NASA Logo

NTRS

NTRS - NASA Technical Reports Server

Back to Results
Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domainCa(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.
Document ID
20040173115
Acquisition Source
Legacy CDMS
Document Type
Reprint (Version printed in journal)
External Source(s)
Authors
Huang, J. F.
(Scripps Research Institute La Jolla, California 92037, United States)
Teyton, L.
Harper, J. F.
Evans, M. L.
Date Acquired
August 22, 2013
Publication Date
October 8, 1996
Publication Information
Publication: Biochemistry
Volume: 35
Issue: 40
ISSN: 0006-2960
Subject Category
Life Sciences (General)
Distribution Limits
Public
Copyright
Other
Keywords
Non-NASA Center
NASA Discipline Plant Biology

Available Downloads

There are no available downloads for this record.
No Preview Available