TAILIEUCHUNG - Báo cáo khoa học: Tyrosine phosphorylation of calponins Inhibition of the interaction with F-actin

The phosphorylation-dephosphorylation of serine and threonine residues of calponin is known tomodulatein vitro its interactionwithF-actin and is thought to regulate several biological processes in cells, involving either of the calponin isoforms. Here, we identify, for the first time, tyrosine-phosphorylated calponin h3 within COS 7 cells, before and after their transfection with the pSV vector containing cDNA encoding the cytoplasmic, Src-related, tyrosine kin-ase, Fyn. | Eur. J. Biochem. 271 2615-2623 2004 FEBS 2004 doi Tyrosine phosphorylation of calponins Inhibition of the interaction with F-actin Julien Abouzaglou1 Christine Benistant1 Mario Gimona2 Claude Roustan3 Rhida Kassab1 and Abdellatif Fattoum1 1 Centre de Recherches de Biochimie Macromoléculaire du CNRS Montpellier France 2Institute of Molecular Biology Salzburg Austria 3 UMR 5539 CNRS UM2 EPHE Montpellier France The phosphorylation-dephosphorylation of serine and threonine residues of calponin is known to modulate in vitro its interaction with F-actin and is thought to regulate several biological processes in cells involving either of the calponin isoforms. Here we identify for the first time tyrosine-phosphorylated calponin h3 within COS 7 cells before and after their transfection with the pSV vector containing cDNA encoding the cytoplasmic Src-related tyrosine kinase Fyn. We then describe the specific tyrosine phosphorylation in vitro of calponin h1 and calponin h3 by this kinase. 32P-labeling of tyrosine residues was monitored by combined autoradiography immunoblotting with a specific phosphotyrosine monoclonal antibody and dephosphorylation with the phosphotyrosine-specific protein phosphatase YOP. PhosphorImager analyses showed the incorporation of maximally and mol of 32P per mol of calponin h3 and calponin h1 respectively. As a result 75 and 68 respectively of binding to F-actin was lost by the phosphorylated calponins. Furthermore F-actin added at a two-or 10-fold molar excess did not protect but rather increased the extent of 32P-labeling in both calponins. Structural analysis of the tryptic phosphopeptides from each 32P-labe-led calponin revealed a single major 32P-peptide in calponin h3 with Tyr261 as the phosphorylation site. Tyr261 was also phosphorylated in calponin h1 together with Tyr182. Collectively the data point to the potential involvement at least in living nonmuscle cells of tyrosine protein kinases and .

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