Protein phosphorylation site study.

Abstract

Protein phosphorylation - dephosphorylation plays a very important role in signal transduction in biological systems. It is a major regulatory mechanism for the proliferation, differentiation, and functions of cells. An understanding of the potential role of a protein in signal transduction and of the significance of its phosphorylation requires elucidation of the mechanism of its phosphorylation in response to various stimuli. Of basic importance, is the identification of the phosphorylated residues. Conventional Edman sequencing method can not routinely provide positive identification of the phosphorylated residues. However methods using mass spectrometry in combination with enzymatic digestion are shown to be a powerful means of obtaining protein structural information, especially when extremely low sample quantities are available. In this study, cellular proteins were first separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), then electrophoretically blotted to a nitrocellulose membrane. Endopeptidase (trypsin) degradation was performed in situ, and microbore HPLC was employed to separate the tryptic peptides. An enzyme that selectively removes the covalently attached phosphate group aided the identification of these modifications. Further proteolytic digests of the tryptic phosphopeptides led to the identification of the phosphorylation sites. In this study we have undertaken the phosphorylation analysis of a highly conserved cytosolic protein OP18, of which the phosphorylation has been implicated in signal transduction in a wide variety of cell types. While phosphorylation at serine 25 and serine 38 was observed, a large increase in phosphorylation involving serine 25 was observed following PMA treatment.