Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Calmodulin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources
PubMed1997

Characterization of substrate phosphorylation and use of calmodulin mutants to address implications from the enzyme crystal structure of calmodulin-dependent protein kinase I.

Calcium/calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to the enzyme and indirectly promotes the phosphorylation and synergistic activation of CaMKI by an exogenous kinase. We have evaluated the initial CaM-dependent activation of the unphosphorylated form of CaMKI. The kinetics of bacterially expressed human CaMKI show that the peptide syntide-2 is a relatively poor substrate, whereas the synapsin site-1 peptide is 17-fold more specific. The peptide ADR1G is 400-fold more specific than syntide-2, and its catalytic rate is among the highest reported for a kinase peptide substrate. To understand how CaM activates CaMKI, we have characterized the activation of the enzyme by CaM mutants with substitutions at hydrophobic residues. The point mutant M124Q located in the C-terminal domain of CaM produced a 57-fold increase in the CaM activation constant for CaMKI and suggests the involvement of methionine 124 in an important hydrophobic interaction with tryptophan 303 of CaMKI. Substituting two, three, and five hydrophobic residues in the N-terminal domain of CaM increased the CaM activation constant for CaMKI by 10-190-fold and lowered the maximal enzyme activity by more than 80%. Two of these N-terminal mutants of CaM do not affect the Km for peptide substrate but instead produce a 5-10-fold higher Km for ATP. This result demonstrates the critical role of the N-terminal domain of CaM in regulating the access of ATP to CaMKI.

Adenosine Triphosphate↗
PubMed2006

Calmodulin isoform-specific activation of a rice calmodulin-binding kinase conferred by only three amino-acids of OsCaM61.

The kinase activity of a Ca(2+)/calmodulin (CaM)-binding serine/threonine protein kinase from rice (Oryza sativa) (OsCBK) has been reported to be unaffected by OsCaM1 binding. In this study, we examined whether other rice CaMs can stimulate OsCBK. It was observed that OsCaM61 stimulated OsCBK in a Ca(2+)-dependent manner. In addition, Ala(111), Gly(123) and Ser(127) were identified as critical residues for OsCBK activation. Mutational study and fluorescent spectroscopy analysis indicated that CaM-binding affinity does not correlate with the kinase activity and that these key amino-acids in OsCaM61 play a vital role in suitable changes of OsCBK conformation for kinase activation.

Alanine↗
PubMed2004

Using capillary electrophoresis with laser-induced fluorescence to study the interaction of green fluorescent protein-labeled calmodulin with Ca2+- and calmodulin-binding protein.

A separation using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was applied to the study of green fluorescent protein tagged calmoldulin (GFP-CaM) that was expressed from Escherichia coli and purified with Ni(2+)-nitrilotriacetate (Ni-NTA) resin column. It was found that GFP-CaM not only has good fluorescence properties under various conditions similar to GFP, but also retains its calcium-binding ability as the native CaM. GFP-CaM was separated and detected by CE-LIF within 10 min with a limit-of-detection (LOD) of 2 x 10(-10) M for an injection volume of 3 nl, higher than that of common chemical fluorescent-tagged protein method. The results indicated that, as a fluorescence probe, GFP could overcome the drawback of inefficient derivatization of chemical fluorescence probes. The interaction between the GFP-CaM and Ca(2+) was studied in detail using affinity capillary electrophoresis with laser-induced fluorescence and the dissociation constant (K(d)) between GFP-CaM and Ca(2+) was determined to be 1.2 x 10(-5), which is in good agreement with the literature values of untagged CaM (10(-6) to 10(-5)M) obtained by conventional method. As a preliminary application, the interaction between GFP-CaM and OsCBK was also investigated. The method makes it possible to screen the trace amounts of target proteins in crude extracts interacting with CaM under physiological conditions.

Base Sequence↗
PubMed2003

A tobacco (Nicotiana tabaccum) calmodulin-binding protein kinase, NtCBK2, is regulated differentially by calmodulin isoforms.

A calcium (Ca2+)/calmodulin (CaM)-binding protein kinase (CBK) from tobacco (Nicotiana tabaccum ), NtCBK2, has been characterized molecularly and biochemically. NtCBK2 has all 11 conserved subdomains of the kinase-catalytic domain and a CaM-binding site as shown by other kinases, including Ca2+-dependent protein kinase and chimaeric Ca2+/CaM-dependent protein kinases. However, this kinase does not contain an EF-hand motif for Ca2+ binding, and its activity was not regulated by Ca2+. Whereas NtCBK2 phosphorylated both itself and other substrates, such as histone IIIS and syntide-2, in a Ca2+/CaM-independent manner, as also shown by OsCBK, a CaM-binding protein kinase from rice (Oryza sativa ), the kinase activity of NtCBK2 was greatly stimulated by Ca2+/CaM, whereas that of OsCBK was not. By molecular dissection analyses, the CaM-binding domain of NtCBK2 has been localized in a stretch of 30 amino acid residues at residue positions 431-460 as a 1-5-10 protein motif. Three tobacco CaM isoforms (NtCaM1, NtCaM3 and NtCaM13) used in the present study have been shown to bind to NtCBK2, but with different dissociation constants ( K(d)s), as follows: NtCaM1, 55.7 nM; NtCaM3, 25.4 nM; and NtCaM13, 19.8 nM, indicating that NtCBK2 has a higher affinity for NtCaM3 and NtCaM13 than for NtCaM1. The enzymic activity of NtCBK2 was also modulated differently by various CaM isoforms. Whereas the phosphorylation activity of NtCBK2 was shown by assay to be enhanced only approximately 2-3-fold by the presence of NtCaM1, the activity could be amplified up to 8-9-fold by NtCaM3 or 10-11-fold by NtCaM13, suggesting that NtCaM3 and NtCaM13 are better activators than NtCaM1 for NtCBK2.

Amino Acid Sequence↗
PubMed1996

Requirements for calcium and calmodulin in the calmodulin kinase activation cascade.

We have previously purified and cloned rat brain Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324). In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr196 by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr196) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1 434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC50 values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM-independent activities of wild-type CaM-K IV, but not the Thr196 --> Ala mutant, upon ionomycin stimulation.

Alanine↗