The zeta subunit of the T cell antigen receptor (TCR) exists primarily as a disulfide-linked homodimer. This receptor subunit is important in TCR-mediated signal transduction and is a substrate for a TCR-activated protein tyrosine kinase. The zeta chain was found to undergo ubiquitination in response to receptor engagement. This posttranslational modification occurred in normal T cells and tumor lines. Both nonphosphorylated and phosphorylated zeta molecules were modified, and at least one other TCR subunit, CD3 delta, was also ubiquitinated after activation of the receptor. These findings suggest an expanded role for ubiquitination in transmembrane receptor function.
PML and Tif1a are fused to RARA and Braf, respectively, resulting in the production of PML-RARalpha and Tif1alpha-B-Raf (T18) oncoproteins. Here we show that PML, Tif1alpha and RXRalpha/RARalpha function together in a transcription complex that is dependent on retinoic acid (RA). We found that PML acts as a ligand-dependent coactivator of RXRalpha/RARalpha. PML interacts with Tif1alpha and CBP. In Pml-/- cells, the RA-dependent induction of genes such as RARB2 and the ability of Tif1alpha and CBP to act as transcriptional coactivators on RA are impaired. We show that both PML and Tif1alpha are growth suppressors required for the growth-inhibitory activity of RA. T18, similar to PML-RARalpha, disrupts the RA-dependent activity of this complex in a dominant-negative manner resulting in a growth advantage. Our data define a new pathway for the control of cell growth and tumorigenesis, and provide a new model for the pathogenesis of acute promyelocytic leukaemia (APL).
CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.
Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptormediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56 lck and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56 lck . The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors.For many transmembrane receptors, including the multisubunit TCR, 1 signaling is initiated by ligand-induced aggregation (1, 2). The earliest obligate intracellular event following TCR aggregation is the activation of the src-family protein tyrosine kinases, Lck (p56 lck ) and Fyn (p59 fyn ). Lck and/or Fyn phosphorylate TCR subunits resulting in the association of a third tyrosine kinase, ZAP-70 (70-kDa -associated protein), with the TCR and to subsequent activation events (3-5). CD45, a tyrosine phosphatase that dephosphorylates key regulatory residues on Lck and Fyn, is also implicated in the initiation of TCR-mediated signaling (6).TCRs consist of six different polypeptides, these include the antigen-recognition element, in most cells an ␣- heterodimer, and a set of invariant signal transducing subunits. The invariant subunits include CD3-␦, -⑀, and -␥ and the structurally distinct TCR-subunit, which exists within the TCR as a disulfide-linked homodimer (4). The minimal signal transducing element of the TCR is the immunoreceptor tyrosine-based activation motif (ITAM) (7). monomers have three ITAMs, and each CD3 subunit has one. ITAMs include two tyrosine residues 10 or 11 amino acids apart that are potential phosphorylation sites. The subunit is a particularly prominent substrate for tyrosine phosphorylation; up to 5% of subunits are phosphorylated on multiple tyrosines upon TCR engagement (8 -11).In addition to being a substrate for tyrosine phosphorylation when cross-linked by antibody or mitogen (11), TCRs also are ubiquitinated. The covalent modification of proteins with chains of ubiquitin, a highly conserved 76-amino acid polypeptide, plays a central role in the targeting of abnormal proteins and a number of regulatory cytosolic and nuclear proteins for degradation in the 26 S proteasome (12-16). Ubiquitination occurs via a multienzyme process involving families of enzymes ter...
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