A rapid separation procedure to analyse phosphorylation of two putative protein kinase C substrates in intact lymphocytes.
Activation of protein kinase C (PKC) has been implicated in the regulation of many biological processes including the regulation of T and B lymphocyte responses. A direct means of studying activation of this kinase is to analyse specific phosphorylation events of cellular substrates. In the present report we describe a fractionation method that allows quantitative analysis of phosphorylation of two distinct PKC substrates using normal human T cells and T-cell tumour lines. This method, which selectively visualizes PKC-dependent phosphorylation of both an 80- and a 19-kDa substrate, involves three simple fractionation steps and allows a large number of samples to be analysed simultaneously. Since specific antibodies to these cellular substrates are not commonly available, the present method provides an alternative approach which makes it feasible to use phosphorylation of the 19- and 80-kDa proteins as a sensitive marker for PKC activation. Finally, in a cellular system where PKC-mediated phosphorylation of these substrates can be studied without prior purification, the present method results in greatly improved resolution of these phosphorylation events.
Unit of Applied Cell and Molecular Biology, University of Umeå, Sweden.
G
G
Brattsand
B
B
Friedrich
M
M
Gullberg
D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology*; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis*; D014407:Tumor Cells, Cultured
D010750:Phosphoproteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C