title: Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation.
abstract: Human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. HEF1 is present at focal adhesions and is involved in integrin signalling mediating cytoskeleton reorganization associated with cell migration, adhesion or apoptosis. HEF1 functions are regulated in part by phosphorylation on tyrosine residues. HEF1 is also phosphorylated on serines/threonines leading to two isoforms refered to as p105 and p115. In most cases, the serine/threonine kinase(s) responsible for HEF1 phosphorylation have not been identified. In the present study, we have investigated HEF1 ser/thr phosphorylation. In the HCT-116 cell line transiently overexpressing Flag-HEF1 we showed that Hesperadin, a synthetic indolinone displaying antiproliferative effect and described as an inhibitor of various kinases including Aurora-B, prevented HEF1 phosphorylation induced by the ser/thr phosphatase PP2A inhibitor: okadaic acid (OA). In addition we showed that conversion of endogenous HEF1 p105 to p115 in HaCaT cells was prevented upon treatment with Hesperadin, resulting in accumulation of p105HEF1. We also identified serine 369 as the target site of phosphorylation by this Hesperadin-inhibited kinase in HCT-116. Finally, we provide evidence that phosphorylation on serine 369 but not phosphorylation on serine 296, triggers HEF1 degradation by the proteasomal machinery. These data suggest that conversion of p105 to p115 results from a ser-369-dependent phosphorylation mediated by an Hesperadin-sensitive kinase and regulates the half-life of HEF1.
authors.affiliation: INSERM U749, Université Paris-sud 11, Faculté de Pharmacie, 5 rue JB Clement, 92296 Chatenay-Malabry, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Hivert
authors.forename: Josiane
authors.initials: J
authors.lastname: Pierre
authors.forename: Joël
authors.initials: J
authors.lastname: Raingeaud
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; D007211:Indoles; C099942:NEDD9 protein, human; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013449:Sulfonamides; D019319:Okadaic Acid; D012694:Serine; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D046988:Proteasome Endopeptidase Complex; C474723:hesperadin