Physiological Role for Phosphatidic Acid in the Translocation of the Novel Protein Kinase C Apl II in Aplysia Neurons

Abstract

In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKC, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.Protein kinase Cs (PKCs) are a family of lipid-activated kinases that mediate a wide variety of cellular processes, including the regulation of synaptic strength in the nervous system (23,42,48). In Aplysia californica, behavioral sensitization is mediated in part by an increase in the strength of the connections between mechanoreceptor sensory neurons and motor neurons (19). This increase, called synaptic facilitation, is mediated by the neurotransmitter serotonin (5HT), which induces facilitation in isolated ganglia as well as in cocultures of sensory neurons and motor neurons (5, 6). Sensitizing stimulation causes the translocation of PKC to neuronal membranes (39, 53). In the Aplysia nervous system, there are two phorbol ester-regulated PKCs: PKC Apl I, which is homologous to the Ca 2ϩ -activated PKC family in vertebrates (␣, ␤1, ␤2, and ␥) that are called conventional or classical PKCs (cPKCs), and PKC Apl II, which is homologous to the Ca 2ϩ -independent epsilon family of PKC in vertebrates (ε and ) that are called novel PKCs (nPKCs) (21,42,43). PKC Apl I and PKC Apl II translocate under different conditions to mediate distinct types of synaptic facilitation (53). PKC Apl II, but not PKC Apl I, is translocated by the application of 5HT to sensory neurons and is required for 5HT-mediated facilitation at synapses that previously have been depressed (24). In contrast, combining 5HT and the firing of the sensory neuron leads to the additional translocation of PKC Apl I, and PKC Apl I is required for the intermediate-term facilitation induced by the combination of sensory neuron firing and 5HT (53).Both cPKCs and nPKCs contain two C1 domains and one C2 domain. However, the C2 domain of nPKCs is located N terminal to the C1 domains and lacks critical aspartic acid residues involved in coordinating Ca 2ϩ ions in cPKCs (2...

PKC Differentially Translocates during Spaced and Massed Training inAplysia

“…Sensory neuron cultures were prepared following published procedures (Zhao et al, 2006;Farah et al, 2008).…”

“…The pNEX3 enhanced green fluorescent protein (eGFP) PKC Apl II has been described previously, as has the injection procedure for expression in sensory neurons (Manseau et al, 2001;Zhao et al, 2006;Farah et al, 2008).…”

“…Live cell imaging was done as described previously (Farah et al, 2008). For spaced training, neurons received five applications of 10 M 5HT (5 min each) at an intertrial interval of 20 min.…”

“…The time series was analyzed using NIH Image J software as previously described (Zhao et al, 2006;Farah et al, 2008). The effect of 5HT was determined by comparing the degree of membrane association (I m /I c ) ratio before and after 5HT (Post/Pre); the fold change is the translocation ratio.…”

“…To monitor PKC activation, we followed the translocation of eGFP-tagged PKC Apl II from the cytoplasm to the plasma membrane in cultured Aplysia sensory neurons (Zhao et al, 2006;Farah et al, 2008). The activity of eGFP-PKC Apl II is indistinguishable from the activity of nontagged PKC Apl II and the level of overexpression under these conditions is modest (Manseau et al, 2001).…”