Anjali M. Teckchandani

Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here we report that mammary epithelial cells require Cullin 5 RING E3 ubiquitin ligase complexes (Cul5-CRLs) to prevent transformation by a Src-Cas signaling pathway. Removing Cul5 stimulates growth factor-independent growth and migration, membrane dynamics, and colony dysmorphogenesis, dependent on the endogenous tyrosine kinase, Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate, p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either SOCS6 knockdown or a degradation-resistant Cas mutant stimulates membrane ruffling but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibiting Src-Cas-induced ruffling through SOCS6.

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain–binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.

Clathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins β1, α1, α2, and α3 but not α5 or αv chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin β1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin β1 and vinculin to the leading edge. By manipulating intracellular and surface integrin β1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.

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.

Oncogene is one of the world’s leading cancer journals. It is published weekly and covers all aspects of the structure and function of Oncogenes. Oncogene also publishes 8 Reviews issues a year, on a broad range of topics.

Oncogene is one of the world’s leading cancer journals. It is published weekly and covers all aspects of the structure and function of Oncogenes. Oncogene also publishes 8 Reviews issues a year, on a broad range of topics.