Mutational analysis of CD45. A leukocyte-specific protein tyrosine phosphatase.
The cytoplasmic domain of murine CD45 has been expressed using an in vitro transcription/translation system. The recombinant protein was isolated by immunoprecipitation with a specific rabbit antiserum and was shown to have protein tyrosine phosphatase (PTPase) activity. Oligonucleotide-directed mutagenesis was then used to investigate the structural requirements for PTPase activity. Although the cysteine crucial for PTPase activity resides in domain I, this domain was not active alone. Both PTPase domains of CD45 and the membrane proximal region of 77 amino acids were required for enzymatic activity. Deletion of 78 residues at the carboxyl terminus of the cytoplasmic region did not influence activity, but an additional deletion of 13 amino acids from PTPase domain II totally abolished activity. Excision of the 21-residue acidic insert in the second PTPase domain resulted in a decrease of activity of approximately 4-fold. Nine conserved residues around the critical cysteine in the first domain were found to be important for activity. Of the 3 conserved tyrosine residues in domain I, only Tyr729 was specifically required for activity.
Johnson
P
P
Department of Cancer Biology, Salk Institute, San Diego, California 92186-5800.
Ostergaard
H L
HL
Wasden
C
C
Trowbridge
I S
IS
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006649:Histocompatibility Antigens / Q000235:genetics*; D017493:Leukocyte Common Antigens; D007962:Leukocytes / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics; D014158:Transcription, Genetic
D015703:Antigens, CD; D006649:Histocompatibility Antigens; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D017493:Leukocyte Common Antigens