Protein kinase C stimulates HuD-mediated mRNA stability and protein expression of neurotrophic factors and enhances dendritic maturation of hippocampal neurons in culture.
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.
Blanchette Rockefeller Neurosciences Institute at West Virginia University, Morgantown, WV 26505, USA. clim@brni.org
Chol Seung
CS
Lim
Daniel L
DL
Alkon
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D051959:ELAV Proteins / Q000378:metabolism*; D000067779:ELAV-Like Protein 4; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006624:Hippocampus / Q000378:metabolism / Q000648:ultrastructure; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D009414:Nerve Growth Factors / Q000096:biosynthesis*; D055495:Neurogenesis / Q000502:physiology*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D011493:Protein Kinase C / Q000378:metabolism*; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology
D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; C000594136:Elavl4 protein, rat; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D011484:Protein-Arginine N-Methyltransferases; C119966:coactivator-associated arginine methyltransferase 1; D011493:Protein Kinase C