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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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The catalytic activity of ERp57 and PDI is Ca2+ independent. Enzymatic activity is measured using the insulin turbidity assay at the indicated Ca2+ concentrations. AbsorbanceOD 650 values were collected in triplicate and plotted. These plots represent four independent experiments. The standard error bars were smaller than the size of the symbols. Input amounts of GST-ERp57 and GST-PDI fusion proteins was 0.8 μM.
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fig6: The catalytic activity of ERp57 and PDI is Ca2+ independent. Enzymatic activity is measured using the insulin turbidity assay at the indicated Ca2+ concentrations. AbsorbanceOD 650 values were collected in triplicate and plotted. These plots represent four independent experiments. The standard error bars were smaller than the size of the symbols. Input amounts of GST-ERp57 and GST-PDI fusion proteins was 0.8 μM.

Mentions: Because the association of ERp57 with the L4 substrate is stronger at higher Ca2+ concentrations, we tested whether the intrinsic activity of ERp57 is Ca2+ dependent. We measured in vitro the catalytic activity of purified GST-ERp57 at the same range of [Ca2+] used in the GST pull-down assay (300, 150, 50, and 10 μM). The activity of ERp57 was only mildly but not significantly dependent on Ca2+ at the concentrations measured (Fig. 6 A). A similar assay was also performed for GST-PDI and in this case we found no Ca2+ dependence of its activity (Fig. 6 B).


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

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

The catalytic activity of ERp57 and PDI is Ca2+ independent. Enzymatic activity is measured using the insulin turbidity assay at the indicated Ca2+ concentrations. AbsorbanceOD 650 values were collected in triplicate and plotted. These plots represent four independent experiments. The standard error bars were smaller than the size of the symbols. Input amounts of GST-ERp57 and GST-PDI fusion proteins was 0.8 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171954&req=5

fig6: The catalytic activity of ERp57 and PDI is Ca2+ independent. Enzymatic activity is measured using the insulin turbidity assay at the indicated Ca2+ concentrations. AbsorbanceOD 650 values were collected in triplicate and plotted. These plots represent four independent experiments. The standard error bars were smaller than the size of the symbols. Input amounts of GST-ERp57 and GST-PDI fusion proteins was 0.8 μM.
Mentions: Because the association of ERp57 with the L4 substrate is stronger at higher Ca2+ concentrations, we tested whether the intrinsic activity of ERp57 is Ca2+ dependent. We measured in vitro the catalytic activity of purified GST-ERp57 at the same range of [Ca2+] used in the GST pull-down assay (300, 150, 50, and 10 μM). The activity of ERp57 was only mildly but not significantly dependent on Ca2+ at the concentrations measured (Fig. 6 A). A similar assay was also performed for GST-PDI and in this case we found no Ca2+ dependence of its activity (Fig. 6 B).

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