title: High-constitutive HuR phosphorylation at Ser 318 by PKC{delta} propagates tumor relevant functions in colon carcinoma cells.
abstract: Overexpression of the messenger RNA (mRNA)-binding protein HuR is an important feature of many tumors and in most cases correlates with a high-grade malignancy. Since phosphorylation of HuR by protein kinase C δ (PKCδ) at serine (Ser) 318 implies an important mode in HuR regulation, we studied its functional role in dysregulated HuR and related functions in colon carcinoma cells. Coimmunoprecipitation experiments revealed a high-constitutive association of nuclear PKCδ with HuR. Using a phospho-Ser 318-specific HuR antibody, we found a strong increase in nuclear HuR phosphorylation in DLD-1 cells when compared with nontransformed CCD 841 colon epithelial cells. Importantly, a strong increase in HuR phosphorylation at Ser 318 was also found in tissue specimen from human colon carcinomas. Employing ribonucleoprotein-immunoprecipitation, we show that DLD-1 cells displayed a strong and constitutive RNA binding of HuR to cyclooxygenase-2 (COX-2) and cyclin A encoding mRNAs that was strongly impaired by rottlerin, an inhibitor of novel PKCs. Accordingly, rottlerin accelerated the decay of COX-2 and cyclin A encoding mRNAs concomitant with a reduced expression of both genes. Functionally, migration and invasion is similarly impaired in PKCδ- or HuR-small interfering RNA-depleted cells and in tumor cells transfected with a nonphosphorylatable serine-to-alanine 318 HuR construct. Conversely, expression of a phosphomimetic Ser 318 aspartic acid (D) HuR caused a significant increase in migration and proliferation of CCD 841 cells. Our data suggest that the increased HuR phosphorylation at Ser 318 by PKCδ reflects an important regulatory paradigm for aberrant HuR functions and emphasize the antitumorigenic potential of PKCδ inhibitory strategies.
authors.affiliation: pharmazentrum frankfurt/ZAFES and University of Frankfurt Medical School, Theodor-Stern-Kai 7, Frankfurt/Main, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Doller
authors.forename: Christine
authors.initials: C
authors.lastname: Winkler
authors.forename: Irina
authors.initials: I
authors.lastname: Azrilian
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schulz
authors.forename: Sylvia
authors.initials: S
authors.lastname: Hartmann
authors.forename: Josef
authors.initials: J
authors.lastname: Pfeilschifter
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eberhardt
mesh_terms: D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D003106:Colon / Q000378:metabolism / Q000473:pathology; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000954:Antigens, Surface; D019925:Cyclin A; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012694:Serine; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D051745:Protein Kinase C-delta