Electrochemical detection of neuronal extracellular phosphorylation by PKA, PKC and Src

• Published in: Analytical biochemistry 2020-11, Vol.608, p.113892, Article 113892
• Main Authors: Ahmad, S., Hossain, M.N., Ahmadi, S., Kerman, K., Kraatz, H.-B.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Biotin - chemistry; Cell Line; Cyclic AMP-Dependent Protein Kinases - metabolism; Dielectric Spectroscopy - methods; Electrochemical Techniques - methods; Electrochemistry; Extracellular Space; Fluorescein-5-isothiocyanate; Humans; Kinase; Microscopy, Electrochemical, Scanning - methods; Microscopy, Fluorescence; Neurons; Neurons - metabolism; Phosphorylation; Protein Kinase C - metabolism; Protein Kinases - metabolism; src-Family Kinases - metabolism
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113892

The focus of this work described here is to establish a method for monitoring and quantifying the extracellular phosphorylation of Human SHSY5Y undifferentiated neuronal cells by three ectokinases PKA, PKC and Src; these are kinases that are known to be present in the extracellular matrix. Here is demonstrated that a combination of different experimental techniques, including microscopy and electrochemistry, can be used to detect extracellular phosphorylations. Phosphorylation profiles of the three ectokinases, PKA, PKC and Src, were investigated using fluorescence microscopy and the number of phosphorylation sites per kinase was estimated using QCM. Finally, the phosphorylation of the extracellular membrane was determined using electrochemistry. Our results clearly demonstrate the extracellular phosphorylation of neuronal cells and the strength of surface electrochemical techniques in the investigation of cellular phosphorylation. [Display omitted] •First demonstration of the electrochemical detection of extracellular membrane phosphorylation of neuronal cells.•Extracellular phosphorylation demonstrated through the use of fluorescence, quartz crystal microbalance and electrochemistry.•Establish electrochemistry as a stand-alone technique for PKA, PKC and Src phosphorylation detection.