ABSTRACT
Depolarization, growth factors, neurotrophins, and other stimuli induce expression of immediate early genes (IEGs) in neurons. We identified a subset of IEGs, IPD-IEGs, which are induced preferentially by depolarization, but not by neurotrophins or growth factors, in PC12 cells. The "promiscuous" IEGs Egr1 and c-fos, induced by growth factors and neurotrophins, in addition to depolarization, require activation of the MAP kinase signaling pathway for induction in response to KCl depolarization in PC12 cells; MEK1/2 inhibitors block KCl-induced Egr1 and c-fos expression. In contrast, MEK1/2 inhibition has no effect on KCl-induced expression of the known IPD-IEGs in PC12 cells. Additional "candidate" IDP-IEGs were identified by a microarray comparison of genes induced by KCl in the presence vs. the absence of an MEK1/2 inhibitor in PC12 cells. Northern blot analyses demonstrated that representative newly identified candidate IPD-IEGs, as with the known IPD-IEGs, are also induced by a MAP kinase- independent pathway in response to depolarization, both in PC12 cells and in rat primary cortical neurons. Nerve growth factor and epidermal growth factor are unable to induce the expression of the Crem/Icer, Nur77, Nor1, Rgs2, Dusp1 (Mkp1), and Dscr1 genes in PC12 cells, validating their identification as IPD-IEGs. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII), calcineurin, or protein kinase A (PKA) activity prevents KCl-induced IPD-IEG mRNA accumulation, suggesting that the IPD-IEG genes are induced by depolarization in neurons via a combination of calcineurin/PKA- and CaMKII-dependent pathways.
Subject(s)
Early Growth Response Protein 1/genetics , Gene Expression/physiology , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Neurons/physiology , Proto-Oncogene Proteins c-fos/genetics , Animals , Blotting, Northern , Calcineurin/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cerebral Cortex/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Electrophysiology , Mitogens/pharmacology , Nerve Growth Factors/pharmacology , Neurons/drug effects , Neurons/metabolism , PC12 Cells/drug effects , PC12 Cells/metabolism , Rats , Reproducibility of Results , Signal Transduction/physiologyABSTRACT
Using a combination of targeted differential display for induced protein kinases and differential library screening, we identified mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2), as a primary response gene whose transcription is stimulated by membrane depolarization and by forskolin in rat PC12 pheochromocytoma cells. MAPKAPK3 was neither induced nor repressed by similar treatments. The increase in MAPKAPK2 mRNA is preceded by an increase in a MAPKAPK2 intron-containing RNA precursor, indicating that the increase in message is due at least in part to increased transcription. The open reading frame of full-length rat MAPKAPK2 cDNA is 99% identical to mouse MAPKAPK2 and 92% identical to human MAPKAPK2. The human MAPKAPK2 predicted protein contains 14 additional amino acids in the proline-rich N-terminal domain, when compared to murine and rat MAPKAPK2 predicted proteins. The MAPKAPK2 form found in PC12 cells corresponds to variant 2 in the human; this ortholog carries a nuclear translocation signal near its C-terminus. MAPKAPK2 message is also induced in the dentate gyrus, CA1, and CA3 of the rat hippocampus between 2-4 hr after the onset of kainic acid-induced seizures.
Subject(s)
Brain/drug effects , Colforsin/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Kainic Acid/pharmacology , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Blotting, Northern/methods , Blotting, Southern/methods , Brain/anatomy & histology , Brain/metabolism , Calcium/metabolism , Cell Differentiation/drug effects , Cloning, Molecular , Cycloheximide/pharmacology , Drug Interactions/physiology , Enzyme Activation , Gene Expression Regulation, Enzymologic/drug effects , In Situ Hybridization/methods , Intracellular Signaling Peptides and Proteins , Male , Molecular Sequence Data , PC12 Cells , Potassium Chloride/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction/methods , Time FactorsABSTRACT
Depolarization-induced vesicle exocytosis is a complex mechanism involving a number of proteins. In this process, synaptotagmins work as members of the Ca(2+)-sensing system that triggers the fusion of the synaptic vesicle with the plasma membrane. Synaptotagmin IV (SytIV), an immediate-early gene induced by depolarization in PC12 pheochromocytoma cells and in the hippocampus, has been suggested to work as a negative regulator of neurotransmitter release. Unlike other synaptotagmins, SytIV has an evolutionarily conserved substitution of an aspartate to a serine in the Ca(2+) coordination site of its C2A domain, preventing SytIV from binding anionic lipids in a Ca(2+)-dependent fashion. We used the secretion of human growth hormone (hGH) as a reporter system with which to examine the effects of overexpressing SytIV and other depolarization-induced immediate-early genes (the protein kinases KID-1, SIK, and PIM-1 and the transcription factors rTLE3 and Nurr1) on depolarization-induced vesicle exocytosis in PC12 cells. SytIV overexpression resulted in decreased depolarization-induced hGH release. However, conversion of the unique serine in SytIV to an aspartate eliminated this inhibitory activity. In addition, rTLE3 overexpression produced only a modest increase in spontaneous vesicle exocytosis, whereas KID-1, SIK, PIM-1, and Nurr1 overexpression had no effect on depolarization-induced exocytosis.
Subject(s)
Exocytosis/physiology , Membrane Glycoproteins/physiology , Membrane Potentials/physiology , Nerve Tissue Proteins/physiology , PC12 Cells/metabolism , Secretory Vesicles/metabolism , Animals , Calcium Signaling/physiology , Calcium-Binding Proteins/physiology , Genes, Immediate-Early/physiology , Human Growth Hormone/metabolism , Mice , NIH 3T3 Cells , Rats , SynaptotagminsABSTRACT
Immediate early genes induced by depolarization are thought to be important in mediating neuronal functional plasticity. We previously identified a group of immediate early genes that are preferentially induced by depolarization and forskolin but not by nerve growth factor or epidermal growth factor in PC12 pheochromocytoma cells. These depolarization-induced genes include synaptotagmin 4; the protein kinases KID-1, PIM-1, and SIK; an orphan transcription factor, Nurr-1; and a transcription corepressor, rTLE-3. All these genes are also induced in the hippocampus in response to kainic-acid induced depolarization. To characterize further the unique functions of these genes in plasticity, we used recombinant proteins to generate and purify antibodies against KID-1 and SIK proteins. Immunoblotting experiments were performed to examine the induced expression of the KID-1 and SIK proteins in PC12 cells. PIM-1 and Nurr-1 protein expression was also examined following stimulation, using commercially available antibodies. There is an increase in synthesis, in PC12 cells, of these four IEG proteins after KCl plus forskolin treatment. Nurr-1 protein peaks between 2 and 4 hr and decreases by 6 hr after the treatment. PIM-1 and KID-1 proteins rise by 1 hr, peak between 2 and 4 hr, and return to their basal levels at 6 hr. SIK protein increases significantly at 2 hr after treatment, peaks between 4 and 6 hr, and returns to the basal level at 8 hr. Immunofluorescence studies demonstrate distinct distribution patterns of each of these depolarization-induced IEG proteins in PC12 cells.
