Immunohistochemical and Immunocytochemical Detection of Phosphoproteins in Striatal Neurons - Drugs of Abuse: Neurological Reviews and Protocols - page 267

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Immunohistochemical and Immunocytochemical
Detection of Phosphoproteins in Striatal Neurons
Limin Mao and John Q. Wang
1. Introduction
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 signal-
ing cascade. For example, the Ca 2+ signal facilitates the phosphorylation of
Ca 2+ /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 fi rst 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 profi les in a given
cellular activity of interest.
Detection of the phosphorylated enzymes and factors in striatal neurons
can be accomplished by immunohistochemical and immunocytochemical
approaches. With recently developed anti-active antibodies specifi c for the phos-
phorylated form of the proteins, the phosphorylation can be visualized in cells