Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts

Filipp, Dominik; Zhang, Jenny; Leung, Bernadine; Shaw, Andréy S.; Levin, Steven D.; Veillette, André; Julius, Michael

doi:10.1084/jem.20022112

Cited by 102 publications

(136 citation statements)

References 25 publications

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“…They may function redundantly and independently. However, Julius and co-workers recently proposed a mechanistic link for activation of each kinase, involving differences in their lipid raft localization (Filipp et al, 2003). After crosslinking primary T cells with anti-TCR and anti-CD4 antibodies, they observed a kinetic discrepancy in kinase activation, with Lck activity peaking early and Fyn lagging behind, all within the first 2 min after stimulation.…”

Section: Model Of Regulation and Activationmentioning

confidence: 99%

“…Fyn activation (both in and out of rafts) markedly increased after initial Lck activity and even increased after Lck activity had waned. Importantly, Filipp et al (2003) showed that Fyn could not be activated in Lck-deficient T cells. This study suggests a model whereby Lck: (1) is initially activated outside of rafts; (2) then translocates into rafts; and (3) then activates Fyn molecules already residing in rafts.…”

Section: Model Of Regulation and Activationmentioning

confidence: 99%

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Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation

2004

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The function of the Src-family kinases (SFKs) Lck and Fyn in T cells has been intensively studied over the past 15 years. Animal models and cell line studies both indicate a critical role for Lck and Fyn in proximal T-cell antigen receptor (TCR) signal transduction. Recruited SFKs phosphorylate TCR ITAMs (immunoreceptor tyrosinebased activation motifs) in the CD3 and f chains, which then serve as docking sites for Syk-family kinases. SFKs then phosphorylate and activate the recruited Syk-family kinase. Lck and Fyn are spatially segregated in cell membranes due to differential lipid raft localization, and may undergo sequential activation. In addition to the CD4 and CD8 coreceptors, a recently described adaptor, Unc119, may link SFKs to the TCR. CD45 and Csk provide positive and negative regulatory control of SFK functions, respectively, and Csk is constitutively bound to the transmembrane adapter protein, PAG/Cbp. TCRbased signaling is required at several stages of T-cell development, including at least pre-TCR signaling, positive selection, peripheral maintenance of naive T cells, and lymphopenia-induced proliferation. SFKs are required for each of these TCR-based signals, and Lck seems to be the major contributor.

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Section: Model Of Regulation and Activationmentioning

confidence: 99%

Section: Model Of Regulation and Activationmentioning

confidence: 99%

Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation

2004

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Section: Introductionmentioning

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].…”

mentioning

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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“…Analysis of mice deficient in Lck, Fyn, or both SFKs has determined that Lck is the predominant SFK required to initiate TCR signaling in thymocytes and mature T cells. However, current models of TCR activation put Fyn downstream of Lck activation, with Lck translocating to lipid rafts upon TCR stimulation to activate raft-associated Fyn (35,36) by an as yet undetermined mechanism. An intriguing possibility is that PTP␣ is a target of Lck, thus promoting PTP␣ function as a Lck effector and a Lck/Fyn signaling intermediate.…”

Section: Discussionmentioning

confidence: 99%

Differential Function of PTPα and PTPα Y789F in T Cells and Regulation of PTPα Phosphorylation at Tyr-789 by CD45

Maksumova

Wang

Wong

et al. 2007

Journal of Biological Chemistry

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CD45 is a major membrane protein tyrosine phosphatase (PTP) expressed in T cells where it regulates the activity of Lck, a Src family kinase important for T cell receptor-mediated activation. PTP␣ is a more widely expressed transmembrane PTP that has been shown to regulate the Src family kinases, Src and Fyn, and is also present in T cells. Here, PTP␣ was phosphorylated at Tyr-789 in CD45؊ T cells but not in CD45 ؉ T cells suggesting that CD45 could regulate the phosphorylation of PTP␣ at this site. Furthermore, CD45 could directly dephosphorylate PTP␣ in vitro. Expression of PTP␣ and PTP␣-Y789F in T cells revealed that the mutant had a reduced ability to decrease Fyn and Cbp phosphorylation, to regulate the kinase activity of Fyn, and to restore T cell receptor-induced signaling events when compared with PTP␣. Conversely, this mutant had an increased ability to prevent Pyk2 phosphorylation and CD44-mediated cell spreading when compared with PTP␣. These data demonstrate distinct activities of PTP␣ and PTP␣-Y789F in T cells and identify CD45 as a regulator of PTP␣ phosphorylation at tyrosine 789 in T cells. Protein tyrosine phosphatases (PTPs) 5, together with protein tyrosine kinases, are responsible for controlling the level of tyrosine phosphorylation within cells. In general, basal tyrosine phosphorylation levels in unstimulated cells are relatively low, suggesting that protein tyrosine phosphatase activity prevails over tyrosine kinase activities under resting conditions. Stimulation of cells via a variety of receptors linked to tyrosine kinases can induce tyrosine phosphorylation cascades that lead to cell activation or modulation of cellular function. In T lymphocytes, CD45 is a major protein tyrosine phosphatase expressed at the T cell membrane where it is thought to contribute over 90% of the membrane-associated PTP activity (1). Recognition of antigen by the T cell receptor (TCR) results in induction of a tyrosine phosphorylation cascade leading to T cell activation and is driven primarily by the activity of the Src family kinase (SFK), Lck (2). Counter-intuitively, CD45 is required for the efficient progression of this TCR-induced tyrosine phosphorylation signal, as it is required to dephosphorylate the negative regulatory site (Tyr-505) of Lck (reviewed in Refs. 3-5). This relieves inhibitory constraints on Lck and primes it for subsequent autophosphorylation at tyrosine 394 and activation. This activating function of CD45 and its requirement for efficient TCR signaling are well established. However, evidence from CD45 Ϫ/Ϫ thymocytes and certain CD45 Ϫ T cell lines indicates that the absence of CD45 can result in enhanced Lck phosphorylation at both the negative regulatory and autophosphorylation sites, suggesting a role for CD45 in both the activation and inactivation of Lck (4, 6, and reviewed in Ref. 7). Thus the function of CD45 may be to keep Lck under control, allowing activation, but preventing sustained activation or inactivation. It has been difficult to examine the role of CD45 in inactivati...

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Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts

Cited by 102 publications

References 25 publications

Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation

Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation

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

Differential Function of PTPα and PTPα Y789F in T Cells and Regulation of PTPα Phosphorylation at Tyr-789 by CD45

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