Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b

K. Pászty, Alan R. Penheiter, Anil K. Verma, Rita Padányi, Adelaida G. Filoteo, John T. Penniston, A. Enyedi

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The role of the plasma membrane Ca2+ pump (PMCA) is to remove excess Ca2+ from the cytosol to maintain low intracellular Ca2+ levels. Asp1080 lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, Á. (2002) J. Biol. Chem. 277, 6822-6829). Asp1080 had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp1080 to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp1080 mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp1077 had no effect on basal activity or calmodulin activation. We propose that the conserved Asp1080, even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.

Original languageEnglish
Pages (from-to)36146-36151
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number39
DOIs
Publication statusPublished - Sep 27 2002

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Calmodulin
Chemical activation
Caspase 3
Catalytic Domain
Pumps
Cell membranes
Cytosol
Protein Isoforms
Cell Membrane
Mutation
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b. / Pászty, K.; Penheiter, Alan R.; Verma, Anil K.; Padányi, Rita; Filoteo, Adelaida G.; Penniston, John T.; Enyedi, A.

In: Journal of Biological Chemistry, Vol. 277, No. 39, 27.09.2002, p. 36146-36151.

Research output: Contribution to journalArticle

Pászty, K. ; Penheiter, Alan R. ; Verma, Anil K. ; Padányi, Rita ; Filoteo, Adelaida G. ; Penniston, John T. ; Enyedi, A. / Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 39. pp. 36146-36151.
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title = "Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b",
abstract = "The role of the plasma membrane Ca2+ pump (PMCA) is to remove excess Ca2+ from the cytosol to maintain low intracellular Ca2+ levels. Asp1080 lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (P{\'a}szty, K., Verma, A. K., Pad{\'a}nyi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, {\'A}. (2002) J. Biol. Chem. 277, 6822-6829). Asp1080 had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp1080 to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp1080 mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp1077 had no effect on basal activity or calmodulin activation. We propose that the conserved Asp1080, even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.",
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AU - Pászty, K.

AU - Penheiter, Alan R.

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AU - Filoteo, Adelaida G.

AU - Penniston, John T.

AU - Enyedi, A.

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N2 - The role of the plasma membrane Ca2+ pump (PMCA) is to remove excess Ca2+ from the cytosol to maintain low intracellular Ca2+ levels. Asp1080 lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, Á. (2002) J. Biol. Chem. 277, 6822-6829). Asp1080 had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp1080 to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp1080 mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp1077 had no effect on basal activity or calmodulin activation. We propose that the conserved Asp1080, even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.

AB - The role of the plasma membrane Ca2+ pump (PMCA) is to remove excess Ca2+ from the cytosol to maintain low intracellular Ca2+ levels. Asp1080 lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, Á. (2002) J. Biol. Chem. 277, 6822-6829). Asp1080 had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp1080 to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp1080 mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp1077 had no effect on basal activity or calmodulin activation. We propose that the conserved Asp1080, even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.

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