Open AccessMolecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation
Author(s) -
Sahin Bogachan,
Kansy Janice W.,
Nairn Angus C.,
Spychala Jozef,
Ealick Steven E.,
Fienberg Allen A.,
Greene Robert W.,
Bibb James A.
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04291.x
Subject(s) - adenosine kinase , phosphorylation , kinase , recombinant dna , gene isoform , protein kinase c , serine , protein kinase a , adenosine , biology , protein phosphorylation , biochemistry , microbiology and biotechnology , gene , adenosine deaminase
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.