Copper specifically regulates intracellular phosphorylation of the Wilson's disease protein, a human copper-transporting ATPase

Copper is a trace element essential for normal cell homeostasis. The major physiological role of copper is to serve as a cofactor to a number of key metabolic enzymes. In humans, genetic defects of copper distribution, such as Wilson's disease, lead to severe pathologies, including neurodegeneration, liver lesions, and behavior abnormalities. Here, we demonstrate that, in addition to its role as a cofactor, copper can regulate important post-translational events such as protein phosphorylation. Specifically, in human cells copper modulates phosphorylation of a key copper transporter, the Wilson's disease protein (WNDP). Copper-induced phosphorylation of WNDP is rapid, specific, and reversible and correlates with the intracellular location of this copper transporter. WNDP is found to have at least two phosphorylation sites, a basal phosphorylation site and a site modified in response to increased copper concentration. Comparative analysis of WNDP, the WNDP pineal isoform, and WNDP C-terminal truncation mutants revealed that the basal phosphorylation site is located in the C-terminal Ser(796)-Tyr(1384) region of WNDP. The copper-induced phosphorylation appears to require the presence of the functional N-terminal domain of this protein. The novel physiological role of copper as a modulator of protein phosphorylation could be central to understanding how copper transport is regulated in mammalian cells.     

The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function.

Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.     

Cobalt-dependent protein phosphatases from human cord blood erythrocytes. II. Further characterization of E2 casein phosphatase

Of three casein phosphatases isolated from the cytosol of human cord blood erythrocytes two were cobalt-dependent, E2 and E3. In the presence of CoCl2, E2 activity was the most prominent. In addition to casein, E2 dephosphorylated phosvitin and p-nitrophenyl phosphate (p-NPP) with pH optima at 6.8-7.2 for proteins and 9.0 for p-NPP. The native enzyme had a molecular weight of 104,000 daltons after AcA-44 Ultrogel filtration. According to SDS/PAGE it consisted of two subunits, 78,000 and 15,000 daltons. The 104,000-dalton form exhibited Michaelis-Menten kinetics and had the greatest affinity for casein between protein substrates tested. Ethanol denaturated the enzyme by 80%. Optimal activation of E2 phosphatase was achieved with 5 mmol/l CoCl2 which did not affect the catalytic properties of the enzyme but did affect the rate of 'E-S' complex formation. Inorganic pyrophosphate was not inhibitory for the 104,000-dalton enzyme. Judging by all these properties the natural substrate for E2 casein phosphatase could be P-pyruvate kinase.     

BAP,a mammalian BiP-associated protein,is a nucleotide exchange factor that regulates the ATPase activity of BiP

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.