Abstract
ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.
Highlights
Grant RO301-69830 from the NHLBI. 1 To whom correspondence should be addressed: California Pacific Medical tion to the A (“actuator”), N (ATP binding), and P domains that are common to other P-type ATPases, the ATP7B protein includes an N metal binding domain (N terminus extension) (NMBD) with six additional copper binding sites
Observing the cells in culture, we found that the level of ATP7B protein and its trafficking are regulated by copper and protein kinase D (PKD)-dependent phosphorylation of serine residues
We found that alkali-stable phosphorylation was totally prevented by specific PKD inhibition with 20 M CID755673, whereas alkali-labile phosphorylation was not affected (Fig. 3B)
Summary
Grant RO301-69830 from the NHLBI. 1 To whom correspondence should be addressed: California Pacific Medical tion to the A (“actuator”), N (ATP binding), and P (intermediate catalytic phosphorylation) domains that are common to other P-type ATPases, the ATP7B protein includes an N metal binding domain (N terminus extension) (NMBD) with six additional copper binding sites (see Fig. 1). 1 To whom correspondence should be addressed: California Pacific Medical tion to the A (“actuator”), N (ATP binding), and P (intermediate catalytic phosphorylation) domains that are common to other P-type ATPases, the ATP7B protein includes an N metal binding domain (N terminus extension) (NMBD) with six additional copper binding sites (see Fig. 1). These sites are referred to with numbers from 1 to 6, starting from the N terminus. We demonstrate the importance of proteasome-mediated degradation in quality control of the nascent ATP7B protein
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