We studied the properties of the ectokinase activity on the outer cell surf
aces of RBL-2H3 cells and examined the phosphorylation of exogenous substra
tes to clarify the substrate specificity of the ectokinases on RBL-2H3 cell
s. Among the several protein substrates tested, casein was the most strongl
y phosphorylated with [gamma-P-32]ATP, and the net incorporation of P-32 in
to casein was 0.65 pmol P/50 mu g/10(6) cells. Casein kinase II peptide was
also phosphorylated with [y-P-32]ATP. The phosphorylation of casein and ca
sein kinase II peptide was almost completely inhibited by the addition of 3
mu g/ml of cell-impermeable K252b. Phosphorylation of casein and casein ki
nase II peptide was also observed by [gamma-P-32]GTP. Western blot analysis
using anti-casein kinase II antibody revealed a 44-kDa casein kinase band
in the membrane fraction and Fc epsilon RI complexes. The immunofluorescenc
e microscopic analysis using anti-casein kinase II antibody showed the exis
tence of casein kinase II on the surface of the cells. This is the first re
port about the existence of ectokinase on mast cells. (C) 1999 Elsevier Sci
ence B.V. All rights reserved.