PP2A phosphatase suppresses function of the mesenchymal invasion regulator NEDD9.

The mesenchymal mode of cancer cell invasion characterized by active adhesion turnover and a polarized actin cytoskeleton, is critically regulated by the adaptor protein NEDD9/HEF1/Cas-L. While it is known that NEDD9 is subject to extensive phosphorylation modification, the molecules that determine NEDD9 phosphorylation to stimulate adhesion turnover and mesenchymal cell morphologies are currently unknown. Earlier studies have suggested that the serine/threonine phosphatase PP2A regulates interconversion between a low molecular mass NEDD9 phosphoform and higher molecular mass phosphoforms. However, previous studies have used chemical inhibitors to block PP2A activity. In the present study we therefore aimed to specifically inhibit PP2A activity via siRNA and dominant negative approaches to investigate the effect of PP2A on interconversion between 115 kDa and 105 kDa NEDD9 and determine the functional consequence of PP2A activity for NEDD9 function. Strikingly, we find that while the phosphatase inhibitor Calyculin A indeed abrogates detachment-induced dephosphorylation of the 115 kDa NEDD9 phosphoform, PP2A depletion does not inhibit 115 kDa to 105 kDa interconversion. Our data suggest instead that PP2A targets discrete NEDD9 phosphorylation modifications separate to the events that mediate interconversion between the two forms. Functionally, PP2A depletion increases NEDD9 mediated cell spreading and mutation of S369 in the serine-rich region of NEDD9 to aspartate mimics this effect. Importantly, mutation of S369 to alanine abrogates the ability of dominant negative PP2A to increase NEDD9-mediated cell spreading. Collectively, our data reveal that the tumour suppressor PP2A may act via S369 to regulated NEDD9-mediated cell spreading.

Estradiol stabilizes the 105-kDa phospho-form of the adhesion docking protein NEDD9 and suppresses NEDD9-dependent cell spreading in breast cancer cells.

Recent data suggest that the adhesion docking protein NEDD9/HEF1/Cas-L is a critical regulator of adhesion-dependent signalling pathways during mammary tumour development. Multiple phosphorylation modifications of NEDD9 regulate interaction with downstream protein partners, thus the regulation of NEDD9 phospho-forms is an important point of control for NEDD9 function. As estradiol (E2) plays a central role in the development and progression of breast cancer, we have investigated NEDD9 phospho-form regulation in MCF-7 estrogen receptor (ER)-positive breast cancer cells in response to estrogen. We find that levels of the 105-kDa NEDD9 phospho-form are significantly increased after 3days of estrogen exposure, and this is suppressed by the anti-estrogen tamoxifen. Analysis of protein decay kinetics following treatment with the protein synthesis inhibitor cycloheximide indicates that increased 105-kDa levels are due to a slower rate of protein decay. Moreover, exogenous expression of NEDD9 failed to induce spreading in the presence of E2, and this was reversed by tamoxifen treatment. Finally, we show that the 105-kDa NEDD9 phospho-form appears to predominate in ER-positive versus ER-negative breast cancer cell lines. Taken together, our results suggest that estradiol may suppress phospho-form-specific functions of NEDD9.

Tropomyosin isoform expression regulates the transition of adhesions to determine cell speed and direction.

The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.