Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation

Abstract

The regulation of adenosine kinase (AK) activity has the potential to control intracellular and interstitial adenosine (Ado) concentrations. In an effort to study the role of AK in Ado homeostasis in the central nervous system, two isoforms of the enzyme were cloned from a mouse brain cDNA library. Following overexpression in bacterial cells, the corresponding proteins were purified to homogeneity. Both isoforms were enzymatically active and found to possess K m and V max values in agreement with kinetic parameters described for other forms of AK. The distribution of AK in discrete brain regions and various peripheral tissues was defined. To investigate the possibility that AK activity is regulated by protein phosphorylation, a panel of protein kinases was screened for ability to phosphorylate recombinant mouse AK. Data from these in vitro phosphorylation studies suggest that AK is most likely not an efficient substrate for PKA, PKG, CaMKII, CK1, CK2, MAPK, Cdk1, or Cdk5. PKC was found to phosphorylate recombinant AK efficiently in vitro. Further analysis revealed, however, that this PKC-dependent phosphorylation occurred at one or more serine residues associated with the N-terminal affinity tag used for protein purification.

Adenosine Kinase: Exploitation for Therapeutic Gain

“…Two isoforms of ADK (ADK long or ADK-L, and ADK short or ADK-S) are present in mammalian cells (Juranka and Chan, 1985;Sahin et al, 1996Sahin et al, , 2004Sakowicz et al, 2001). Both isoforms are identical except for the amino acids encoded by their first exons (exon 1 and exon 1A) with exon 1A being located in the intron between exon 1 and 2.…”

“…The long isoform of ADK, ADK-L, contains 21 additional N-terminal amino acids (MAAAEEEPKPKKLKVEAPQAL in human ADK-L), which replace four N-terminal amino acids of the short isoform ADK-S (MTSV in human ADK). Both isoforms are enzymatically functional and show no obvious differences in their kinetic properties (Sakowicz et al, 2001;Sahin et al, 2004).…”

“…A screen of a panel of protein kinases for their ability to phosphorylate recombinant mouse ADK yielded negative results (Sahin et al, 2004). Accordingly, ADK is most likely not an efficient substrate for PKA, PKC, PKG, CaMKII, CK1, CK2, MAPK, Cdk1, or Cdk5 (Sahin et al, 2004).…”

Cdk5 Modulates Cocaine Reward, Motivation, and Striatal Neuron Excitability

“…Images were captured and analyzed using a scanning laser confocal microscope. Immunoblotting was performed as described previously (Sahin et al, 2004). Tissue homogenates were prepared from control and Cdk5 cKO mice, and equal amounts of total protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes.…”

Regulation of ΔFosB Stability by Phosphorylation

“…In the cell culture and brain slice phosphorylation studies, the values obtained for the 32 Plabeled protein were then divided by the values obtained for total ⌬FosB, and expressed as a ratio. In the in vitro phosphorylation studies, the amount of 32 P-labeled ⌬FosB per mole of ⌬FosB (stoichiometry) was calculated as described previously (Sahin et al, 2004). All measurements were taken within the linearity range of the instrument used.…”