in T cells with low Lck levels

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