Cl. Rowerendleman et J. Eichberg, P-0 PHOSPHORYLATION IN NERVES FROM NORMAL AND DIABETIC RATS - ROLE OFPROTEIN-KINASE-C AND TURNOVER OF PHOSPHATE GROUPS, Neurochemical research, 19(8), 1994, pp. 1023-1031
The effects of phorbol ester and forskolin on the net phosphorylation
and turnover of P-0 phosphate groups was studied in normal and experim
entally diabetic rats. In sciatic nerve segments isolated from normal
rats and incubated with [P-32]-inorganic phosphate, phosphorylation of
the major peripheral myelin protein, P-0, was increased 2-5 fold in a
time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). Thi
s increase was blocked by the protein kinase inhibitors, H-7 and staur
osporine. Both the basal and PDB-stimulated phosphorylation of P-0 wer
e significantly greater in segments of sciatic nerve from streptozotoc
in-induced diabetic rats. Prolonged exposure of nerve segments to PDB
abolished the stimulated phosphorylation of P-0 and immunoblots of ner
ve proteins revealed a decrease in the content of the protein kinase C
alpha-isoform. The adenylate cyclase activator, forskolin, had no aff
ect on the PDB-stimulated phosphorylation of P-0 in normal nerve but d
ecreased phosphorylation in diabetic nerve. To measure turnover of P-0
phosphate groups, nerves were incubated with P-32 and incorporated la
bel was then chased in radioactivity-free medium for up to 4 hours. P-
0 from normal nerve prelabeled under basal conditions lost 25% of its
radioactivity during this time. In contrast, nearly all of the additio
nal phosphate groups prelabeled in the presence of PDB disappeared aft
er 2 hours of chase. P-0 phosphate groups from diabetic nerve displaye
d similar turnover kinetics. When forskolin was added to the chase med
ium, the turnover of P-0 phosphate moieties was accelerated in normal,
but not in diabetic nerve. These findings clearly establish a promine
nt role for protein kinase C in P-0 phosphorylation, provide evidence
for heterogeneous turnover of P-0 phosphate groups and suggest that cy
clic AMP-mediated processes may modulate P-0 phosphorylation. Further,
these results indicate that the metabolism of P-0 phosphate moieties
is perturbed in nerve from diabetic animals.