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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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ERp57 reduces the frequency of Ca2+ oscillations mediated by SERCA 2b. (A) Confocal images of Ca2+ oscillations in Xenopus oocytes expressing SERCA 2b alone (n = 22) or coexpressing ERp57 with SERCA 2b (n = 22). Traces represent changes in fluorescence (ΔF/F) as a function of time. Each plot represents two independent experiments with 11 oocytes per group. Individual Ca2+ wave images are presented at the indicated time. The white square in each and subsequent images represents a 5 × 5 pixel area used in determining ΔF/F. Histogram plots of the period between oscillations and the decay time (t1/2) are for individual oscillations at peak activity, defined as the highest frequency for a 100-s window of time. Asterisks indicate statistical significance (P < 0.05, t test). (B) Western blots of SERCA 2b and ERp57. One oocyte equivalent was loaded per lane and proteins were resolved through 10% SDS-PAGE. SERCA 2b migrates ∼110 kD and ERp57 ∼60 kD as expected. The loading control lane in each and subsequent gels represents an invariant Xenopus protein that migrates at ∼42 kD. These gels represent three independent Western blots. Bar, 100 μm.
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fig1: ERp57 reduces the frequency of Ca2+ oscillations mediated by SERCA 2b. (A) Confocal images of Ca2+ oscillations in Xenopus oocytes expressing SERCA 2b alone (n = 22) or coexpressing ERp57 with SERCA 2b (n = 22). Traces represent changes in fluorescence (ΔF/F) as a function of time. Each plot represents two independent experiments with 11 oocytes per group. Individual Ca2+ wave images are presented at the indicated time. The white square in each and subsequent images represents a 5 × 5 pixel area used in determining ΔF/F. Histogram plots of the period between oscillations and the decay time (t1/2) are for individual oscillations at peak activity, defined as the highest frequency for a 100-s window of time. Asterisks indicate statistical significance (P < 0.05, t test). (B) Western blots of SERCA 2b and ERp57. One oocyte equivalent was loaded per lane and proteins were resolved through 10% SDS-PAGE. SERCA 2b migrates ∼110 kD and ERp57 ∼60 kD as expected. The loading control lane in each and subsequent gels represents an invariant Xenopus protein that migrates at ∼42 kD. These gels represent three independent Western blots. Bar, 100 μm.

Mentions: We used a confocal Ca2+ oscillation assay as a tool to investigate the modulation of the SERCA 2b pump activity. In this assay, increases in Ca2+ oscillation frequency (shorter period between oscillations and/or decay time [t1/2] of individual waves) reflect increased Ca2+ ATPase activity and vice versa (Camacho and Lechleiter, 1993, 1995, 2000; John et al., 1998; Roderick et al., 2000; Falcke et al., 2003). To test whether ERp57 modulates SERCA 2b activity in Xenopus oocytes, we coexpressed SERCA 2b with ERp57. We found that coexpression reduced the frequency of Ca2+ oscillations compared with overexpression of SERCA 2b by itself. A histogram of period and t1/2 shows that both were significantly prolonged suggesting that Ca2+ uptake into the ER is reduced (Fig. 1 A). Western blots demonstrated that expression levels of SERCA 2b were the same regardless of whether ERp57 was coexpressed with the pump (Fig. 1 B). These results are consistent with the hypothesis that ERp57 modulates ER thiol groups in SERCA 2b.


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

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

ERp57 reduces the frequency of Ca2+ oscillations mediated by SERCA 2b. (A) Confocal images of Ca2+ oscillations in Xenopus oocytes expressing SERCA 2b alone (n = 22) or coexpressing ERp57 with SERCA 2b (n = 22). Traces represent changes in fluorescence (ΔF/F) as a function of time. Each plot represents two independent experiments with 11 oocytes per group. Individual Ca2+ wave images are presented at the indicated time. The white square in each and subsequent images represents a 5 × 5 pixel area used in determining ΔF/F. Histogram plots of the period between oscillations and the decay time (t1/2) are for individual oscillations at peak activity, defined as the highest frequency for a 100-s window of time. Asterisks indicate statistical significance (P < 0.05, t test). (B) Western blots of SERCA 2b and ERp57. One oocyte equivalent was loaded per lane and proteins were resolved through 10% SDS-PAGE. SERCA 2b migrates ∼110 kD and ERp57 ∼60 kD as expected. The loading control lane in each and subsequent gels represents an invariant Xenopus protein that migrates at ∼42 kD. These gels represent three independent Western blots. Bar, 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171954&req=5

fig1: ERp57 reduces the frequency of Ca2+ oscillations mediated by SERCA 2b. (A) Confocal images of Ca2+ oscillations in Xenopus oocytes expressing SERCA 2b alone (n = 22) or coexpressing ERp57 with SERCA 2b (n = 22). Traces represent changes in fluorescence (ΔF/F) as a function of time. Each plot represents two independent experiments with 11 oocytes per group. Individual Ca2+ wave images are presented at the indicated time. The white square in each and subsequent images represents a 5 × 5 pixel area used in determining ΔF/F. Histogram plots of the period between oscillations and the decay time (t1/2) are for individual oscillations at peak activity, defined as the highest frequency for a 100-s window of time. Asterisks indicate statistical significance (P < 0.05, t test). (B) Western blots of SERCA 2b and ERp57. One oocyte equivalent was loaded per lane and proteins were resolved through 10% SDS-PAGE. SERCA 2b migrates ∼110 kD and ERp57 ∼60 kD as expected. The loading control lane in each and subsequent gels represents an invariant Xenopus protein that migrates at ∼42 kD. These gels represent three independent Western blots. Bar, 100 μm.
Mentions: We used a confocal Ca2+ oscillation assay as a tool to investigate the modulation of the SERCA 2b pump activity. In this assay, increases in Ca2+ oscillation frequency (shorter period between oscillations and/or decay time [t1/2] of individual waves) reflect increased Ca2+ ATPase activity and vice versa (Camacho and Lechleiter, 1993, 1995, 2000; John et al., 1998; Roderick et al., 2000; Falcke et al., 2003). To test whether ERp57 modulates SERCA 2b activity in Xenopus oocytes, we coexpressed SERCA 2b with ERp57. We found that coexpression reduced the frequency of Ca2+ oscillations compared with overexpression of SERCA 2b by itself. A histogram of period and t1/2 shows that both were significantly prolonged suggesting that Ca2+ uptake into the ER is reduced (Fig. 1 A). Western blots demonstrated that expression levels of SERCA 2b were the same regardless of whether ERp57 was coexpressed with the pump (Fig. 1 B). These results are consistent with the hypothesis that ERp57 modulates ER thiol groups in 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