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Protein kinase G (PKG): Involvement in Promoting Neural Cell Survival, Proliferation, Synaptogenesis, and Synaptic Plasticity and the Use of New Ultrasensitive Capillary-Electrophoresis-Based Methodologies for Measuring PKG Expression and Molecular Actions

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Protein Kinase Technologies

Part of the book series: Neuromethods ((NM,volume 68))

Abstract

Cyclic GMP-dependent protein kinase (protein kinase G, PKG) plays an important role as a key protein kinase mediating the neuroprotective effects of nitric oxide (NO) at low, physiological levels (10 pM–10 nM) and by the natriuretic peptides, atrial natriuretic peptide (ANP) and brain (B-type) natriuretic peptide (BNP). The NO-, ANP-, and BNP-induced stimulation of PKG kinase (serine/threonine-phosphorylating) activity promotes the survival of neural cells, preventing or minimizing both spontaneous apoptosis and the apoptosis induced by neurotoxins [e.g., minimizing the apoptotic cell death caused by high/toxic levels of NO (>100 nM NO)]. PKG is also involved in promoting/regulating cell proliferation and migration of neural progenitor cells and neural cancer cells, synaptogenesis (promoting formation of filopodia and regulating the guidance of growth cones), synaptic plasticity [contributing to memory consolidation, including long-term potentiation (LTP) in hippocampus and long-term depression (LTD) in cerebellum], nociception, and circadian rhythm. Studies from our laboratory suggest that the pro-growth and pro-survival actions of PKG, specifically the PKG-Iα isoform, involve the downstream phosphorylation of specific target proteins, including BAD (an apoptosis-regulating protein), CREB (a transcription factor involved in memory consolidation and neuroprotection), c-Src (a tyrosine kinase that promotes cell proliferation and survival in both normal and cancerous cells), and vasodilator-stimulated phosphoprotein (VASP, a protein that regulates actin filament formation and focal adhesions). This chapter highlights the use of capillary electrophoresis (CE), coupled with either LED-induced fluorescence or chemoluminescence detection, for providing ultrasensitive, highly quantitative measurements of (1) apoptotic DNA fragmentation, (2) RT-PCR products (determining mRNA expression levels) in a multiplexed analysis, and (3) levels of protein expression and site-specific phosphorylation. The ultrasensitive measurement of protein expression/phosphorylation levels utilizes the recently developed NanoPro100 system [ProteinSimple (originally named Cell Biosciences, Inc.), Santa Clara, CA, USA], an automated CE-chemoluminescence-based immunoquantification instrument, which provides exceedingly high sensitivity (e.g., femtogram quantities of protein) and much better phosphoprotein resolving power and quantification, compared with conventional Western blot analysis. These novel methodologies can now provide neuroscientists with valuable new tools for studying the expression and phosphorylation of lower abundance proteins, such as PKG, which in the past have been difficult to measure because of the relatively low-level sensitivity and inadequate quantification of conventional techniques. These new CE-based methodologies also allow the accurate quantification of apoptotic DNA fragmentation, multiplexed mRNA levels, and protein expression/phosphorylation levels in very small biological samples, potentially containing fewer than 1,000 mammalian cells, thus ideal for studies of primary cell cultures and isolated stem/progenitor cells.

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Acknowledgments

Funding for this project was provided by startup funding for the Fiscus Laboratory in the Center for Diabetes & Obesity Prevention, Treatment, Research & Education at the Roseman University of Health Sciences (formerly the University of Southern Nevada), Henderson and Las Vegas, NV, USA.

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Authors and Affiliations

  1. Center for Diabetes and Obesity Prevention, Treatment, Research, and Education, College of Pharmacy, Roseman University of Health Sciences (Formerly the University of Southern Nevada), Henderson, NV, USA

    Ronald R. Fiscus

  2. Center for Diabetes and Obesity Prevention, Treatment, Research, and Education, The College of Pharmacy, Roseman University of Health Sciences (Formerly the University of Southern Nevada), Henderson, NV, USA

    Mary G. Johlfs

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  1. Ronald R. Fiscus
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Correspondence to Ronald R. Fiscus .

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  1. Biosignal Research Center, Kobe University, Rokkodai 1-1, Kobe, 657-8501, Japan

    Hideyuki Mukai

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Fiscus, R.R., Johlfs, M.G. (2012). Protein kinase G (PKG): Involvement in Promoting Neural Cell Survival, Proliferation, Synaptogenesis, and Synaptic Plasticity and the Use of New Ultrasensitive Capillary-Electrophoresis-Based Methodologies for Measuring PKG Expression and Molecular Actions. In: Mukai, H. (eds) Protein Kinase Technologies. Neuromethods, vol 68. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-824-5_18

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  • DOI: https://doi.org/10.1007/978-1-61779-824-5_18

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