[22] Isolation and characterization of bovine brain calcineurin: A calmodulin-stimulated protein phosphatase

Klee, Claude B.; Krinks, Marie; Manalan, Allan S.; Cohen, Philip; Stewart, Alexander A.

doi:10.1016/s0076-6879(83)02024-8

Cited by 140 publications

(87 citation statements)

References 21 publications

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“…Klee et al [28] discussed the point that the calmodulin binding activity of some CaMbinding proteins was often lost following partial degradation by an endogenous calcium-dependent protease. In the case quoted, it was found that the protease could be inhibited by leupeptin [29]. We employed this protease inhibitor at the recommended concentration during C kinase purification without any success.…”

Section: Discussionmentioning

confidence: 99%

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase

Bakrim

Echevarrı́a

Crétin

et al. 1992

European Journal of Biochemistry

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The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C, plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the fecdback inhibitor L-malate and an increase in V,,,. Feeding detached leaves various photosynthetic inhibitors, i. e. 3-(3,Cdichlorophenyl)-l, 1-dimethylurea, gramicidin and DL-glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin-Benson cycle as components in the signal-transduction chain. Two proteine-serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light-adapted leaves. One was isolated on a calmodulin-Sepharose column; it was calcium-dependent but did not require calmodulin for activity. The other was purified on a bluedextran-agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv-carboxylase-kinase. Desalted extracts from light-adapted leaves possessed a considerably grealer phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin-LR, which suggests that the kindse is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light-adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue-dextranagarose. However, no information is available with respect to the specific nature of the link between the above-mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (SerS) in the N-terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non-photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.Correspondence to J. Vidal, Laboratoire dc Physiologie Vegetale Molecubdire, blt. 430,

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Section: Discussionmentioning

confidence: 99%

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase

Bakrim

Echevarrı́a

Crétin

et al. 1992

European Journal of Biochemistry

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“…Except when indicated otherwise, the Ca2÷-dependent activity was measured in the presence of 0.66 mM Ca 2÷ and 0.33 mM EGTA. The enzyme activity was calculated as described [7].…”

Section: Protein Phosphatase Assaymentioning

confidence: 99%

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

et al. 1995

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The Ca2+-dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin-stimnlated protein phosphatase, calcineurin. It is stimulated more than 200-fold by Ca 2+ and inhibited by the calmodnlin-binding peptide, MI3, and by the immunosuppressive drug, FKS06. It is insensitive to rapamycin at concentrations up to 1 pM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti-bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time-, temperature-, and Ca2+/ calmodulin-dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight-containing fraction elnted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).

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“…Native CN was prepared from bovine brain as described previously [20]. The eDNA gene for the tz isoform o1" rat CNA [21,22] was obtained as a gift from Drs.…”

Section: En'-yme Preparaffonmentioning

confidence: 99%

“…B. Perrino and T. Soderling. Recombinant CNA (rCNA) was overexpressed in g. coil using a T7 sy~rem and purified by a method similar to that for native bovine brain CN [20] (Haddy, A,, Swanson, S.K.-H. and Rusnak, F., in preparation). CNB was purified from native bovine brain CN by gel filtration chromatography in the presence of SDS [17] and was essentially devoid of phosphatase activity ( <1% of the native enzyme).…”

Section: En'-yme Preparaffonmentioning

confidence: 99%

Inhibition of calcineurin by cyclosporin A‐cyclophilin requires calcineurin B

et al. 1992

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The interaction of the immunosuppressive complex cyclosporin A‐cyclophilin (CsA‐CyP) with the Ca2+/calmodulin‐dependent protein phosphatase calcineurin is investigated using a recombinant form of the A subunit of calcineurin (rCNA). Only in the presence of purified calcineurin B (CNB) does rCNA show the response of native calcineurin, i.e. 50% inhibition of rCNA phosphatase activity at 6 nM human cyclophilin B and 0.6 μM human cyclophilin A using [32P]casein as substrate, yet stimulation of activity with p‐nitrophenyl phosphate as substrate. This study demonstrates that the B subunit is necessary to confer sensitivity of calcineurin to CsA‐CyP.

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[22] Isolation and characterization of bovine brain calcineurin: A calmodulin-stimulated protein phosphatase

Cited by 140 publications

References 21 publications

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

Inhibition of calcineurin by cyclosporin A‐cyclophilin requires calcineurin B

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[22] Isolation and characterization of bovine brain calcineurin: A calmodulin-stimulated protein phosphatase

Cited by 140 publications

References 21 publications

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase

Factors responsible for the Ca2+‐dependent inactivation of calcineurin in brain

Inhibition of calcineurin by cyclosporin A‐cyclophilin requires calcineurin B

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Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain