The Research Of In Vitro Phosphorylation Of NDPK-A And Its Mutants By AMPK And Their Interaction Protein

Objective: Construct the eukaryotic expression vector of pMO-NDPKA wide type and a seriesof pMO-NDPKA mutants (C4S、C109S、C145S、C4/109/145S),in order to provide the basictools for NDPKA protein interaction investigation. Studied in vitro phosphorylation activityof wide type NDPK-A and mutants (S120and H118) by AMPKs,which AMPK-KD is akinase dead mutant and AMPK-CA is a consistent active mutant, that could lay a goodfoundation to reveal the phosphorylation sites of NDPKA and activation mechanism. Andexplore the possible interaction of NDPK-A、AMPK and CFTR,which could give a new insightinto NDPK-A’s biological functions. It suggested that NDPK-A participates in regulation ofchloride channel and ion transport of epithelial cells.Methods: Exploit the plasmid recombinant gene cloning technology to construct thepMO-NDPKA wild-type and mutant eukaryotic expression vector with V5labeled as well asthe transient transfection method to make these plasmids expressed in HEK293cells.Co-immunoprecipitation method combined with isotope-labeled was used to establish thephosphorylation assay in vitro in order for the study of the in vitro phosphorylation activitybetween AMPK and NDPK-A. And Co-immunoprecipitation, immunoprecipitation withGST-pulldown methods were preformed to explore the potential interaction among the CFTR,NDPK and AMPK.Results: The recombinant plasmid pMO-NDPKA WT and its mutant pMO-NDPKs(C4S、C109S、C145S、C4/109/145S) were constructed successfully, and these plasmids were allexpressed after transfected into HEK293cells. The wild-type NDPKA couldautophosphorylation and improve the phosphorylation activity with presence of the consistentactivation of AMPK(AMPK-CA), but the kinase dead AMPK(AMPK-KD)would notsignificantly enhance the WT-NDPKA autophosphorylation. And in the3mutants of S120, theS120A phosphorylation activity was slightly enhanced than WT, but there was no significantlydifference; The autophosphorylation activity of S120D/S120E was declined and H118F mutanthad no phosphorylation activity; AMPKalpha could combine with CFTR and NDPKA, butthere was no direct binding or interaction between CFTR and NDPKA.Conclusion: The autophosphorylation of NDPK-A happened on the site H118, that means, the autophosphorylation of NDPK-A depended on the activity of its histidine kinase. NDPK-Acould accept the activated phosphate from AMPK, thus to increase the autophosphorylation ofNDPK-A,but this enhancement depended on the activation of AMPK, only combining withAMPK could not increase the autophosphorylation of NDPK-A.Results of immunoprecipitationshowed that AMPKalpha can respectively interact with CFTR and NDPK-A. Maybe NDPK-Acould indirect control the activity of CFTR by AMPK,thus regulate the ion transport ofepithelial cells.