Nucleoside diphosphate kinase (NDPK) is involved in multiple cellular processes including development, G-protein regulation, cellular energy production and K⁺ channel regulation. Previous findings in our laboratory identified NDPK as a member of a [Na⁺] and [K⁺]-regulated histidine phosphorylation cascade in ovine tracheal membranes (Marshall et al. 1999). This ion-dependent regulation was shown to be defective in mice, which are null for the CFTR protein (Riemen et al. 1998). However, the methods by which NDPK activity is regulated are poorly understood. Here we describe a novel interaction between NDPK and the adenosine monophosphate activated kinase (AMPK) that may be regulatory towards NDPK activity.

Data presented previously (Best et al. 2000) using cAMP-affinity purification and overlay assays identified a 62 kDa protein that interacts with both cAMP and NDPK. Preliminary data also suggested that NDPK autophosphorylation (with γ[³²P] ATP as nucleotide donor) is augmented in the presence of the 62 kDa protein. Literature searches identified the α-subunit of the hetero-trimeric metabolic sensor kinase, AMPK, as a possible candidate. Immunoprecipitation (IP) using antibodies against AMPK (a gift from Professor G. Hardie, Dundee and D. Carling, Hammersmith) demonstrated that NDPK and the α/β-subunits of AMPK co-precipitate from ovine tracheal membranes.

Findings by Hallows et al. (2000) showed that the α-subunit of AMPK binds to CFTR. Preliminary data suggest that NDPK and CFTR also co-IP in membranes from ovine tracheal epithelium. We conclude that CFTR, a nucleotide-gated ion channel, may associate with a nucleotide generator (NDPK) and a mononucleotide activated protein kinase (AMPK). Our data suggest a novel mechanism by which the transfer of phosphate to NDPK is controlled by a second kinase and provides the first evidence of NDPK regulation.