Phosphorylation and protein turnover regulate epithelial cell migration and membrane ruffling

Abstract

Cell migration is crucial for the development and maintenance of multi-cellular animal life. This complex, dynamic process must be coordinated properly, as disruption of mechanisms and signaling pathways that regulate migration contribute to diseases such as cancer. Cullin5-RING E3 ubiquitin ligase (CRL5) and substrate adaptors from the suppressors of cytokine signaling (SOCS) protein family serve to negatively regulate tyrosine phosphorylated proteins through ubiquitination and degradation. We have shown that Cul5, in combination with SOCS2, SOCS4, SOCS5, and SOCS6, inhibits epithelial cell transformation, including inhibition of migration and membrane ruffling. The turnover of focal adhesion (FA) adaptor protein, p130Cas (Cas), is regulated by CRL5-SOCS6 at the leading edge of migrating cells to inhibit FA turnover and membrane ruffling. However, overexpression of Cas alone does not stimulate migration, suggesting that turnover of other CRL5-SOCS substrates contribute to this mechanism. In an effort to characterize novel substrates of CRL5-SOCS, we identified breast cancer antiestrogen resistance 3 (BCAR3) as a candidate, as two BCAR3 phosphopeptides are increased in cells lacking functional CRL5. BCAR3 is a Cas binding partner in FAs and has been shown to regulate migration and invasion in breast cancer cell lines. However, little is known about the regulation of BCAR3. I found that the turnover of BCAR3 is regulated by CRL5-SOCS6, dependent on the proteasome. Additionally, CRL5-SOCS6-mediated regulation of BCAR3 is independent of Cas, as BCAR3-SOCS6 association occurs in the absence of Cas and under conditions where BCAR3 cannot bind Cas. Binding studies using BCAR3 and SOCS6 mutants show that BCAR3-SOCS6 association require a functional SOCS6 SH2 domain and BCAR3 Y117, suggesting that pY117-BCAR3 is bound by SOCS6 through the SOCS6 SH2 domain. Consistent with these data, BCAR3 Y117F protein levels are not regulated by CRL5. Next, I determined whether BCAR3 regulates migration in normal epithelial cells. While BCAR3 is important for individual cell migration and invasion towards epidermal growth factor (EGF) independent of CRL5, BCAR3 is required for the increased collective migration and membrane ruffling of Cul5-deficient cells in the absence of EGF. While I hypothesized that BCAR3 Y117F would be a gain-of-function mutant because it is not degraded through CRL5, BCAR3 Y117F is not functional. Expressing BCAR3 Y117F does not induce membrane ruffling, as wildtype BCAR3 does and expressing BCAR3 Y117F at normal levels in the absence of endogenous BCAR3 does not support the increased migration and membrane ruffling observed in Cul5-deficient cells. Collectively, these data suggest that BCAR3 Y117 is required for both degradation and function. In addition to Y117, I also found that the BCAR3 SH2 domain and Cas-binding sites are required for BCAR3 function in regulating migration and membrane ruffling. Interestingly, these three sites are all required to promote tyrosine phosphorylation of Cas at the leading edge of migrating cells, which is known to stimulate membrane ruffling. Taken together, these data identify and characterize novel mechanisms through which BCAR3 is regulated and through which BCAR3 regulates pY-Cas and collective migration. In addition to investigating BCAR3, I also studied FA protein exchange and FA structure and stability. FA proteins are dynamic and constantly associate with and disassociate from the adhesion. This protein exchange is rapid, with 40-80% of FA protein exchanging over a couple minutes and the remaining protein being considered immobile. Such rapid dynamics raise questions about how the adhesion maintains its structure and propagates downstream signals over long periods of time. I found that the population of FA protein previously considered immobile exchanges slowly with cytoplasmic protein over longer periods of time and hypothesize that this slow-exchanging population is actively regulated and participates in signaling. Collectively, my results establish novel regulatory mechanisms of FA proteins BCAR3 and Cas, FA structure and the leading edge of migrating cells.