Isoform Specificity of PKC Translocation in Living Aplysia Sensory Neurons and a Role for Ca 2 ϩ -Dependent PKC APL I in the Induction of Intermediate-Term Facilitation

Department of Psychiatry and Biobehavioral Science, University of California at Los Angeles, Los Angeles, California 90095-1606, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 09/2006; 26(34):8847-56. DOI: 10.1523/JNEUROSCI.1919-06.2006
Source: PubMed


Protein kinase Cs (PKCs) are important effectors of synaptic plasticity. In Aplysia, there are two major phorbol ester-activated PKCs, Ca2+-activated PKC Apl I and Ca2+-independent PKC Apl II. Functional Apl II, but not Apl I, in sensory neurons is required for a form of short-term facilitation induced at sensorimotor synapses by the facilitatory transmitter serotonin (5-HT). Because PKCs are activated by translocating from the cytoplasm to the membrane, we used fluorescently tagged PKCs to determine the isoform and cell-type specificity of translocation in living Aplysia neurons. In Sf9 cells, low levels of diacylglycerol translocate Apl II, but not Apl I, which requires calcium for translocation at low concentrations of diacylglycerol. Accordingly, application of 5-HT to Aplysia sensory neurons in the absence of neuronal firing translocates Apl II, but not Apl I, consistent with the role of Apl II in short-term facilitation. This translocation is observed in sensory neurons, but not in motor neurons. Apl I translocates only if 5-HT is coupled to firing in the sensory neuron; firing alone is ineffective. Because combined 5-HT and firing are required for the induction of one type of intermediate-term facilitation at these synapses, we asked whether this form of synaptic plasticity involves activation of Apl I. We report here that dominant-negative Apl I, but not Apl II, blocks intermediate-term facilitation. Thus, different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity: Ca2+-independent Apl II is involved in short-term facilitation, and Ca2+-dependent Apl I contributes to intermediate-term facilitation.

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Available from: Carole Abi Farah, Aug 20, 2014
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    • "To specifically test the role of PKM Apl III, we used the kinase-dead mRFP–PKM D392A as a dominant negative. Kinase-dead dominant negatives have been previously used to demonstrate specific roles for PKC Apl I and PKC Apl II (Manseau et al., 2001; Zhao et al., 2006; Lorenzetti et al., 2008; Hu et al., 2010). In motor neurons injected with the mRFP–PKM D392A, but not motor neurons injected with mRFP, the later phase of ITF seen after a 10 min application of 5-HT was reduced (Fig. 6). "
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    ABSTRACT: A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist in Aplysia californica despite the fact that inhibitors of PKMζ erase memory in Aplysia in a fashion similar to rodents. We have previously shown that, in Aplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron processes. Furthermore, a dominant-negative PKM Apl III expressed in the motor neuron blocked the late phase of intermediate-term facilitation in sensory-motor neuron cocultures induced by 10 min of serotonin. In summary, we provide evidence that PKC Apl III is cleaved into PKM Apl III during memory formation, that the requirements for cleavage are the same as the requirements for the plasticity, and that PKM in the motor neuron is required for intermediate-term facilitation.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2012; 32(42):14630-14640. DOI:10.1523/JNEUROSCI.3026-11.2012 · 6.34 Impact Factor
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    • "Thus, to examine phosphorylation of PKC Apl II at Ser36 during physiological activation of the kinase, we expressed eGFP-PKC Apl II or eGFP-PKC Apl II S36A in sensory neurons and examined phosphorylation at Ser36 following 5 min or 45 min 5HT treatment followed by fixation and immunocytochemistry using a phospho Ser36 antibody previously described (Pepio and Sossin 2001). We have previously shown that PKC Apl II translocates to the plasma membrane in response to 5HT, not to the cytoskeleton, in Aplysia sensory neurons (Zhao et al. 2006; Farah et al. 2008; Nagakura et al. 2010; Farah and Sossin 2011a). Both eGFP-PKC Apl II and eGFP-PKC Apl II S36A translocated significantly to the membrane in response to 5HT (Fig. 2a and c and data not shown). "
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    ABSTRACT: Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine-glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine-alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation-dependent regulation of translocation.
    Journal of Neurochemistry 08/2012; 123(3):360-72. DOI:10.1111/j.1471-4159.2012.07930.x · 4.28 Impact Factor
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    • "PKC-dependent reductions in AP output occurred within minutes of agonist application (Fig. 3, 7). While protein kinases can translocate upon activation (Zhao et al., 2006), this time scale limits the distance PKC could move before acting on AIS T-type channels. PKA has been shown to translocate from dendritic shaft to spine within seconds of PKA activation (Zhong et al., 2009), but longer distances (e.g., from nucleus to plasma membrane in HEK 293 cells, ~5 μm) require > 30 min (O'Flaherty et al., 2001). "
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    ABSTRACT: Action potentials initiate in the axon initial segment (AIS), a specialized compartment enriched with Na(+) and K(+) channels. Recently, we found that T- and R-type Ca(2+) channels are concentrated in the AIS, where they contribute to local subthreshold membrane depolarization and thereby influence action potential initiation. While periods of high-frequency activity can alter availability of AIS voltage-gated channels, mechanisms for long-term modulation of AIS channel function remain unknown. Here, we examined the regulatory pathways that control AIS Ca(2+) channel activity in brainstem interneurons. T-type Ca(2+) channels were downregulated by dopamine receptor activation acting via protein kinase C, which in turn reduced neuronal output. These effects occurred without altering AIS Na(+) or somatodendritic T-type channel activity and could be mediated by endogenous dopamine sources present in the auditory brainstem. This pathway represents a new mechanism to inhibit neurons by specifically regulating Ca(2+) channels directly involved in action potential initiation.
    Neuron 11/2010; 68(3):500-11. DOI:10.1016/j.neuron.2010.09.026 · 15.05 Impact Factor
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