CD45, an integral membrane protein tyrosine phosphatase. Characterization of enzyme activity.
Although CD45 resembles the low Mr protein tyrosine phosphatases (PTPases) from human placenta in its specificity for phosphotyrosyl residues and absolute dependence on sulfhydryl compounds for activity, it also exhibits a number of distinguishing features. Most notably, it displayed substrate specificity in vitro, preferentially dephosphorylating myelin basic protein, over the other substrates tested, with high specific activity. Limited trypsinization of CD45 generated active fragments of approximately 65 kDa that were apparently derived exclusively from the intracellular segment of the molecule. These retained high activity against myelin basic protein, suggesting that this is an intrinsic feature of the PTPase domains and not the result of secondary interactions between the substrate and the putative ligand binding structure. With reduced carboxamidomethylated and maleylated lysozyme as substrate, CD45 was stimulated up to 12-fold by basic compounds such as spermine; divalent metal ions were also stimulatory, most notably Zn2+, which was previously identified as a potent inhibitor of the low Mr PTPases. CD45 was phosphorylated to high stoichiometry by casein kinase-2 (up to 1.5 mol/mol) and also by glycogen synthase kinase 3 (approximately 0.3 mol/mol) and protein kinase C (approximately 0.1 mol/mol); in all cases, no alteration in enzyme activity was detected following these modifications. Autophosphorylated preparations of epidermal growth factor receptor, insulin receptor, and p56lck protein tyrosine kinases were also substrates for CD45 in vitro.
Tonks
N K
NK
Department of Biochemistry, University of Washington, Seattle 98195.
Diltz
C D
CD
Fischer
E H
EH
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000302:isolation & purification*; D000943:Antigens, Differentiation / Q000302:isolation & purification*; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D005260:Female; D006649:Histocompatibility Antigens / Q000302:isolation & purification*; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D017493:Leukocyte Common Antigens; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D017027:Protein Tyrosine Phosphatases; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D015703:Antigens, CD; D000943:Antigens, Differentiation; D006649:Histocompatibility Antigens; D007106:Immune Sera; D008562:Membrane Glycoproteins; D010455:Peptides; D010749:Phosphoprotein Phosphatases; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases; D014357:Trypsin