Abstract
The hypothesis that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, contains an autoinhibitory domain was tested using synthetic peptides corresponding to regions of the carboxyl-terminus of calcineurin. Of the several peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein phosphatase activity. Using [32P]myosin light chain as substrate an IC50 of about 10 μM was obtained with either native calcineurin, assayed in the presence of Ca2+/calmodulin, or with calcineurin subjected to partial proteolysis which converts it to a fully active phosphatase when assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 μM was observed. Studies with overlapping peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of phosphatase activity was not competitive with respect to [32P]myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/ calmodulin-dependent protein kinase II. These results indicate that amino acids within this sequence of calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal phosphatase activity in the absence of Ca2+/calmodulin.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1924-1927 |
| Number of pages | 4 |
| Journal | Journal of Biological Chemistry |
| Volume | 265 |
| Issue number | 4 |
| State | Published - Feb 5 1990 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Identification of an autoinhibitory domain in calcineurin. / Hashimoto, Yoshiaki; Perrino, Brian A.; Soderling, Thomas.
In: Journal of Biological Chemistry, Vol. 265, No. 4, 05.02.1990, p. 1924-1927.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of an autoinhibitory domain in calcineurin
AU - Hashimoto, Yoshiaki
AU - Perrino, Brian A.
AU - Soderling, Thomas
PY - 1990/2/5
Y1 - 1990/2/5
N2 - The hypothesis that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, contains an autoinhibitory domain was tested using synthetic peptides corresponding to regions of the carboxyl-terminus of calcineurin. Of the several peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein phosphatase activity. Using [32P]myosin light chain as substrate an IC50 of about 10 μM was obtained with either native calcineurin, assayed in the presence of Ca2+/calmodulin, or with calcineurin subjected to partial proteolysis which converts it to a fully active phosphatase when assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 μM was observed. Studies with overlapping peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of phosphatase activity was not competitive with respect to [32P]myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/ calmodulin-dependent protein kinase II. These results indicate that amino acids within this sequence of calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal phosphatase activity in the absence of Ca2+/calmodulin.
AB - The hypothesis that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, contains an autoinhibitory domain was tested using synthetic peptides corresponding to regions of the carboxyl-terminus of calcineurin. Of the several peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein phosphatase activity. Using [32P]myosin light chain as substrate an IC50 of about 10 μM was obtained with either native calcineurin, assayed in the presence of Ca2+/calmodulin, or with calcineurin subjected to partial proteolysis which converts it to a fully active phosphatase when assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 μM was observed. Studies with overlapping peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of phosphatase activity was not competitive with respect to [32P]myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/ calmodulin-dependent protein kinase II. These results indicate that amino acids within this sequence of calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal phosphatase activity in the absence of Ca2+/calmodulin.
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UR - http://www.scopus.com/inward/citedby.url?scp=0024991506&partnerID=8YFLogxK
M3 - Article
C2 - 2153670
AN - SCOPUS:0024991506
VL - 265
SP - 1924
EP - 1927
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 4
ER -