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

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.