Phosphorylated ITIMs enable ubiquitylation of an inhibitory cell surface receptor.

Immune responses are modulated by activating and inhibitory receptors that traffic to and from the cell surface. Ligands that bind to inhibitory receptors induce phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails, followed by recruitment of inhibitory signaling molecules. Mechanisms that control the surface levels of inhibitory receptors are largely unexplored. Here, we show, using CD33/sialic acid-binding immunoglobulin-like lectin (Siglec)-3 as a paradigm, that ITIMs can bind to the ubiquitin ligase Cbl and that ITIMs are ubiquitylated following Src family kinase-mediated tyrosine phosphorylation. Ubiquitylation is a known signal for endocytosis. Accordingly, cells expressing CD33 mutants that cannot become ubiquitylated show significantly increased cell surface expression of CD33 and have impaired CD33 internalization, whereas in-frame fusion of ubiquitin to CD33 reverses this phenotype. Our results identify a novel function of ITIMs and demonstrate that phosphorylation-dependent ubiquitylation regulates cell surface expression and internalization, and thus possibly function, of CD33/Siglec-3, suggesting an important role of ubiquitin in endocytosis of ITIM-bearing inhibitory immunoreceptors.

c-Cbl regulates migration of v-Abl-transformed NIH 3T3 fibroblasts via Rac1.

Cellular events like cell adhesion and migration involve complex rearrangements of the actin cytoskeleton. We have previously shown that the multidomain adaptor protein c-Cbl facilitates actin cytoskeletal reorganizations that result in the adhesion of v-Abl-transformed NIH 3T3 fibroblasts. In this report, we demonstrate that c-Cbl also enhances migration of v-Abl-transformed NIH 3T3 fibroblasts. This effect of c-Cbl depends on its tyrosine phosphorylation, specifically on phosphorylation of its Tyr-731, which is required for binding of PI-3' kinase to c-Cbl. Furthermore, we demonstrate that the effect of c-Cbl on migration of v-Abl-transformed fibroblasts is mediated by active PI-3' kinase and the small GTPase Rac1. Our results also indicate that ubiquitin ligase activity of c-Cbl is required, while spatial localization of c-Cbl to the pseudopodia is not required for the observed effects of c-Cbl on cell migration.

The multidomain protooncogenic protein c-Cbl binds to tubulin and stabilizes microtubules.

The protooncogenic protein c-Cbl is known to regulate the actin cytoskeleton. In this study, we present results indicating that c-Cbl can also regulate the microtubular network. We have shown that c-Cbl binds to tubulin and microtubules through its tyrosine kinase binding (TKB) domain. However, the character of the interactions described in this report is novel, since the G306E mutation, which disrupts the ability of c-Cbl's TKB to bind to tyrosine-phosphorylated proteins, does not affect the observed interaction between c-Cbl and microtubules. Furthermore, overexpression of c-Cbl in human pulmonary artery endothelial cells and COS-7 cells leads to microtubule stabilization. We demonstrate that this effect of c-Cbl is mediated by TKB, and, like c-Cbl binding to microtubules, is independent of the ability of TKB to bind to tyrosine-phosphorylated proteins. Finally, we have shown that c-Cbl directly polymerizes microtubules in vitro, and that TKB is necessary and sufficient for this effect of c-Cbl. In this last phenomenon, as well as in the previous ones, the effect of TKB is not sensitive to the inactivating G306E mutation. Overall, the results presented in this report suggest a novel function for c-Cbl-microtubule binding and stabilization.

TULA: an SH3- and UBA-containing protein that binds to c-Cbl and ubiquitin.

Downregulation of protein tyrosine kinases is a major function of the multidomain protein c-Cbl. This effect of c-Cbl is critical for both negative regulation of normal physiological stimuli and suppression of cellular transformation. In spite of the apparent importance of these effects of c-Cbl, their own regulation is poorly understood. To search for possible novel regulators of c-Cbl, we purified a number of c-Cbl-associated proteins by affinity chromatography and identified them by mass spectrometry. Among them, we identified the UBA- and SH3-containing protein T-cell Ubiquitin LigAnd (TULA), which can also bind to ubiquitin. Functional studies in a model system based on co-expression of TULA, c-Cbl, and EGF receptor in 293T cells demonstrate that TULA is capable of inhibiting c-Cbl-mediated downregulation of EGF receptor. Furthermore, modulation of TULA concentration in Jurkat T-lymphoblastoid cells demonstrates that TULA upregulates the activity of both Zap kinase and NF-AT transcription factor. Therefore, our study indicates that TULA counters the inhibitory effect of c-Cbl on protein tyrosine kinases and, thus, may be involved in the regulation of biological effects of c-Cbl. Finally, our results suggest that TULA-mediated inhibition of the effects of c-Cbl on protein tyrosine kinases is caused by TULA-induced ubiquitylation and degradation of c-Cbl.