We compared the phosphorylation-dependent regulation of three mammalian
Na$^+$/Ca$^2+$ exchanger isoforms (NCX1-NCX3) expressed in
CCL39 fibroblasts that have little endogenous activity. Na$^+$i-dependent
45Ca$^2+$ uptake into NCX1- or NCX3-expressing cells, but not
that into NCX2-expressing cells, was significantly enhanced by phorbol
12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB,
which was abolished by pretreatment of cells with calphostin C or
a prior long exposure to PMA. This suggests that NCX1 or NCX3, but
not NCX2, is stimulated by a pathway involving protein kinase C (PKC).
Immunoprecipitation experiments using 32Porthophosphate-labeled
cells revealed that both NCX2 and NCX3 proteins were phosphorylated
to a much lesser extent than the NCX1 protein in unstimulated cells
and that the extent of phosphorylation was not increased by treatment
with PKC activators, although NCX1 phosphorylation was enhanced significantly.
Using site-directed mutagenesis, we identified three phosphorylation
sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249,
Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated.
We found that the NCX1 mutant with these serine residues substituted
with alanine still maintained a normal response to PMA. In contrast,
the NCX1 or NCX3 mutant, with the large central cytoplasmic loop
deleted, lost the responsiveness to PMA. These results suggest that
the PKC-dependent regulation of NCX1 or NCX3 requires the central
cytoplasmic loop but does not require the direct phosphorylation
of the exchanger.