Immunohistochemical and Immunocytochemical Detection of Phosphoproteins in Striatal Neurons

Phosphorylation of protein kinases and transcription factors represents a major biological mechanism to activate those imperative intracellular effectors. Examination of such phosphorylation in response to extracellular stimulation can assess the functional participation of those proteins in a particular signaling cascade. For example, the Ca²⁺ signal facilitates the phosphorylation of Ca²⁺ /calmodulin-dependent protein kinase II (CaMKII), a multifunctional protein kinase regulating diverse cellular functions (1 ). Phosphorylated CaMKII (pCaMKII) can in turn phosphorylate a nuclear transcription factor, cAMP response element-binding protein (CREB), to regulate target DNA transcription (2 ). The first subclass of the mitogen-activated protein kinase (MAPK), the extracellular signal-regulated kinase (ERK), is another protein kinase that is catalyzed by MAPK kinase (MEK) to undergo the phosphorylation (3 ). Once phosphorylated, pERK further induces the phosphorylation of a nuclear transcription factor, Elk-1 (a member of the ternary complex factor family of Ets domain proteins that bind serum response elements), to alter DNA transcription (4 ). Apparently, detection of the phosphorylation state of those proteins can provide valuable information on their functional profiles in a given cellular activity of interest.