PMID- 8407954
OWN - NLM
STAT- MEDLINE
DCOM- 19931124
LR  - 20210210
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 268
IP  - 28
DP  - 1993 Oct 5
TI  - Ca(2+)-dependent and thapsigargin-inhibited phosphorylation of Na+,K(+)-ATPase 
      catalytic domain following chimeric recombination with Ca(2+)-ATPase.
PG  - 21185-92
AB  - Two chimeric proteins comprising the Na,K-ATPase catalytic domain (large 
      cytosolic loop) and the two flanking regions of the Ca-ATPase were obtained by 
      transient or stable expression in mammalian cells transfected with recombinant 
      DNA. In the first chimera (CpNC), a large portion (containing the 
      nucleotide-binding site) of the cytosolic loop between putative membrane spans M4 
      and M5 of the sarcoendoplasmic reticulum Ca2+ (SERCA) 1 (fast muscle) ATPase was 
      replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. In the 
      second chimera (CNpC), an even larger portion (containing the nucleotide-binding 
      site and the phosphorylation site) of the analogous cytosolic loop of the SERCA2 
      (cardiac muscle) ATPase was replaced by the corresponding portion of the 
      Na,K-ATPase alpha 1 subunit. Steady state Ca2+ transport and coupled ATP 
      hydrolysis by the chimeric proteins were negligible as compared to those obtained 
      with SERCA enzymes. Nevertheless, the chimeric proteins were able to utilize ATP 
      to form phosphoenzyme levels equal to those formed by SERCA ATPases. Chimeric and 
      SERCA enzymes exhibited an identical Ca2+ requirement for ATP utilization and 
      sensitivity to thapsigargin (TG) which is a specific inhibitor of SERCA ATPase 
      and not of Na,K-ATPase. Furthermore, both SERCA and chimeric enzymes could be 
      phosphorylated with P(i), and this reaction required removal of Ca2+. In 
      comparative experiments, the functional pattern of seemingly unaffected 
      phosphoenzyme formation and inhibited Ca2+ transport was produced in the SERCA 
      ATPase even by single mutation of Pro337 to Ala, evidently due to defective 
      protein conformation. Retention of Ca2+ and TG sensitivity by the chimeric 
      proteins demonstrates that the Ca(2+)- and TG-binding domains do not reside 
      within the cytosolic loop replaced by chimeric substitution and strongly support 
      previous studies suggesting that binding of calcium required for enzyme 
      activation occurs within the membrane-bound region of the SERCA ATPases (Clarke 
      et al., 1989a; Sumbilla et al., 1991).
FAU - Sumbilla, C
AU  - Sumbilla C
AD  - Department of Biological Chemistry, University of Maryland, Baltimore 21201.
FAU - Lu, L
AU  - Lu L
FAU - Lewis, D E
AU  - Lewis DE
FAU - Inesi, G
AU  - Inesi G
FAU - Ishii, T
AU  - Ishii T
FAU - Takeyasu, K
AU  - Takeyasu K
FAU - Feng, Y
AU  - Feng Y
FAU - Fambrough, D M
AU  - Fambrough DM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (DNA, Single-Stranded)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Terpenes)
RN  - 67526-95-8 (Thapsigargin)
RN  - 9DLQ4CIU6V (Proline)
RN  - EC 7.2.2.10 (Calcium-Transporting ATPases)
RN  - EC 7.2.2.13 (Sodium-Potassium-Exchanging ATPase)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Base Sequence
MH  - Biological Transport
MH  - Calcium/*metabolism
MH  - Calcium-Transporting ATPases/antagonists & inhibitors/*genetics
MH  - Cells, Cultured
MH  - Chickens
MH  - DNA, Single-Stranded
MH  - Hydrolysis
MH  - Mice
MH  - Microsomes/metabolism
MH  - Molecular Sequence Data
MH  - Mutation
MH  - Phosphorylation
MH  - Proline/genetics
MH  - Recombinant Fusion Proteins/genetics/metabolism
MH  - Recombination, Genetic
MH  - Sarcoplasmic Reticulum/enzymology
MH  - Sequence Homology, Amino Acid
MH  - Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors/genetics/*metabolism
MH  - Terpenes/*pharmacology
MH  - Thapsigargin
MH  - Transfection
EDAT- 1993/10/05 00:00
MHDA- 1993/10/05 00:01
CRDT- 1993/10/05 00:00
PHST- 1993/10/05 00:00 [pubmed]
PHST- 1993/10/05 00:01 [medline]
PHST- 1993/10/05 00:00 [entrez]
AID - S0021-9258(19)36908-X [pii]
PST - ppublish
SO  - J Biol Chem. 1993 Oct 5;268(28):21185-92.