Demonstration of a Direct Interaction between p56 and the Cytoplasmic Domain of CD45 in Vitro

Abstract

p56lck is a potential in vivo substrate for the tyrosinespecific phosphatase, CD45. In this study, recombinant purified p56 lck was found to specifically associate with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of another related tyrosine phosphatase, receptor protein-tyrosine phosphatase ␣. Under equilibrium binding conditions, the binding was saturable and occurred at a 1:1 molar stoichiometry. A fusion protein containing only the amino-terminal region of p56 lck (residues 34 -150) also bound to recombinant CD45, and further analysis of this region indicated that glutathione S-transferase fusion proteins of the unique amino-terminal region and the SH2 domain, but not the SH3 domain of p56 lck , bound to recombinant CD45. The SH2 domain protein bound with a higher affinity than the amino-terminal region, but both were able to compete for the binding of p56 lck to CD45, and when added together worked synergistically to compete for p56 lck binding. The SH2 domain interaction with CD45 was specific as glutathione S-transferase-SH2 fusion proteins from p85␣ subunit of phosphatidylinositol 3-kinase and SHC did not bind to CD45. In addition, this interaction occurred in the absence of any detectable tyrosine phosphorylation on CD45, suggesting a nonconventional SH2 domain interaction. p56lck and CD45 (see Ref. 1 for review) are both required to generate an effective T cell antigen receptor (TCR) 1 -mediated signal, being required for the earliest detectable event to occur upon TCR stimulation, the tyrosine phosphorylation of cellular proteins (2, 3). p56lck is a member of the Src family of tyrosine kinases and is thought to be regulated by tyrosine phosphorylation at the autophosphorylation and negative regulatory sites. Tyrosine phosphorylation at the carboxyl-terminal negative regulatory site is believed to result in an intramolecular interaction with its SH2 domain which results in an inactive or inaccessible kinase (4). In the presence of CD45 this negative regulatory tyrosine residue in p56 lck is dephosphorylated (5-9), and p56lck is then thought to be able to participate in TCRmediated signal transduction events (10 -12). Exactly how dephosphorylation of p56 lck results in effective TCR-mediated signal transduction remains unclear as recent data indicate that this dephosphorylation event may not necessarily lead to increased kinase activity (13). Dephosphorylation of these kinases may also result in the release of the intramolecular binding of the carboxyl-terminal tyrosine to the SH2 domain, which would then allow the unoccupied SH2 domain to bind to other tyrosine-phosphorylated proteins. Hence CD45 may act to regulate the SH2 domain interactions of p56 lck , which itself could be crucial in mediating a TCR induced signal. Consistent with such a model is the demonstration that the SH2 domain of p56 lck also plays an active role in T cell activation (14). CD45 is a major lymphocyte glycoprotein and a transmembrane two domain tyrosine-specific phosphatase (reviewed in Ref. ...

Dynamic Association of CD45 with Detergent-Insoluble Microdomains in T Lymphocytes

“…This is in contrast to the predominant interaction between p56 lck and CD45, which is unaffected by MCD and is therefore unlikely to be DRM dependent. This is consistent with previous reports showing that recombinant p56 lck can bind to recombinant cytoplasmic region of CD45 in vitro (33).…”

Interactions of CD45-associated Protein with the Antigen Receptor Signaling Machinery in T-lymphocytes

“…In part, this interaction is probably indirect, as a consequence of the capacity of p56 lck to associate with CD4 and CD8 through its unique region (42,56) and possibly with CD45 via its unique and SH2 domains (52). Nonetheless, our transient expression experiments in COS-1 cells, and those in HeLa cells reported by others (25), showed that an association between CD45-AP and Lck could take place in the absence of other lymphoid-specific components.…”

“…Although the basis for this association was not determined, it was shown to happen in the absence of CD4 and CD8 expression (15). Further experiments with recombinant proteins suggested that this interaction was possibly direct, as a result of an association between the cytoplasmic portion of CD45 and the unique and SH2 domains of p56 lck (52). Nevertheless, recent analyses of CD45-AP-deficient Tcells suggested that the association between CD45 and Lck may be facilitated by CD45-AP, especially in TCR-stimulated cells (30).…”

CD148, a membrane protein tyrosine phosphatase, is able to induce tyrosine phosphorylation on human lymphocytes

“…In fact, it has been shown that p56 lck co-immunoprecipitates with CD45 thus establishing a link between these two molecules [9]. Although we have no data on the effect of CD148 on p56 lck , no clear phosphorylation of substrates around 56 Kd was detected after CD148 stimulation.…”