Phosphotyrosine phosphatases acting on band 3 in human erythrocytes of different age: PTP1B processing during cell ageing.
The phosphorylation of tyrosine residues of human red blood cell (RBC) band 3 is regulated in vivo by constitutively active tyrosine-kinases (PTKs) and phosphotyrosine-phosphatases (PTPs), identified so far as, respectively, p72(syk) and p56/53(lyn), and PTP1B and SHPTP-2. Tyr-phosphorylation of band 3 increases upon reduction of cell volume as in hypertonic media or during Ca(2+)-induced membrane vesiculation. We show here that old RBCs display higher Tyr-phosphorylation levels of band 3 than younger cells under hypertonic conditions, at least in part due to the reduced cell volume of old RBCs, a condition of lowered threshold for activation of volume-sensitive PTKs. We have also analysed the membrane-bound PTP activity and the relative abundance of PTP1B (as the main membrane-associated PTP) in RBCs of different age. Immunodetection of PTP1B in purified ghost membranes revealed that the catalytic, N-terminal domain of the PTP is partially cleaved, in an age-dependent manner, from the membrane-bound domain, and it is lost during the preparation of ghost membranes. This suggests that erythrocytes may undergo in vivo activation of the Ca(2+)-dependent calpain system that proteolytically regulates PTP1B activity, as already documented for other cell types. On the other hand, the assay of the PTP activity that remains associated with the membranes of RBCs of different age indicated that the PTP undergoes oxidative inactivation that can be further differentiated into reversible and irreversible components.
Ciana
Annarita
A
Università di Pavia, Dipartimento di Biochimica "A. Castellani", Sezione di Scienze, via Bassi, 21, 27100 Pavia, Italy.
Minetti
Giampaolo
G
Balduini
Cesare
C
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods; D004229:Dithiothreitol / Q000494:pharmacology; D004905:Erythrocyte Aging; D004912:Erythrocytes / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D006982:Hypertonic Solutions; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D004229:Dithiothreitol