Background: Spf1 belongs to the least characterized group of P5-ATPases. Results: GFP-Spf1 hydrolyzes ATP and forms a phosphoenzyme that rapidly decays in the presence of ADP. Conclusion: The Spf1 performs well the E 1 steps of the reaction cycle, but progression to the E 2 forms is slow. Significance: The study extends the understanding of the catalytic mechanism of P5-ATPases.

Background:The calmodulin-stimulated human plasma membrane Ca 2ϩ pump is regulated by autoinhibition. Results: The E99K mutation deregulates the pump increasing its maximal activity at saturating concentrations of Ca 2ϩ . Conclusion:The cytosolic portion of the M1 transmembrane segment is critical for inhibition by the extreme C-terminal autoinhibitory domain. Significance: This work presents new insights into the structure and the mechanism of human PMCA autoinhibition.

P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeast Saccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg 2؉ , and EGTA. . Halfmaximal phosphorylation was attained at 8 M Mg 2؉, but higher concentrations partially protected from Ca 2؉ inhibition. In conditions similar to those used for phosphorylation, Ca 2؉ had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.P5-ATPases comprise a group of proteins that are classified as P-ATPases based on the presence of the characteristic PATPase motifs in their primary sequence (1, 2). P5-ATPases have been found only in eukaryotes and have been recently proposed to play an essential role in the endosomal-lysosomal system (3, 4). The yeast Saccharomyces cerevisiae contains two genes coding for P5-ATPases: YEL031W, coding for Spf1p (also called Cod1p), and YOR291W, coding for Ypk9. Spf1p (sensitivity to Pichia farinosa killer toxin) was initially isolated from a mutation protecting Saccharomyces from the effect of a Pichia toxin (5). The protein was also independently identified as required for the controlled degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the endoplasmic reticulum (ER) 3 (6). These and later studies have shown that Spf1p is located in the yeast ER and that deletion of Spf1p leads to phenotypes related to ER stress (7-9). In humans, five genes (ATP13A1-A5) code for P5-ATPases (10). Mutations in ATP13A2 have been linked to an early onset autosomal recessive form of Parkinson disease (Kufor-Rakeb syndrome) and neuronal ceroid lipofuscinosis, whereas mutations in ATP13A4 have been associated with autism spectrum disorder (11-13). P-ATPases are a large group of enzymes that couple the hydrolysis of ATP with the active transport of ions (14, 15). During the transport cycle, they transiently form a phosphoenzyme (EP) that plays a key role in the active transport mechanism. P-ATPases comprise a membrane domain (M) and a soluble portion with nucleotide binding (N), phosphorylation (P), and actuator (A) domains. These domains are involved in a kinasephosphatase reaction cycle through two major conformations, E 1 -E 2 , and the transient formation of a catalytic EP. The binding of the transported ion to the E 1 form prompts the assembly of the phosphorylation site between the ATP-bound N domain and the P domain, whereas the A domain directs the occlusion of the bound ion. When the phosphorylation reaction occurs, it initially generates the high energy E 1 ϳP intermediate and releases ADP. E 1 ϳP then changes to E 2 P, and the A domain associates with the N-...

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Ang (angiotensin) 1–7 MasR (Mas receptor) and D2R (dopamine D2 receptor) stimulation is coupled to anti-inflammatory responses. In the present work, we investigated the hypothesis that the anti-inflammatory action mediated by both receptors results from MasR-D2R heteromerization. Human monocyte (THP-1) cells differentiated to macrophages and exposed to lipopolysaccharide were employed. Ang (1–7) and the D2R agonist SUM (sumanirole) induced a decrease in proinflammatory IL (interleukin) 6 release in human macrophages exposed to a proinflammatory stimulus. The Ang (1–7)–induced decrease in IL-6 was blocked by the D2R antagonist. Conversely, the SUM induced decrease in IL-6 was prevented by the MasR antagonist and when MasR expression was downregulated, suggesting MasR-D2R interaction. Co-immunoprecipitation assay in THP-1 cells and in human monocyte differentiated macrophages from peripheral blood mononuclear cells confirmed MasR-D2R interaction. To avoid the influence from other receptors, MasR-D2R interaction was characterized in transfected human embryonic kidney 293T cells. Fluorescence resonance energy transfer analysis showed that MasR and D2R formed a constitutive heteromer, which was not modified by their agonists. Ang (1–7) and dopamine stimulated ERK (extracellular signal-regulated kinase) 1/2 and Akt (protein kinase B) phosphorylation only in cells expressing MasR-D2R heteromers, but not in cells expressing each receptor alone. Ang (1–7)–stimulated ERK1/2 and Akt phosphorylation was prevented by D2R blockade while the effect of dopamine was prevented by MasR blockade, reinforcing the fact that MasR-D2R heteromers are involved in ERK1/2 and Akt activation induced by their agonists. Our findings provide new evidence regarding the mechanisms underlying the cross-talk between the Ang (1–7)/MasR axis and the dopaminergic system in response to a proinflammatory process.

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca 2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl 2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D 487 N) also exhibited Ca 2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a copurifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca 2+ or Zn 2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca 2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca 2+. These results indicate that the reported effects of Ca 2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.

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