Identification of AKAP9 as a cCMP- and cUMP-binding protein using cCMP and cUMP coupled to agarose and biotin matrices

The biological roles of cAMP and cGMP are well understood. They regulate many intracellular processes like cell growth and differentiation. Cytosine 3’,5’-cyclic monophosphate (cCMP) and uridine 3’,5’-cyclic monophosphate (cUMP) fulfill the criteria for second messengers. Previously, PKARIa, PKARIIa and PKG were identified as a cCMP- and cUMP-binding proteins. AKAPs are scaffold proteins which bind to the regulatory subunits of PKA and other signaling proteins. Here, we show the identification of Yotiao, the smallest splice variant of AKAP9, as a cCMP- and cUMP-binding protein.

Extracts of A549, HeLa cells and mouse lung tissue were generated and incubated with cUMP or cCMP attached to agarose matrices or linked to biotin. cCMP or cUMP were added to compete for protein binding. Bound proteins were separated by SDS-PAGE or digested with trypsin. Further binding proteins were identified by LC-MS and data base searches. 293 cells were transiently transfected with myc-tagged Yotiao cDNA. Western blots were used to verify the protein binding.

Binding of PKARIa, PKARIIa and PKG to cCMP and cUMP were verified using agarose and biotin matrices. Various AKAP9 isoforms were identified as cCMP- and cUMP-binding proteins. Binding of Yotiao to cCMP as well to cUMP was confirmed by western blotting in transiently transfected 293 cells. Successful competition with cCMP and cUMP confirmed the specificity of the binding to Yotiao.

Affinity chromatography coupled with LC-MS constitutes a feasible approach to identify cCMP- and cUMP-binding proteins. cCMP and cUMP agarose and biotin matrices are suitable for identifying new – currently unknown – binding proteins.