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Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

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Enzymatic activity of ERp57 plays a critical role in modulating SERCA 2b. (A) Confocal images of Ca2+ oscillations shown in oocytes overexpressing proteins as labeled: SERCA 2b alone (n = 23); SERCA 2b + ERp57 (n = 24); SERCA 2b + ERp57-T1 (n = 20); SERCA 2b + ERp57-T2 (n = 23); and SERCA 2b + ERp57-T1T2 (n = 32). Traces represent two independent experiments with 11–15 oocytes per group. (B) Histograms of period and t1/2 for the experiment in A. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and SERCA 2b + ERp57 or SERCA 2b + ERp57-T2. (C) Western blots of SERCA 2b and ERp57 from lysates of experimental oocytes. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent three independent Western blots. (D) In vitro translations of ERp57 and mutants in rabbit reticulocytes in the absence or presence of canine pancreatic microsomes. Note that the signal peptide was proteolytically cleaved in reactions supplemented with microsomes. This gel is representative of four independent experiments. Bar, 100 μm.
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fig4: Enzymatic activity of ERp57 plays a critical role in modulating SERCA 2b. (A) Confocal images of Ca2+ oscillations shown in oocytes overexpressing proteins as labeled: SERCA 2b alone (n = 23); SERCA 2b + ERp57 (n = 24); SERCA 2b + ERp57-T1 (n = 20); SERCA 2b + ERp57-T2 (n = 23); and SERCA 2b + ERp57-T1T2 (n = 32). Traces represent two independent experiments with 11–15 oocytes per group. (B) Histograms of period and t1/2 for the experiment in A. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and SERCA 2b + ERp57 or SERCA 2b + ERp57-T2. (C) Western blots of SERCA 2b and ERp57 from lysates of experimental oocytes. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent three independent Western blots. (D) In vitro translations of ERp57 and mutants in rabbit reticulocytes in the absence or presence of canine pancreatic microsomes. Note that the signal peptide was proteolytically cleaved in reactions supplemented with microsomes. This gel is representative of four independent experiments. Bar, 100 μm.

Mentions: To determine the relationship between ERp57 catalytic activity with Ca2+ pump activity, we coexpressed wild-type or mutant ERp57 with SERCA 2b. As expected, ERp57 had the highest effect in reducing the frequency of Ca2+ oscillations (i.e., reduced pump activity). The ERp57-T2 mutant that had 70% enzymatic activity in the insulin assay exhibited intermediate levels of pump inhibition. Neither ERp57-T1 (30% enzyme activity) nor ERp57-T1T2 (devoid of enzyme activity) affected the Ca2+ wave period and t1/2 that was enhanced by SERCA 2b overexpression (Fig. 4, A and B). Western blots demonstrated that ERp57 and its mutants as well as SERCA 2b expression levels were similar throughout expression groups (Fig. 4 C). Independent confirmation that all ERp57 mutants were correctly processed in the ER was provided by in vitro translations supplemented with canine microsomes (Fig. 4 D). Together, these results strongly suggest that ERp57 oxidoreductase activity is responsible for the modulation of Ca2+ uptake by SERCA 2b.


Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

Enzymatic activity of ERp57 plays a critical role in modulating SERCA 2b. (A) Confocal images of Ca2+ oscillations shown in oocytes overexpressing proteins as labeled: SERCA 2b alone (n = 23); SERCA 2b + ERp57 (n = 24); SERCA 2b + ERp57-T1 (n = 20); SERCA 2b + ERp57-T2 (n = 23); and SERCA 2b + ERp57-T1T2 (n = 32). Traces represent two independent experiments with 11–15 oocytes per group. (B) Histograms of period and t1/2 for the experiment in A. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and SERCA 2b + ERp57 or SERCA 2b + ERp57-T2. (C) Western blots of SERCA 2b and ERp57 from lysates of experimental oocytes. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent three independent Western blots. (D) In vitro translations of ERp57 and mutants in rabbit reticulocytes in the absence or presence of canine pancreatic microsomes. Note that the signal peptide was proteolytically cleaved in reactions supplemented with microsomes. This gel is representative of four independent experiments. Bar, 100 μm.
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Related In: Results  -  Collection

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fig4: Enzymatic activity of ERp57 plays a critical role in modulating SERCA 2b. (A) Confocal images of Ca2+ oscillations shown in oocytes overexpressing proteins as labeled: SERCA 2b alone (n = 23); SERCA 2b + ERp57 (n = 24); SERCA 2b + ERp57-T1 (n = 20); SERCA 2b + ERp57-T2 (n = 23); and SERCA 2b + ERp57-T1T2 (n = 32). Traces represent two independent experiments with 11–15 oocytes per group. (B) Histograms of period and t1/2 for the experiment in A. Asterisks indicate statistical significance (P < 0.05, t test) between SERCA 2b and SERCA 2b + ERp57 or SERCA 2b + ERp57-T2. (C) Western blots of SERCA 2b and ERp57 from lysates of experimental oocytes. One oocyte equivalent was loaded per lane and proteins were resolved through 12% SDS-PAGE. Gels represent three independent Western blots. (D) In vitro translations of ERp57 and mutants in rabbit reticulocytes in the absence or presence of canine pancreatic microsomes. Note that the signal peptide was proteolytically cleaved in reactions supplemented with microsomes. This gel is representative of four independent experiments. Bar, 100 μm.
Mentions: To determine the relationship between ERp57 catalytic activity with Ca2+ pump activity, we coexpressed wild-type or mutant ERp57 with SERCA 2b. As expected, ERp57 had the highest effect in reducing the frequency of Ca2+ oscillations (i.e., reduced pump activity). The ERp57-T2 mutant that had 70% enzymatic activity in the insulin assay exhibited intermediate levels of pump inhibition. Neither ERp57-T1 (30% enzyme activity) nor ERp57-T1T2 (devoid of enzyme activity) affected the Ca2+ wave period and t1/2 that was enhanced by SERCA 2b overexpression (Fig. 4, A and B). Western blots demonstrated that ERp57 and its mutants as well as SERCA 2b expression levels were similar throughout expression groups (Fig. 4 C). Independent confirmation that all ERp57 mutants were correctly processed in the ER was provided by in vitro translations supplemented with canine microsomes (Fig. 4 D). Together, these results strongly suggest that ERp57 oxidoreductase activity is responsible for the modulation of Ca2+ uptake by SERCA 2b.

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

Show MeSH
Related in: MedlinePlus