Rapid degradation of protein tyrosine phosphatase 1B in sickle cells: Possible contribution to sickle cell membrane weakening

Abstract

Although both protein tyrosine phosphatases and kinases are constitutively active in healthy human red blood cells (RBCs), the preponderance of phosphatase activities maintains the membrane proteins in a predominantly unphosphorylated state. We report here that unlike healthy RBCs, proteins in sickle cells are heavily tyrosine phosphorylated, raising the question regarding the mechanism underpinning this tyrosine phosphorylation. Upon investigating possible causes, we observe that protein tyrosine phosphatase 1B (PTP1B), the major erythrocyte tyrosine phosphatase, is largely digested to a lower molecular weight fragment in sickle cells. We further find that the resulting truncated form of PTP1B is significantly less active than its intact counterpart, probably accounting for the intense tyrosine phosphorylation of Band 3 in sickle erythrocytes. Because this tyrosine phosphorylation of Band 3 promotes erythrocyte membrane weakening that causes release of both membrane vesicles and cell free hemoglobin that in turn initiates vaso-occlusive events, we conclude that cleavage of PTP1B could contribute to the symptoms of sickle cell disease. We further posit that methods to inhibit proteolysis of PTP1B could mitigate symptoms of the disease.

Keywords: Band 3 tyrosine phosphorylation; PTP1B; calpain proteolysis; cleavage of PTP1B; phosphatase activity; sickle cells.

FIGURE 1

Tyrosine phosphorylation of erythrocyte membrane proteins in healthy individuals and sickle cell disease patients. Erythrocyte membrane proteins from healthy volunteers and sickle cell patients were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with anti-phosphotyrosine antibodies (left panel). Anti-actin immunostaining was used as a loading control. Coomassie staining was used to evaluate the integrity of the major membrane proteins (right panel).

FIGURE 2

Effect of different tyrosine phosphatase inhibitors on tyrosine phosphorylation of Band 3. Erythrocytes from healthy donors were treated with different protein tyrosine phosphatase inhibitors, after which the extent of Band 3 tyrosine phosphorylation was determined by Western blotting. The phosphatase inhibitors examined were: (A) PTPV (Calbiochem CAS #314291-83-3) and PTPXVIII (CAS #1229246-07-4) that have moderate specificity for protein tyrosine phosphatase 1B (PTP1B), (B) PTP1B inhibitor (Cayman #15782) that has high specificity for PTP1B, (C) SHP-1 inhibitor (Axon Medchem #2723), and (D) SHP-2 inhibitor (Axon Medchem #2633).

FIGURE 3

Comparison of the abilities of tyrosine phosphatases protein tyrosine phosphatase 1B (PTP1B) and SHP2 to dephosphorylate Syk phosphorylated Band 3 cytoplasmic domain. Equal units of activity of full length PTP1B and SHP2 enzymes were incubated with tyrosine phosphorylated Band 3 (1–379). Cbd3, cytoplasmic domain of band 3; cdb3-PO4, phosphorylated cdb3.

FIGURE 4

(A) Assessment of protein tyrosine phosphatase 1B (PTP1B) cleavage in healthy and sickle erythrocytes by anti-PTP1B immunoblotting. (B) Examination of the correlation between PTP1B cleavage and Band 3 tyrosine phosphorylation in each blood sample. (C) Examination of the correlation between PTP1B cleavage and percent HbS in each blood sample. Blood from healthy control (C), and sickle cell patients treated with hydroxyurea (SCD3) or repeated transfusions (SCD4) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunostained with mouse anti-phosphotyrosine (A, top panel) or anti-PTP1B antibody (A, bottom panel) antibodies. Actin content was used as a loading control (A, bottom panel).

FIGURE 5

Impact of protein tyrosine phosphatase 1B (PTP1B) cleavage on its phosphatase activity. Catalytic activities of the 42 kDa short and 50 kDa full length PTP1B enzymes were analyzed using 4-nitrophenyl phosphate as a substrate (A), yielding the results shown in panel (B).

FIGURE 6

Correlation between reticulocyte content and protein tyrosine phosphatase 1B (PTP1B) cleavage in whole blood samples from healthy donors and sickle cell patients. (A) Immunoblot assessing the content of cleaved and intact PTP1B in healthy volunteer and sickle cell patient blood samples. (B) Total amount of the sum of intact plus cleaved PTP1B relative to the amount measured in sample AA1. Also shown is the percent of total PTP1B in each sample that is cleaved.