Cell Biology

Sustained by Degradation

Science Signaling  19 Apr 2011:
Vol. 4, Issue 169, pp. ec111
DOI: 10.1126/scisignal.4169ec111

Binding of cAMP (cyclic adenosine monophosphate) to the regulatory (R) subunit of PKA (protein kinase A) releases the active catalytic subunit (PKAc). One way in which specificity of PKA phosphorylation is achieved is by the tethering of the regulatory subunit to A-kinase anchoring proteins (AKAPs), which bind to different sets of substrates. Lignitto et al. performed a yeast two-hybrid screen of a rat cDNA library with mouse RIIα as bait and identified the RING domain–containing E3 ubiquitin ligase praja as a binding partner. Transfected praja2 interacted with endogenous RIIα or RIIβ (RIIα/β) in human embryonic kidney (HEK) 293 cells. Immunocytochemistry indicated that endogenous praja2 and RIIβ partially colocalized at postsynaptic sites in primary hippocampal neurons. In transfected HEK293 cells, praja2 ubiquitinated endogenous RIIα/β, an effect that was enhanced by pharmacological manipulations (such as forskolin treatment) that increase cAMP concentrations. Activation of PKA by cAMP induced binding of praja2 to PKAc and phosphorylation of praja2 by PKAc, which in turn enhanced binding of praja2 to the PKA holoenzyme. PKAc phosphorylates and activates the transcription factor CREB (cAMP response element–binding protein), and in human neuroblastoma SHSY cells, depletion of endogenous praja2 by siRNA decreased phosphorylation of CREB in response to forskolin and reduced the abundance of c-fos, a transcriptional target of CREB. These latter effects were rescued by expression of wild-type but not a catalytically inactive form of praja2. Intracerebral infusion of praja2 siRNA into rats attenuated CREB phosphorylation, c-fos transcription, and the degree of PKA-dependent long-term potentiation. Thus, by promoting degradation of the regulatory subunit of PKA, praja2 increases PKA signaling.

L. Lignitto, A. Carlucci, M. Sepe, E. Stefan, O. Cuomo, R. Nisticò, A. Scorziello, C. Savoia, C. Garbi, L. Annunziato, A. Feliciello, Control of PKA stability and signalling by the RING ligase praja2. Nat. Cell Biol. 13, 412–422 (2011). [PubMed]