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Acta Biochimica et Biophysica Sinica Advance Access originally published online on August 30, 2009
Acta Biochimica et Biophysica Sinica 2009 41(10):846-851; doi:10.1093/abbs/gmp071
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Regulation of membrane band 3 Tyr-phosphorylation by proteolysis of p72Syk and possible involvement in senescence process

Luciana Bordin1, Cristina Fiore2, Marcantonio Bragadin3, Anna Maria Brunati1 and Giulio Clari1,*

1 Dipartimento di Chimica Biologica, Università di Padova, Viale G. Colombo 3, 35121 Padova, Italy
2 Dipartimento di Scienze Mediche Chirurgiche-Endocrinologia, Università di Padova, Via Ospedale 105, 35100 Padova, Italy
3 Dipartimento di Scienze Ambientali, Università di Venezia, Calle Larga S. Marta 2137, 30123 Venezia, Italy

* Correspondence address. Tel: +39-49-8276113; Fax: +39-49-8073310; E-mail: labclari{at}mail.bio.unipd.it


  Abstract

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation (Tyr-P) of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa (p72Syk) to the 36 kDa (p36Syk) isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32P uptake into the cytoplasmic domain of band 3 after addition of p72Syk or p36Syk. Effect of pre-phosphorylation of erythrocyte membrane band 3 protein by p36Syk on p72Syk-mediated phosphorylation and the effect of addition of a protease inhibitor (leupeptin) on p72Syk-mediated phosphorylation were studied by autoradiographic visualization of 32P uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36Syk had a higher band 3 tyrosine phosphorylating activity compared with p72Syk. Pre-phosphorylation with p36Syk or p72Syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72Syk but not p36Syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72Syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton.

Keywords    band 3 Tyr-phosphorylation; Syk; proteolysis; human erythrocyte

Received: April 26, 2009; Accepted: June 6, 2009
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