A Method for the Purification of Phospho(Tyr)calmodulin Free of Nonphosphorylated Calmodulin

Palomo-Jiménez, Paloma I.; Hernández-Hernando, Silvia; Garciá-Nieto, R.M.; Villalobo, Antonio

doi:10.1006/prep.1999.1092

Cited by 15 publications

(14 citation statements)

References 26 publications

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“…Thus, the potential differential role exerted by P-(Tyr)-CaM as compared with nonphosphorylated CaM has been assessed in vitro on systems such as phosphodiesterase 1 (PDE1), nitric oxide synthase (NOS) and the plasma membrane Ca 2 + -ATPase among others [32]. CaM phosphorylated by the EGFR was incapable of activating PDE1 from bovine heart [36], whereas insulin receptor-phosphorylated CaM has no differential effect as compared to non-phosphorylated CaM when tested toward PDE1 isolated from rat hepatocytes [52]. P-(Tyr99)-CaM tested on NOS had a tremendous effect on its activity, increasing the V max of the enzyme more than 3-fold compared with non-phosphorylated CaM [53].…”

Section: Discussionmentioning

confidence: 99%

“…One unit of c-Src transfers 2.6 pmoles of phosphate per hour to 375 μM of Src substrate peptide using the Src assay kit provided by the manufacturer. The formed 32 P-(Tyr)-CaM was purified as described earlier [36] in three steps: (i) isolation of the CaM fraction containing 32 P-(Tyr)-CaM and non-phosphorylated CaM using Ca 2 + -dependent hydrophobic chromatography in a phenylSepharose column; (ii) purification of 32 P-(Tyr)-CaM, free of non-phosphorylated CaM, by using a column of immobilized anti-phospho-tyrosine antibody covalently linked to agarose upon elution with phenyl-phosphate; and (iii) removal of phenylphosphate by filtration chromatography by using a Bio-Gel P-2 column [36]. Quantification of the purified 32 P-(Tyr)-CaM was done by measuring the densitometry of the band observed by Western blot using an anti-CaM antibody and known amounts of purified pig brain CaM as standards.…”

Section: Cam Phosphorylation and Purification Of 32 P-(tyr)-cammentioning

confidence: 99%

“…To directly test the potential capacity of P-(Tyr)-CaM to enhance the EGF-dependent activation of the isolated EGFR, we phosphorylated CaM with recombinant c-Src in vitro and prepared purified P-(Tyr)-CaM free of non-phosphorylated CaM by using the method previously described [36]. Figure 3(A) shows that addition of purified 32 P-(Tyr)-CaM to the assay system strongly enhanced the EGF-dependent phosphorylation of PGT mediated by the EGFR, whereas no significant effect of 32 P-(Tyr)-CaM was detected in the absence of EGF.…”

Section: Purified P-(tyr)-cam Enhances Egf-dependent Egfr Activationmentioning

confidence: 99%

“…We have previously demonstrated that the EGFR phosphorylates CaM in the absence of Ca 2 + and presence of different cationic polypeptides with a stoichiometry close to 1 (mol/mol), preferentially at Tyr 99 as compared with Tyr 138 [17,18,[33][34][35][36][37]]. Hence, we tested whether phospho-(Tyr)-CaM exerts an effect aimed at modulating the ligand-dependent activation of EGFR.…”

Section: Introductionmentioning

confidence: 99%

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The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

Stateva

Salas

Benguría

et al. 2015

Biochemical Journal

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The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca 2 + , but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca 2 + . This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca 2 + , absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized antiphospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

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Section: Discussionmentioning

confidence: 99%

Section: Cam Phosphorylation and Purification Of 32 P-(tyr)-cammentioning

confidence: 99%

Section: Purified P-(tyr)-cam Enhances Egf-dependent Egfr Activationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

Stateva

Salas

Benguría

et al. 2015

Biochemical Journal

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“…CaM possesses only two tyrosine residues, Tyr 99 and Tyr 138 both of which are known to be phosphorylated [4]. Evidence show that most of the phosphorylation (90%) occurs at Tyr 99 and only 10% at Tyr 138 [5][6][7]. In the proposed study, we focus on investigating the hypoxiainduced phosphorylation of CaM at Tyr 99 in the cerebral cortex of newborn piglets.…”

Section: Introductionmentioning

confidence: 94%

Mechanism of Increased Tyrosine (Tyr99) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

Mishra

Ashraf

Delivoria‐Papadopoulos

2009

Neurochem Res

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The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr(99) phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F(i)O(2) of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr(99) and total) of calmodulin determined by Western blot using anti-phospho-(pTyr(99))-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr(99) calmodulin (ODxmm(2)) was 78.55 +/- 10.76 in Nx, 165.05 +/- 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 +/- 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 +/- 13.69 in Nx, 156.17 +/- 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 +/- 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr(99) phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr(99) phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr(99) phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr(99) phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.

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The Epidermal Growth Factor Receptor and the Calcium Signal

Villalobo

Ruano

Palomo-Jiménez

et al. 2000

Calcium: The Molecular Basis of Calcium Action in Biology and Medicine

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A Method for the Purification of Phospho(Tyr)calmodulin Free of Nonphosphorylated Calmodulin

Cited by 15 publications

References 26 publications

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

Mechanism of Increased Tyrosine (Tyr99) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

The Epidermal Growth Factor Receptor and the Calcium Signal

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