Xiaotang Fan

Characterization of an RNA Granule from Developing Brain

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding β-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.

5-HT stimulates eEF2 dephosphorylation in a rapamycin-sensitive manner in Aplysia neurites

In Aplysia, serotonin mediates behavioral sensitization by increasing the strength of the synapse between sensory and motor neurons, a process known as facilitation. The retention of long‐term facilitation is blocked by rapamycin, an inhibitor of a specific translational pathway. One possible rapamycin‐sensitive target is the increased translation of 5′‐terminal oligopyrimidine mRNAs. These transcripts encode components of the translational machinery and have been proposed to be important for retention of long‐term facilitation. We have cloned the 5′‐terminal oligopyrimidine mRNA encoding eukaryotic elongation factor 2 and shown that serotonin increased its translation in synaptosomes. Another possible rapamycin‐sensitive target is the inactivation of eukaryotic elongation factor 2 kinase. Eukaryotic elongation factor 2 kinase phosphorylates and inactivates eukaryotic elongation factor 2, blocking translational elongation. Serotonin application decreased eukaryotic elongation factor 2 phosphorylation in synaptosomes and in isolated neurites, and this was blocked by rapamycin. We propose a role for the rapamycin‐sensitive pathway in neurons. Stimulation blocks translation by inducing calcium entry and phosphorylation of eukaryotic elongation factor 2. This block is reversed through activation of the rapamycin‐sensitive system and dephosphorylation of eukaryotic elongation factor 2.

Serotonin-Induced Cleavage of the Atypical Protein Kinase C Apl III in Aplysia

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.

Compartment‐specific, differential regulation of eukaryotic elongation factor 2 and its kinase within Aplysia sensory neurons

J. Neurochem. (2011) 117, 841–855.

Protein Kinase C Phosphorylated at a Conserved Threonine Is Retained in the Cytoplasm

Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 inAplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation.

The role of C2 domains in Ca2+-activated and Ca2+-independent protein kinase Cs in aplysia.

In the nervous system of the marine molluskAplysia there are two protein kinase C (PKC) isoforms, the Ca2+-activated PKC Apl I and the Ca2+-independent PKC Apl II. PKC Apl I, but not PKC Apl II is activated by a short-term application of the neurotransmitter serotonin. This may be explained by the fact that purified PKC Apl II requires a higher mole percentage of phosphatidylserine to stimulate enzyme activity than does PKC Apl I. In order to understand the molecular basis for this difference, we have compared the ability of lipids to interact with the purified kinases and with regulatory domain fusion proteins derived from the kinases using a variety of assays including kinase activity, phorbol dibutyrate binding, and liposome binding. We found that a C2 domain fusion protein derived from PKC Apl I binds to lipids constitutively, while a C2 domain fusion protein derived from PKC Apl II does not. In contrast, fusion proteins containing the C1 domains of PKC Apl I and PKC Apl II showed only small differences in lipid interactions. Thus, while the presence of a C2 domain assists lipid-mediated activation of PKC Apl I, it inhibits activation of PKC Apl II.

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, PKCε, 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.

Novel calpain families and novel mechanisms for calpain regulation in Aplysia

Calpains are a family of intracellular proteases defined by a conserved protease domain. In the marine mollusk Aplysia californica, calpains are important for the induction of long-term synaptic plasticity and memory, at least in part by cleaving protein kinase Cs (PKCs) into constitutively active kinases, termed protein kinase Ms (PKMs). We identify 14 genes encoding calpains in Aplysia using bioinformatics, including at least one member of each of the four major calpain families into which metazoan calpains are generally classified, as well as additional truncated and atypical calpains. Six classical calpains containing a penta-EF-hand (PEF) domain are present in Aplysia. Phylogenetic analysis determined that these six calpains come from three separate classical calpain families. One of the classical calpains in Aplysia, AplCCal1, has been implicated in plasticity. We identify three splice cassettes and an alternative transcriptional start site in AplCCal1. We characterize several of the possible isoforms of AplCCal1 in vitro, and demonstrate that AplCCal1 can cleave PKCs into PKMs in a calcium-dependent manner in vitro. We also find that AplCCal1 has a novel mechanism of auto-inactivation through N-terminal cleavage that is modulated through its alternative transcriptional start site.

The Ca V 2α1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of Ca V 2 channels

The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVβ, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2α1, CaVβ, and CaVα2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.