SPECIFIC INTERACTION OF THE CD45 PROTEIN-TYROSINE-PHOSPHATASE WITH TYROSINE-PHOSPHORYLATED CD3 ZETA-CHAIN

The CD45 transmembrane protein-tyrosine phosphatase (PTPase, EC 3.1.3. 48) plays an essential role in T-cell activation by activating the Lck and/or Fyn protein-tyrosine kinases. However, numerous experiments ha ve indicated that CD45 may have both stimulatory and inhibitory roles in T-cell activation. Thus, it is unlikely that the two kinases are th e sole substrates of the CD45 PTPase. Furthermore, the complex regulat ion of the alternative splicing of the extracellular domain in various leukocyte lineages also suggests additional roles for the CD45 PTPase . To identify such functions, it is necessary to identify physiologica lly relevant substrates of the CD45 PTPase other than the two protein- tyrosine kinases. To this end, we searched for high-affinity substrate s of the CD45 PTPase among the tyrosine-phosphorylated T-cell proteins by using purified glutathione S-transferase-CD45 fusion molecules. Th e enzymatically inactive CD45 C828S mutant protein, in which the cyste ine residue at the catalytic center was changed to a serine residue, b ound tightly to the phosphorylated CD3 zeta chain. This binding was sp ecific to CD45 PTPase, as neither the leukocyte common antigen-related molecule (LAR) PTPase nor the CD45-LAR hybrid PTPases bound the phosp horylated CD3 zeta chain. Furthermore, phosphorylated CD3 zeta chain w as preferentially dephosphorylated by the wild-type CD45 PTPase under conditions that did not significantly dephosphorylate other cellular p roteins. Thus, the phosphorylated CD3 zeta chain is a specific and hig h-affinity substrate of the CD45 PTPase. These results suggest that CD 45 is involved in the termination of the T-cell response via dephospho rylation of CD3 zeta chain.