Protein kinase C stimulates HuD‐mediated mRNA stability and protein expression of neurotrophic factors and enhances dendritic maturation of hippocampal neurons in culture

Abstract

HuD protein is an RNA-binding protein involved in post-transcriptional regulation of gene expression for synaptogenesis, neuronal differentiation, and learning and memory, and is up-regulated and redistributed by a protein kinase C (PKC)-dependent pathway in neurons. Here, we show a PKC-regulated mechanism on HuD-mediated mRNA stability and expression of several neurotrophic factors (NTFs) in cultured hippocampal neurons. HuD pull-down assays showed that HuD is associated with brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin (NT)-3 mRNAs. Reduction of HuD expression with short hairpin RNAs decreased BDNF, NGF, and NT-3 mRNAs and NTFs expression. Bryostatin, a PKC activator, treatment enhanced their association with HuD and increased these transcripts' stability. Bryostatin induced HuD phosphorylation, which was inhibited by Ro 32-0432, a specific PKC inhibitor. Activated PKC specifically phosphorylated coactivator-associated arginine methyltransferase 1 (CARM1), which methylates HuD and negatively modulates HuD-mRNA interactions during neuronal differentiation, and inhibited its methyltransferase activity, resulting in decrease in CARM1-mediated HuD methylation. Furthermore cotreatment of bryostatin and AMI-1, a specific CARM1 inhibitor, potentiated PKC-dependent HuD-mRNA interactions and enhanced dendritic arborization. These results demonstrate that PKC may play an important role in neuronal differentiation and synaptogenesis via stimulating HuD-mediated mRNA stability and inhibiting CARM1 in hippocampal neurons.

Emerging complexity of the HuD/ELAVl4 gene; implications for neuronal development, function, and dysfunction

“…A recent report found a direct link between the antagonistic effects of PKC and CARM1 on HuD function and cell fate (Lim and Alkon 2012). In hippocampal neurons, the study demonstrated using pathway-specific inhibitors that activated PKC isoforms interact and phosphorylate CARM1, which leads to decreased CARM1 methyltransferase activity.…”

“…Numerous other ARE-harboring transcripts, such as N-myc and c-myc, were later identified as targets of HuD using similar in vitro approaches, indicating that HuD binds short-lived mRNAs whose protein product functions in cell proliferation (Liu et al 1995;Ross et al 1997). In addition to these transcripts, HuD has been shown to bind and regulate several other mRNAs in vitro and in vivo, many of which encode proteins with important functions in neuronal differentiation such as cell cycle arrest (p21 cip1/waf1 ) (Joseph et al 1998), neuroblast proliferation (MSI-1) (Ratti et al 2006), neuron migration (MARCKS) (Wein et al 2003), neurite extension ([GAP-43] , Tau [Aranda-Abreu et al 1999], and AChE [Cuadrado et al 2003;Deschenes-Furry et al 2006;), synapse formation (NOVA-1) (Ratti et al 2008), and neuronal growth and survival (NGF, BDNF, and NT-3) ( Table 2; Lim and Alkon 2012).…”

“…Currently, there are only three known pathways that regulate the abundance or function of HuD: protein kinase C (PKC) Pascale et al 2005;Lim and Alkon 2012); coactivatorassociated arginine methyltransferase 1 (CARM1) (Fujiwara et al 2006;Hubers et al 2010); and protein kinase B (PKB/ AKT) (Fujiwara et al 2011a). …”

“…Moreover, stimulation of PKCα promotes its interaction with nELAV proteins resulting in their phosphorylation at threonine residues, redistribution to the neuronal cytoskeletal and membrane fractions, and stabilization of GAP-43 mRNA. In addition, a recent study demonstrated that PKCε also interacts with HuD, and activation of both PKC isoforms by bryostatin-1 results in HuD phosphorylation on nine residues (Lim and Alkon 2012). Importantly, HuD is a focal downstream target of this neurite-extending pathway since nerve growth factor (NGF) or phorbol ester treatment of PC12 cells following HuD knockdown prevents neurite outgrowth .…”

Signaling pathways controlling activity-dependent local translation of BDNF and their localization in dendritic arbors

“…Translation of neuronal mRNAs localized in dendrites is controlled by signaling cascades activated by glutamate receptors and BDNF itself (Bramham and Wells, 2007;Leal et al, 2014). Translation of BDNF mRNA requires interaction with the binding protein HuD (also known as ELAVL4), which is mediated by a protein kinase C (PKC)-dependent pathway (Lim and Alkon, 2012), and phosphorylation of the eukaryotic elongation factor (eEF2) by the eEF2 kinase (eEF2K), two key pathways involved in synaptic plasticity (Verpelli et al, 2010). However, it is still unclear how the different signaling cascades controlling translation can regulate BDNF and where these signaling pathways are active in the different subcellular regions.…”