title: Regulation of urokinase receptor expression by phosphoglycerate kinase is independent of its catalytic activity.
abstract: Posttranscriptional regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA involves the interaction of a uPAR mRNA coding region sequence with phosphoglycerate kinase (PGK), a 50-kDa uPAR mRNA binding protein. PGK catalyzes a reversible transfer of a phosphoryl group from 1,3-biphosphoglycerate to ADP in the glycolytic pathway. Our previous studies showed that overexpression of PGK in uPAR-overproducing H157 lung carcinoma cells results in decreased cytoplasmic uPAR mRNA and cell surface uPAR protein expression through destabilization of the mRNA. In order to determine the role of PGK enzymatic activity on uPAR mRNA stability we mutated PGK by changing amino acid P204H and amino acid D219A. The mutant proteins were expressed in Epicurian coli BL21 cells, and the purified proteins were analyzed for PGK activity. We found that mutation of amino acid P204H and D219A reduced PGK activity by 99 and 83%, respectively. By gel mobility shift and Northwestern assay, we found that the mutant proteins were able to bind to uPAR mRNA as effectively as wild-type PGK. Overexpression of mutant, inactive PGK in H157 cells reduced cell surface uPAR protein as well as uPAR mRNA expression. Run-on transcription analysis indicated that overexpression of mutant PGKs fails to alter the rate of synthesis of uPAR mRNA, whereas transcription chase experiments demonstrated that both mutants and wild-type PGK reduce the stability of the uPAR mRNA transcripts to a similar extent. Overexpression of mutant PGK also inhibited the rate of DNA synthesis and the invasion-migration ratio. These results demonstrate that uPAR mRNA binding activity as well as PGK-mediated regulation of uPAR mRNA are independent of PGK enzymatic activity.
authors.affiliation: Department of Medicine, University of Texas Health Center, Tyler, TX 75708, USA. sreerama.shetty@uthct.edu
authors.forename: Sreerama
authors.initials: S
authors.lastname: Shetty
authors.forename: Malathesha
authors.initials: M
authors.lastname: Ganachari
authors.forename: Ming-Cheh
authors.initials: MC
authors.lastname: Liu
authors.forename: Ali
authors.initials: A
authors.lastname: Azghani
authors.forename: Harish
authors.initials: H
authors.lastname: Muniyappa
authors.forename: Steven
authors.initials: S
authors.lastname: Idell
mesh_terms: D002294:Carcinoma, Squamous Cell; D002384:Catalysis; D002455:Cell Division; D045744:Cell Line, Tumor; D002465:Cell Movement; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator
chemical_list: C524898:PLAUR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010735:Phosphoglycerate Kinase