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J Biol Chem, Vol. 273, Issue 32, 20029-20035, August 7, 1998
From the Medizinische Universitätsklinik, The development and functional analysis of
a monoclonal antibody (16C2) are reported; the antibody recognizes
vasodilator-stimulated phosphoprotein (VASP; an established substrate
of both cAMP- and cGMP-dependent protein kinase) only when
serine 239 is phosphorylated. VASP serine 239 represents one of the
best characterized cGMP-dependent protein kinase
phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP
constructs with mutated phosphorylation sites (S157A, S239A, T278A) and
experiments with intact cells (human/rat platelets and other cells)
treated with cyclic nucleotide-elevating agents demonstrated the
specificity of the monoclonal antibody 16C2. Quantitative analysis of
the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2
monoclonal antibody (VASP serine 239 phosphorylation) in human
platelets stimulated by selective protein kinase activators confirmed
that serine 239 is the VASP phosphorylation site preferred by
cGMP-dependent protein kinase in intact cells.
Immunofluorescence experiments with human platelets treated with cGMP
analogs showed that the 16C2 monoclonal antibody also detects VASP
serine 239 phosphorylation in situ at established
intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method
presently available to measure cGMP-dependent protein
kinase activation in intact cells. Also, the 16C2 antibody promises to
be an excellent tool for the evaluation of VASP function in intact
cells.
Analysis and Regulation of Vasodilator-stimulated Phosphoprotein
Serine 239 Phosphorylation in Vitro and in Intact Cells
Using a Phosphospecific Monoclonal Antibody
, and nanoTools
Antikörpertechnik,
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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