Fyn, Yes, and Syk Phosphorylation Sites in c-Cbl Map to the Same Tyrosine Residues That Become Phosphorylated in Activated T Cells

Feshchenko, Elena A; Langdon, Wallace Y.; Tsygankov, Alexander Y.

doi:10.1074/jbc.273.14.8323

Cited by 150 publications

(190 citation statements)

References 68 publications

(72 reference statements)

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“…Jurkat and COS-7 cell lines were obtained from American Type Culture Collection (ATCC). Cells were grown and stimulated with pervanadate, where indicated, as described previously (Feshchenko et al, 1998(Feshchenko et al, , 1999. COS-7 cells were transfected using DMRIE-C reagent (Invitrogen/Life Technologies, Carlsbad, CA, USA) with an efficiency of B75%, as determined using co-transfection with an expression plasmid encoding for green fluorescent protein.…”

Section: Methodsmentioning

confidence: 99%

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c-Cbl-facilitated cytoskeletal effects in v-Abl-transformed fibroblasts are regulated by membrane association of c-Cbl

2007

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The multi-functional protein c-Cbl is an important modulator of actin cytoskeletal dynamics in diverse biological systems. We had previously reported that c-Cbl facilitates cell spreading and adhesion and suppresses anchorage-independent growth of v-Abl-transformed fibroblasts. To assess the importance of membrane localization of c-Cbl for the observed effects of c-Cbl in v-Abl-3T3 cells, we first mapped the membrane interactive domain(s) of c-Cbl. Our studies indicate that localization of c-Cbl to the membrane is likely to be mediated by the tyrosine kinase binding (TKB) domain and the proline-rich region of c-Cbl, whereas C-terminal tyrosine phosphorylation does not play a role. The association of v-Cbl, which encompasses the TKB domain, with the membrane was unusual as it was not entirely dependent on SH2-phosphotyrosine interactions. Our studies further demonstrate that Src-like adaptor protein (SLAP), which binds to v-Cbl in a tyrosine phosphorylation-independent manner, facilitates membrane association of Cbl. The interaction between c-Cbl and SLAP in v-Abl-3T3 cells positively influenced c-Cbl-mediated spreading and adhesion of these cells. SLAP appears to exert its effects not simply by increasing the amount of c-Cbl in the membrane but by facilitating binding of p85-phosphatidylinositol-3-kinase (PI3K) with membrane-associated c-Cbl.

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Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation and immunoblotting were carried out as described previously (Feshchenko et al, 1998). For immunoprecipitation of c-Cbl from sub-cellular fractions, the membrane pellet obtained by ultracentrifugation, was solubilized using 2% octyl D-glucoside (ODG) lysis buffer.…”

Section: Immunoprecipitation and Immunoblottingmentioning

confidence: 99%

c-Cbl-facilitated cytoskeletal effects in v-Abl-transformed fibroblasts are regulated by membrane association of c-Cbl

2007

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Section: Introductionmentioning

confidence: 99%

“…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].…”

mentioning

confidence: 99%

“…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].…”

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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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Role of Syk in Fcγ receptor-coupled tyrosine phosphorylation of Cbl in a manner susceptible to inhibition by protein kinase C

Hazeki

Matsuo

et al. 1999

Eur. J. Immunol.

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Fcγ receptors (FcγR) of guinea pig neutrophils were ligated and anti‐Cbl immunoprecipitates prepared therefrom were assayed for the associated protein tyrosine kinase activity, which increased upon ligation of FcγR. The increases were overcome upon activation of cellular protein kinase C by simultaneous addition of phorbol 12‐myristate 13‐acetate (PMA) to the ligated cells. Syk proved to be the most important tyrosine kinase bound to Cbl that served as the major substrate; essentially no tyrosine phosphorylation occurred in the anti‐Cbl immunoprecipitates prepared from the cell lysate that had been depleted of Syk by prior immunoprecipitation with anti‐Syk antibodies. Exposure of the 32P‐labeled cells to PMA resulted in phosphorylation of cellular Cbl on serine residues. Thus, protein kinase C‐induced serine phosphorylation of Cbl suppressed its tyrosine phosphorylation by Syk as a result of tyrosine kinase inhibition by unknown mechanisms, leading to inhibition of Cbl‐mediated signaling such as phosphatidylinositol 3‐kinase activation.

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Fyn, Yes, and Syk Phosphorylation Sites in c-Cbl Map to the Same Tyrosine Residues That Become Phosphorylated in Activated T Cells

Cited by 150 publications

References 68 publications

c-Cbl-facilitated cytoskeletal effects in v-Abl-transformed fibroblasts are regulated by membrane association of c-Cbl

c-Cbl-facilitated cytoskeletal effects in v-Abl-transformed fibroblasts are regulated by membrane association of c-Cbl

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

Role of Syk in Fcγ receptor-coupled tyrosine phosphorylation of Cbl in a manner susceptible to inhibition by protein kinase C

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