Regulation of membrane band 3 Tyr-phosphorylation by proteolysis of p72(Syk) and possible involvement in senescence process.
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 (p72(Syk)) to the 36 kDa (p36(Syk)) isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of (32)P uptake into the cytoplasmic domain of band 3 after addition of p72(Syk) or p36(Syk). Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36(Syk) on p72(Syk)-mediated phosphorylation and the effect of addition of a protease inhibitor (leupeptin) on p72(Syk)-mediated phosphorylation were studied by autoradiographic visualization of (32)P uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36(Syk) had a higher band 3 tyrosine phosphorylating activity compared with p72(Syk). Pre-phosphorylation with p36(Syk) or p72(Syk) increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72(Syk) but not p36(Syk) 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 p72(Syk) is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton.
Dipartimento di Chimica Biologica, Università di Padova, Viale G. Colombo 3, Padua, Italy.
Luciana
L
Bordin
Cristina
C
Fiore
Marcantonio
M
Bragadin
Anna Maria
AM
Brunati
Giulio
G
Clari
D000375:Aging / Q000502:physiology*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000502:physiology*; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D010447:Peptide Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D010447:Peptide Hydrolases