title: PP2A phosphatase suppresses function of the mesenchymal invasion regulator NEDD9.
abstract: 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.
authors: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, NSW, 2145 Australia.
authors: Peta
authors: P
authors: Bradbury
authors: Maha
authors: M
authors: Mahmassani
authors: Jessie
authors: J
authors: Zhong
authors: Kylie
authors: K
authors: Turner
authors: Andre
authors: A
authors: Paul
authors: Nicole M
authors: NM
authors: Verrills
authors: Geraldine M
authors: GM
authors: O'Neill
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000378:metabolism; D008387:Marine Toxins; D008648:Mesoderm / Q000166:cytology / Q000502:physiology*; D010080:Oxazoles / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008387:Marine Toxins; C099942:NEDD9 protein, human; D010080:Oxazoles; D010750:Phosphoproteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C059041:calyculin A; D054648:Protein Phosphatase 2; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde