PIPKIgamma ubiquitination in breast cancer cell migration and metastasis

Recent approval of bortezomib, a drug that inhibits the 26S proteasome, has underscored the importance of the ubiquitin proteasome pathway in cancer therapy. Protein ubiquitination has been implicated in focal adhesion dynamics and cell migration, key steps in cancer metastasis. However, the key ubiquitin pathways that govern this process remain to be identified. The long-term goal of this project is to identify novel ubiquitin pathways for cancer therapeutical approaches. The objective of this proposal is to characterize a novel ubiquitin pathway that regulates cell adhesion dynamics, cancer invasion and metastasis. HECTD1 is a 280 kDa HECT domain E3 ubiquitin ligase essential for cell migration. Our preliminary data shows that HECTD1 is highly expressed in breast cancer tissues and regulates FA dynamics. However, its substrates, cellular functions and regulatory mechanisms remain to be elucidated. Phosphatidylinositol 4 phosphate 5-kinase type I ã (PIPKIã), an enzyme that produces phosphatidylinositol 4,5-bisphosphate (PIP2), is essential for FA dynamics and cancer cell invasion. PIPKIã and the â integrin tail compete for the same binding site on talin, the activator of integrins. In preliminary studies we found that HECTD1 ubiquitinated PIPKIã ubiquitination leading to our hypothesis that HECTD1-mediated PIPKIã ubiquitination and subsequent degradation mediate FA assembly/disassembly, cancer cell migration and metastasis by modulating talin-â integrin interaction. We will investigate the roles of HECTD1-PIPKIã pathway in regulating FA dynamics, cancer cell migration and metastasis and dissect the downstream pathways of PIPKIã ubiquitination and the mechanism of HECTD1 regulation by addressing the following SPECIFIC AIMS: Aim 1. Define the role of PIPKIã ubiquitination by HECTD1 in regulating PIP2 dynamics, integrin activation and cancer metastasis. Our preliminary data showed that PIPKIã ubiquitination by HECTD1 regulated focal adhesion dynamics, cell migration and invasion. We hypothesize that PIPKIã ubiquitination and consequent degradation regulate PIP2 dynamics spatially and temporarily and are required for dynamical integrin activation and cancer metastasis. Aim 2. Dissect the molecular mechanisms by which PIPKIã ubiquitination regulates breast cancer cell migration and metastasis. PIPKIã strongly binds talin and competes with the â integrin tail for the same binding site on talin. Our preliminary data showed that the PIPKIã-talin interaction is responsible for the inhibition of PIPKIãK97R, a ubiquitination-resistant mutant, on focal adhesion dynamics and cell migration, and that PIPKIã ubiquitination by HECTD1 regulated PIP2 and PIP3 levels in breast cancer cells. We hypothesize that PIPKIã ubiquitination and consequent degradation allow the â integrin tail to bind talin, whereas reduction in PIP2 (caused by PIPKIã ubiquitination) facilitates the dissociation of talin with the integrin. Aim 3. Determine how HECTD1 is regulated by Akt1 during cell migration and metastasis. Our preliminary data demonstrated that HECTD1 is a substrate of Akt1, a key regulator of cell migration. We hypothesize that Akt1-mediated HECTD1 phosphorylation inhibits the activity of HECTD1, thus regulating focal adhesion dynamics and cell migration. We will dissect Akt-mediated HECTD1 phosphorylation and define its role in regulating focal adhesion dynamics, cell migration and invasion. Our findings will identify a novel pathway that accounts for a central mechanism for focal adhesion dynamics, cell migration and invasion, and will significantly advance our understanding of the molecular mechanisms of cancer cell migration and invasion. Although bortezomib was approved for myeloma treatment and several proteasome inhibitors are being evaluating in clinical trials, to minimize the potential side effects, one future direction should focus on developing inhibitors against specific ubiquitin ligases. Thus, HECTD1 could be a potential target for designing and screening therapeutic compounds. We have extensive experience in using advanced molecular, biochemical and imaging techniques to study protein phosphorylation and ubiquitination as well as cell migration. Our lab is a pioneer in studying E3 ubiquitin ligases in regulating focal adhesion dynamics and cell migration. We have state of art equipment (such as Nikon TIRF microscope and Biostaion) for live-cell focal adhesion and migration analysis. Our experience in studying large proteins (such as talin) will be invaluable to our study on HECTD1 (280 kDa). Thus, we are well-positioned to make progress on the proposed studies.