The MBP fusion protein restores the activity of the first phosphatase domain of CD45.
CD45 is a receptor-like protein tyrosine phosphatase critically involved in the regulation of initial effector functions in B- and T-cells. The protein comprises two phosphatase (PTP) domains in its cytoplasmic region. However, whether each PTP domain has enzyme activity by itself or whether both domains are required to build up a functional enzyme is unclear. We have studied different constructions of human CD45 comprising the two PTP domains, both separately and as a single protein, fused to maltose-binding protein (MBP). In apparent contrast with previous studies, we show that the first PTP domain of CD45 (when fused to MBP) may be a viable phosphatase in the absence of the second domain. Phosphatase activity resides in the monomeric form of the protein and is lost after proteolytic cleavage of the fusion partner, indicating that MBP specifically activates the first PTP domain. Furthermore, changes in the optimal pH for activity with respect to wild-type CD45 suggest that protein-protein interactions involving residues in the neighbourhood of the catalytic site mediate enzyme activation.
Lorenzo
H K
HK
Unité d'Immunologie Structurale, Institut Pasteur, Paris, France.
Farber
D
D
Germain
V
V
Acuto
O
O
Alzari
P M
PM
D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015951:Factor Xa / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011993:Recombinant Fusion Proteins; C008644:nitrophenylphosphate; D017493:Leukocyte Common Antigens; D015951:Factor Xa