Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation.
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.
Sahin
Bogachan
B
The University of Texas Southwestern Medical Center, Dallas, TX 75390-9070, USA.
Kansy
Janice W
JW
Nairn
Angus C
AC
Spychala
Jozef
J
Ealick
Steven E
SE
Fienberg
Allen A
AA
Greene
Robert W
RW
Bibb
James A
JA
D000241:Adenosine / Q000378:metabolism; D000248:Adenosine Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000502:physiology; D003001:Cloning, Molecular; D006706:Homeostasis; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014018:Tissue Distribution
D007527:Isoenzymes; D011994:Recombinant Proteins; D000248:Adenosine Kinase; D000241:Adenosine