Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells.

Ota, Yasuo; Beitz, Laurie; Scharenberg, Andrew M.; Donovan, Jerald A.; Kinét, Jean Pierre; Samelson, Lawrence E.

doi:10.1084/jem.184.5.1713

Cited by 89 publications

(56 citation statements)

References 55 publications

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“…How Syk and Cbl might act in concert to regulate Hrs monoubiquitination? Syk and Cbl have been previously reported to be constitutively associated in RBL-2H3 cells [30]. FcεRI engagement promotes Syk/Cbl membrane recruitment and the subsequent activation of both enzymes, being the kinase activity of Syk required for Cbl ligase activity [17].…”

Section: Discussionmentioning

confidence: 99%

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Syk‐dependent regulation of Hrs phosphorylation and ubiquitination upon FcεRI engagement: Impact on Hrs membrane/cytosol localization

et al. 2012

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Several lines of evidence suggest that Syk controls immune receptor endocytic trafficking. However, the Syk substrates that regulate this process are not currently known. Here, we demonstrate that Syk knockdown prevents the trafficking of engaged high affinity IgE receptor (FcεRI) to a degradative compartment in mast cells. We then concentrate our attention on hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) as potential Syk substrate, since it serves as critical regulator for FcεRI entry into lysosomes. We show that Hrs undergoes antigen-dependent tyrosine phosphorylation and ubiquitination, and identify Syk as the kinase responsible for Hrs phosphorylation. Syk was also required for Hrs ubiquitination catalyzed by c-Cbl E3 ligase. Syk-dependent regulation of Hrs covalent modifications, without affecting protein stability, controlled Hrs localization. The majority of phosphorylated Hrs forms were observed only in membrane compartments, whereas ubiquitinated Hrs was predominantly cytosolic, suggesting that both modifications might impact on Hrs function. Together, these findings provide a major step forward in understanding how Syk orchestrates endocytosis of engaged immune receptors.Keywords: FcεRI r Hrs r Phosphorylation r Syk kinase r Ubiquitination Supporting Information available online IntroductionThe Syk/ZAP-70 family of protein tyrosine kinases (PTKs) plays an essential role in signaling through a variety of immune receptors (IRs), including the TCR and BCR, the high-affinity receptor for IgE (FcεRI), and the widely distributed receptors for IgG [1]. All these IRs contain multiple subunits; some, unique for each receptor, are used for ligand binding whereas others share a conserved ITAM Correspondence: Prof. Rossella Paolini e-mail: rossella.paolini@uniroma1.it that is rapidly phosphorylated by PTKs of the Src family upon IR aggregation, thus allowing signal propagation [2,3].IR-mediated signals also lead to a negative-feedback regulation by the internalization and delivery of engaged receptor complexes to lysosomes for degradation [4][5][6][7][8][9][10][11].In the past years, we have concentrated our interest on the molecular mechanisms responsible for ligand-induced endocytosis of IRs, mainly focusing on the FcεRI that is constitutively expressed on the membrane of mast cells and basophils. FcεRI is composed of an IgE-binding α chain, and the ITAM-containing β and γ subunits [12]. Upon FcεRI cross-linking, the β chainassociated Src family PTK Lyn, phosphorylates β and γ-chain We have previously demonstrated that upon antigen stimulation FcεRI β and γ subunits are ubiquitinated through the combined enzymatic activities of the PTK Syk and the Ub ligase c-Cbl [17]. More recently, we provided evidence that this modification controls receptor internalization and sorting along the endocytic compartments through the action of Ub-binding adapters [11,18,19]. Notably, we have envisaged a critical role for the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) in controlling the...

