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

Abstract

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...

TCR activation kinetics and feedback regulation in primary human T cells

“…Figure 5A shows that cells expressing low amount of Lck displayed prolonged Erk1/2 activation. These observations are in line with previous studies showing that knockdown of Lck in Jurkat and primary human T cells prolonged Erk phosphorylation and transcriptional activation [22,23]. …”

“…This hypothesis is also supported by our observations and previously published data showing that suppression of Lck expression by RNAi strongly impaired the activation of the inhibitory molecules SHP-1 and c-Cbl and also prolonged downstream signaling (i.e. pErk, NFAT/AP-1) induced by soluble CD3 stimulation [22,23]. Thus, strong Lck activation may have inhibitory effects on T-cell activation.…”

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

“…Lck and Fyn are the primary tyrosine kinases that phosphorylate ITAMs of the CD3 complex in T cells (10,11). Consistent with previous results (10), we found reduced phosphorylation and degradation of ZIP WT in the Lck-deficient cell line J.CaM1, suggesting that the reporter is a substrate for tyrosine phosphorylation by Lck (Fig.…”

The Late Endosomal Transporter CD222 Directs the Spatial Distribution and Activity of Lck

“…Because Lck represents a very powerful kinase that is involved in various T cell signaling pathways (41)(42)(43)(44), its transport and regulation has been investigated extensively (45)(46)(47)(48)(49). One important molecule implicated in the transport of Lck is the myelin and lymphocyte protein (MAL).…”