Identification of UNC119 as a Novel Activator of SRC-type Tyrosine Kinases

Abstract

Lyn, an Src-type tyrosine kinase, is associated with the interleukin (IL)-5 receptor in eosinophils. The mechanism of its activation is unknown. Through yeast twohybrid screening we have cloned and characterized a new signaling molecule, Unc119, that associates with IL-5R␣ and Src family tyrosine kinases. Unc119 induces the catalytic activity of these kinases through interaction with Src homology 2 and 3 domains. IL-5 stimulation of eosinophils increases Unc119 association with Lyn and induces its catalytic activity. Lyn is important for eosinophil survival. Eosinophils that are transduced with Unc119 have increased Lyn activity and demonstrate prolonged survival in the absence of IL-5. Inhibition of Unc119 down-regulates eosinophil survival. To our knowledge Unc119 is the first receptor-associated activator of Src family tyrosine kinases.One of the fundamental goals of cell biology is to understand the mechanism of signal generation by receptors. Many receptors rely upon kinases, especially tyrosine kinases, for receptor phosphorylation and activation of signaling cascades. The Src family tyrosine kinases (SrcTKs) 1 frequently serve as the trigger mechanism for cytosolic signals (1). Receptor-associated SrcTKs exist in a non-active conformation and become transiently activated following ligand binding and receptor oligomerization (2). How receptor oligomerization leads to the activation of SrcTKs is unknown.Two intramolecular interactions tightly regulate structural conformation and enzymatic activity of SrcTKs. One is through Src homology 2 (SH2) and the other is through the SH3 domain. All cellular SrcTKs have a C-terminal regulatory tyrosine residue (Tyr 527 for Src). Phosphorylated Tyr 527 forms an intramolecular interaction with the SH2 domain of the kinase (3). This interaction decreases the kinase activity. Dephosphorylation of this tyrosine residue by phosphatases such as CD45 is required but is not sufficient for kinase activation (4). Receptor-bound SrcTKs are frequently found to be dephosphorylated under basal conditions, yet lack appreciable catalytic activity (5). CD45-negative cells have variable effects on SrcTKs. CD45 negatively regulates Lyn by dephosphorylating both its negative and positive regulatory tyrosine residues (6). The activity of Lyn and Hck is increased in macrophages (7) and B cells (8) lacking CD45.Regulation of SrcTK activity through other intramolecular interactions was realized after the crystal structures of Src (9) and Hck (10) were solved. The linker region that is located between the SH2 and the kinase domains binds to the SH3 domain of the kinase rendering a non-active conformation. Furthermore, a short amino acid stretch connecting the SH2 and SH3 domains has recently been identified as an additional negative regulator of Hck and Src activation (11). The SH3 domain of SrcTKs prefers to bind to the RXXPXXP motif (a motif is defined as a short stretch of amino acid residues that binds to a signaling domain) (12, 13). Although the linkers of SrcTK bind to the SH3 domain, ...

Uncoordinated 119 Preferentially Induces Th2 Differentiation and Promotes the Development of Asthma

“…T cells are probably not the only cells that upregulate Unc119 expression under inflammatory conditions. Unc119 regulates other inflammatory cells: eosinophils and myofibroblasts (11,16). We have previously reported that an elevation of the Unc119 protein level via protein transduction protects human eosinophils from apoptosis (11).…”

“…Rabbit polyclonal anti-phospho-Crk (Tyr221) Ab was acquired from Cell Signaling Technology (Danvers, MA). The generation of the rabbit polyclonal anti-Unc119 Ab and the affinity purification was described previously (11,14,15,17). For cell culture, the following Abs were used: Armenian hamster monoclonal anti-mouse CD3 (clone 145-2C11), Syrian hamster monoclonal anti-mouse CD28 (clone 37.51), mouse monoclonal anti-human CD3 (clone OKT3), mouse monoclonal anti-human CD28 (clone CD28.2), rat monoclonal anti-mouse IL-12/IL-23 (p40 subunit; clone C17.8), rat monoclonal anti-mouse IFN-g (clone XMG1.2; all from eBioscience, San Diego, CA), and rat monoclonal anti-mouse IL-4 (clone 11B.11; National Cancer Institute Preclinical Repository).…”

“…We and others cloned the adaptor protein uncoordinated 119 (Unc119) (11)(12)(13). We subsequently showed that Unc119 controls the constitutive transport of Lck to the plasma membrane via endosomes.…”

Synaptic transmission at retinal ribbon synapses

“…A late onset conerod dystrophy is produced by mutations in HRG4 which is highly enriched at photoreceptor ribbon synapses and which appears to be associated with synaptic vesicles (Kobayashi et al, 2000). Although the function of this protein at ribbon synapses is unclear, its C. elegans orthologue, UNC119, has been shown to activate SRC tyrosine kinases (Cen et al, 2003). Xlinked congenital stationary night blindness arises from mutations in the Ca v 1.4 calcium channel (CACNA1F) found in synaptic terminals of rods and rod bipolar cells (Bech-Hansen et al, 1998).…”

C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

“…There is indirect evidence linking human C5orf30 with immune function via its association with intracellular UNC119 (13); the latter increasing both T-cell activation by up-regulating Lck/Fyn activity and Src kinases regulating macrophages activation (14,15). There are, however, no studies of the biological functions of human C5orf30 and, in view of the genetic association with RA susceptibility and severity, we have undertaken in silico analysis and both in vitro and in vivo experiments to determine its functional activities in RA.…”