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Section: Discussionmentioning

confidence: 99%

“…In RBL-2H3 cells c-Cbl constitutively associates with Syk [30], and its ligase activity is rapidly induced upon receptor engagement [17]. (Fig.…”

Section: Syk-dependent Hrs Monoubiquitination Is Mediated By the E3 Lmentioning

confidence: 99%

Syk‐dependent regulation of Hrs phosphorylation and ubiquitination upon FcεRI engagement: Impact on Hrs membrane/cytosol localization

et al. 2012

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“…This suggests that the extent of the tyrosine phosphorylation of Cbl does not regulate histamine release. The tyrosine phosphorylation of Cbl in the Syk Y317F-expressing cells could be due to Cbl interacting with Syk at sites besides Tyr 317 (36,43,63). The Y317F Syk could still be coimmunoprecipitated with Cbl from COS-1 cells (data not shown) (63).…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies indicate that Cbl interacts with Syk and can downregulate the function of Syk (42)(43)(44). Studies with the yeast twohybrid system suggest that Tyr 317 is a candidate binding site for the truncated transforming form of Cbl (36).…”

Section: Role Of Tyr 317 In Syk In Tyrosine Phosphorylation Of Cblmentioning

confidence: 99%

Point Mutation of a Tyrosine in the Linker Region of Syk Results in a Gain of Function

Sada

Zhang

Siraganian

2000

The Journal of Immunology

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The protein tyrosine kinase Syk plays an essential role in FcεRI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the FcεRI-mediated tyrosine phosphorylation of phospholipase C-γ and a dramatic enhancement of histamine release. The in vivo FcεRI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the FcεRI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The FcεRI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.

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“…Although flotillin-1 is often used as a marker of caveolae or lipid rafts, its physiological role has not yet been established. Flotillin-1 and its associated proteins, CAP and Cbl, play a crucial role in the signal transduction of insulin-stimulated glucose transport (33)(34)(35)(36) plays a lesser role in mast cells. After stimulation, the association of Lyn with flotillin-1 increased (Fig.…”

Section: Discussionmentioning

confidence: 99%

Flotillin-1 Regulates IgE Receptor-Mediated Signaling in Rat Basophilic Leukemia (RBL-2H3) Cells

Kato

Nakanishi

Hirashima

2006

The Journal of Immunology

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Cross-linking of high-affinity IgE receptors by multivalent Ag on mast cells (rat basophilic leukemia (RBL)-2H3) induces the phosphorylation of ITAM motifs of an IgE receptor by Src family tyrosine kinase, Lyn. The phosphorylation of IgE receptors is followed by a series of intracellular signals, such as Ca2+ mobilization, MAPK activation, and degranulation. Therefore, Lyn is a key molecule in the activation of mast cells, but the molecular mechanisms for the activation of Lyn are still unclear. Recently, it is suggested that the localization of Lyn in lipid rafts is critical for its activation in several cell lines, although the precise mechanism is still unknown. In this study, we found that flotillin-1, which is localized in lipid rafts, is involved in the process of Lyn activation. We obtained flotillin-1 knockdown (KD)2 rat basophilic leukemia (RBL)-2H3 cells, which express a low level of flotillin-1. In the flotillin-1 KD cells, we observed a significant decrease in Ca2+ mobilization, the phosphorylation of ERKs, tyrosine phosphorylation of the γ-subunit of IgE receptor, and IgE receptor-mediated degranulation. We also found that flotillin-1 is constitutively associated with Lyn in lipid rafts in RBL-2H3 cells, and Ag stimulation induced the augmentation of flotillin-1 binding to Lyn, resulting in enhancement of kinase activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE receptor-mediated mast cell activation, and regulates the kinase activity of Lyn in lipid rafts.

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Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells.

Cited by 89 publications

References 55 publications

Syk‐dependent regulation of Hrs phosphorylation and ubiquitination upon FcεRI engagement: Impact on Hrs membrane/cytosol localization

Syk‐dependent regulation of Hrs phosphorylation and ubiquitination upon FcεRI engagement: Impact on Hrs membrane/cytosol localization

Point Mutation of a Tyrosine in the Linker Region of Syk Results in a Gain of Function

Flotillin-1 Regulates IgE Receptor-Mediated Signaling in Rat Basophilic Leukemia (RBL-2H3) Cells

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