Copper is necessary for all organisms since it acts as a cofactor in different
enzymes, although toxic at high concentrations. ATP7B is one of two
copper-transporting ATPases in humans, its vital role being manifested in
Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving
protein kinase C (PKC) modulate ATP7B activity. Different
isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-
ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-
ATPase activity by 60%, whereas
calphostin C and
U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of
phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca(2+), whereas PMA plus
EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after
phosphatase λ treatment, showing that the ɛ
isoform is essential for activation of the
enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for
ATP and
copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of
copper by ATP7B, thus evidencing a central role for this specific
kinase-mediated mechanism in hepatic
copper handling.