CD45 and RPTPalpha display different protein tyrosine phosphatase activities in T lymphocytes.
To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPalpha, RPTPalpha was expressed in a CD45(-), T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPalpha did not fully restore either proximal or distal TCR-mediated signalling events. RPTPalpha was unable to reconstitute the phosphorylation of CD3zeta and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPalpha did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56(lck) or p59(fyn), suggesting that RPTPalpha does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPalpha was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56(lck) as substrates indicated that CD45 was consistently more active than RPTPalpha, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPalpha, which provides one reason why RPTPalpha cannot effectively dephosphorylate p56(lck) and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.
Ng
D H
DH
Department of Microbiology and Immunology, 300-6174 University Boulevard, University of British Columbia, Vancouver, B.C., Canada V6T 1Z3.
Jabali
M D
MD
Maiti
A
A
Borodchak
P
P
Harder
K W
KW
Brocker
T
T
Malissen
B
B
Jirik
F R
FR
Johnson
P
P
D000818:Animals; D015153:Blotting, Western; D017252:CD3 Complex / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology / Q000502:physiology; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured
D017252:CD3 Complex; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C494960:Fyn protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases; C516843:Ptpra protein, mouse; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4