Transcriptional co-activator LEDGF interacts with Cdc7-activator of S-phase kinase (ASK) and stimulates its enzymatic activity.
Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK.
Division of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, United Kingdom and.
Siobhan
S
Hughes
Victoria
V
Jenkins
Mohd Jamal
MJ
Dar
Alan
A
Engelman
Peter
P
Cherepanov
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D064111:Minichromosome Maintenance Complex Component 2; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012694:Serine / Q000378:metabolism; D013499:Surface Properties; D015534:Trans-Activators / Q000378:metabolism*
D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; C119380:lens epithelium-derived growth factor; D012694:Serine; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2