Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme, which in the inactive form consists of two regulatory (R) subunits and two catalytic (C) subunits. PKA is a key enzyme in cellular signal transmission systems and an important regulatory factor in the activation of T-lymphocytes. The diversity of cellular responses due to signalling through the cAMP-PKA system can be explained by the existence of multiple R and C isoforms and splicing variants that can form a PKA holoenzyme.An important aspect of cell signalling is subcellular localization of components in intracellular signalling pathways. Lipid areas, also called lipid rafts, are specific domains in the cell membrane. They work as unifying platforms for a variety of external stimuli that the cell is exposed to. Subcellular localization of PKA R and C isoforms may affect their role in regulating the process of T-cell activation.
In the present work our major objectives were to establish a method to investigate the localization and activity of PKA catalytic subunits Calphal, Cbeta2, and Cbeta4 as control in the cell membrane using transfected HEK 293T cells as a model system, and compare our results with findings in human T-lymphocytes. Moreover, we wanted to examine the effect of increased cAMP concentration on PKA Calphal, Cbeta2 and Cbeta4 as control, with special attention to their association and cAMP induced activity of those in the lipid rafts. We showed that a proportion of PKA Calphal and Cbeta2 catalytic units can be detected in lipid rafts in both cell model systems. There was also an enrichment of the neurospecific C subunit Cbeta4 that was transfected and used as control in the HEK 293T cells. Localization in the lipid rafts was confirmed by showing co-fractioning of C subunits with lipid-raft marker flotillin-1. We showed in addition that PKA Calphal, Cbeta2 and Cbeta4 units were catalytically active in the lipid rafts assayed by phosphorylation of the PKA-specific substrate Kemptide in vitro in both HEK 293T and T-cells. We also showed that the localization of PKA C subunits in lipid rafts was differentiated. Although all investigated C subunits could be detected in lipid raft-rich fractions, the relative proportion of active Cbeta2 subunits compared to proportion of active Calpha1 or Cbeta4 units was higher in the lipid raft fractions than in non-lipid raft fractions. Finally, we could demonstrate relatively higher cAMP induced increase in the activity of Cbeta2 compared with Calpha1 or Cbeta4 associated with lipid rafts.
We concluded that our method and our model systems are suited to detect catalytically active PKA C subunits associated with the lipid rafts, and can be useful as the basis for developing future studies that wish to describe and compare the regulation of processes that occur after the activation of human T-lymphocytes with different ligands or drugs.