Welcome to the Cooper Lab

Signal transduction

We investigate the roles of two types of post-translational modification, protein-tyrosine phosphorylation and ubiquitination, in cell and developmental biology. We have found that protein tyrosine-phosphorylation and phosphotyrosine-directed ubiquitination regulate cell proliferation, transformation and migration in normal and cancer cells. We use the tools of molecular, cellular and developmental biology to understand the mechanisms involved.

Phosphoproteins can be inactivated by protein phosphatases or targeted for ubiquitination and degradation by phospho-specific ubiquitin ligases. In the case of phosphoproteins containing phosphotyrosine, Cullin5-RING ligases (CRL5s) target specific phosphotyrosine proteins for polyubiquitination and degradation. Substrate-recognition (adaptor) components of CRL5, known as SOCS proteins, determine which phosphoproteins are ubiquitinated. While dephosphorylation can be followed by re-phosphorylation, proteasomal destruction is irreversible. Thus protein phosphatases set the steady state level of phosphorylation but CRL5s set the phosphoprotein half-life, creating a negative feedback loop. This has important implications for the spatiotemporal dynamics of signaling and the cellular response. Complete exhaustion of a signaling protein may create a refractory period during which re-stimulation is not possible, and local depletion within a specific subcellular region may restrict the response to a small part of the cell.

Currently we study two systems:



Immature neurons migrate during mammalian brain development to create layers of neurons with similar functions. Some migrations are regulated by external signals provided by a secreted protein, Reelin. Reelin signals are transduced by specific receptors, Src kinases, and an adaptor protein, Dab1. Dab1 phosphorylation stimulates various small GTPases, including Rap1A, Ral, Rac and Cdc24, and protein kinases including PKB/Akt, and promotes cell polarization and migration. Altered trafficking of N cadherin is required for the correct cell responses. We are investigating how Rap1A and Ral regulate N cadherin traffic and how N cadherin traffic causes cell polarization. Reelin-Dab1 signaling is down-regulated when activated Dab1 is ubiquitinated and degraded, dependent on SOCS-CRL5. This down-regulation blocks transduction of the Reelin signal and allows cells to stop moving and complete differentiation. We are studying how the regulated expression of different SOCS genes during development modulates the strength of Reelin-Dab1 signaling at different stages of neuron migration.

Epithelial cells can either migrate as sheets of interconnected cells or undergo the epithelial-mesenchymal transition and migrate individually. We found that sheet migration is stimulated by removal of CRL5. Increased activity of the tyrosine kinase Src in CRL5-deficient cells is necessary but not sufficient for the increase in migration. This suggests that proteins that are phosphorylated by Src and ubiquitinated by CRL5 may stimulate migration and proliferation. Our evidence shows that several such Src/CRL5 substrates combine to stimulate migration. Detailed study of one substrate shows that a SOCS-CRL5 complex relocalizes within the cell to locally inactivate the substrate during cell migration. We are working to understand the signals for SOCS-CRL5 relocalization and how localized substrate ubiquitination regulates cytoskeletal dynamics. We have also found that CRL5 inhibits cell proliferation, and are identifying the CRL5 substrates and molecular mechanisms.

Time lapse movie of cortical neurons in slice culture. Neurons were labeled three days previously by in utero electroporation. Many cells express a red fluorescent nuclear marker and a subset expresses a green actin marker. Neurons that have finished migrating appear to move downwards because the top of the slice was fixed during the movie. Note the protrusive activity. (Susumu Antoku)