Expression of neurogranin tagged with enhanced green fluorescence protein in HEK293 cells and its effects on neuronal signaling

Abstract

Neurogranin (Ng) is a postsynaptic protein kinase C substrate, whose phosphorylation and oxidation may be related to learning and memory. In order to explore the physiological functions of Ng, cellular localization of Ng and its mutants (S36A, S36D, I33Q) in fusion with Enhanced Green Fluorescence Protein (EGFP) was investigated in HEK293 cells. The results showed EGFP-Ng wild-type and its mutants localized to both the cytoplasm and nucleus, with significantly higher intensity in the nucleus. No observable difference in the distribution pattern was detected between the wild-type and mutants. Secondly, higher ERK1/2 phosphorylation was observed in EGFP-Ng transfected cells induced by PMA, suggesting PMA induced PKC-mediated Ng phosphorylation affects ERK MAPK pathway in the cells. Finally, an Isotope-Coded Affinity Tags (ICAT) analysis on protein profiling of Ng expressed N2A cells suggested Ng may be linked to neurite formation by affecting expression of several microtubule related proteins.