Regulatory subunits of cAMP-dependent protein kinases are degraded after conjugation to ubiquitin: a molecular mechanism underlying long-term synaptic plasticity.

Abstract

In Aplysia, behavioral sensitization of defensive reflexes and the underlying presynaptic facilitation of sensory-to-motor neuron synapses lasts for several minutes (short term) or days to weeks (long term). Short-term sensitization has been explained by modulation ofion-channel function through cAMP-dependent protein phosphorylation. Long-term facilitation requires additional molecular changes including protein synthesis. A key event is the persistent activation of the cAMP-dependent protein kinase at baseline concentrations of cAMP. This activation is due to selective loss of regulatory (R) subunits ofPKA without any change in catalytic (C) subunits. To understand the molecular mechanisms that produce the loss of R subunits in long-term facilitation, we investigated how R subunits are degraded in extracts of Aplysia nervous tissue and in rabbit reticulocyte lysates. Degradation ofAplysia R subunits requires ATP, ubiquitin, and a particulate component that appears to be the proteasome complex. Degradation is blocked by hemin, which causes the accumulation of high molecular weight derivatives of R subunits that are likely to be ubiquitin conJugates of R subunits and intermediates in the degradative pathway. We also show that vertebrate RI and RI, subunits can be degraded through the ubiquitin pathway. We suggest that degradation is initiated by cAMP, which causes the holoenzyme to dissociate and, further, that the altered R-to-C ratio inAplysia sensory neurons is maintained in long-term facilitation by newly synthesized proteins that help target R subunits for accelerated degradation.In sensory-to-motor neuron synapses in Aplysia, cAMPdependent protein kinase (PKA) plays an important role in the presynaptic facilitation that underlies both short-term (1, 2) and long-term (3, 4) sensitization of defensive reflexes. In short-term facilitation, PKA modulates the function of ion channels directly (2,5). Several additional changes are needed to produce long-term facilitation, however, including protein synthesis (6,7). A key molecular event is the persistent activation of PKA in sensory neurons at basal concentrations of cAMP. Greenberg et al. (8) and Bergold et al. (9) showed that the amounts of regulatory (R) subunits of PKA are diminished in trained animals or after treatments that produce long-term facilitation in isolated ganglia. Since the amount of catalytic (C) subunits is not altered under these conditions, the loss of R subunits should lead to enhanced protein phosphorylation for prolonged periods (8,9). Earlier studies suggested that loss of R subunits results from accelerated proteolysis rather than from reduced synthesis (8-10). We therefore examined how R protein is degraded in Aplysia neurons.

Drosophila Orb2 targets genes involved in neuronal growth, synapse formation, and protein turnover

“…On the other hand, activation of the proteosomal pathway and proteases might remove inhibitory constraints at the synapse to create and maintain a permissive environment for synaptic growth. The involvement of Uch and ubiquitin-mediated protein-turnover pathways in synaptic plasticity has previously been observed in Aplysia and in mice (24)(25)(26)(27)(28). Because ubiquitination can act as a posttranslational modification in addition to a signal for degradation, the ubiquitination pathway can, in principle, modulate stability, function, or membrane distribution of proteins at the activated synapse.…”

The ubiquitin-proteasome pathway and synaptic plasticity

“…The first discovery of ubiquitin-proteasome-mediated degradation of a substrate relevant to synaptic plasticity in the nervous system was that of R subunits of PKA (Hegde et al 1993). Since then several substrates of the UPP in the nervous system have been identified (Hegde 2010).…”

“…What is the mechanism of R subunit degradation? Hegde et al (1993) found through a series of biochemical experiments that R subunits were substrates for ubiquitination and proteasome-mediated degradation. Moreover, a UCH ([Ap-uch] Aplysia ubiquitin C-terminal hydrolase ) that interacts with the proteasome was found to be induced by serotonin, the neurotransmitter that induces LTF.…”

Critical role for protein kinase A in the acquisition of gregarious behavior in the desert locust

“…Degradation of the R subunits can then result in sustained PKA activity that outlasts the cAMP signal (44). RNAi against PKAR1A has recently been found to increase basal and stimulated PKA activity in HEK293 cells (45).…”