Characterization of the Interaction between CD45 and CD45-AP

CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56 lck , a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56 lck . The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56 lck could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56 lck . Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56 lck . Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.Activation of T-lymphocytes by antigen is initiated by protein tyrosine phosphorylation (1-4). Although the T-cell antigen receptor (TCR) 1 and the associated CD3 and subunits are devoid of intrinsic protein tyrosine kinase activity, they can recruit two classes of cytoplasmic protein tyrosine kinases to mediate this response, the Src family and the Syk/Zap-70 family. The Src-related enzymes Lck and FynT initiate TCR-mediated signals by phosphorylating a signaling motif in the cytoplasmic domain of CD3 and termed ITAM (for immunoreceptor tyrosine-based activation motif). Following this phosphorylation, the Syk/Zap-70 family kinases are activated through binding of their tandem Src homology 2 (SH2) domains to doubly phosphorylated ITAMs. Together with Src family kinases, Zap-70 and Syk are responsible for subsequent tyrosine phosphorylation of several signal transduction molecules including phospholipase C-␥1, Cbl, Vav, Slp-76, and Lat.CD45 is a 180 -220-kDa transmembrane protein tyrosine phosphatase expressed on all nucleated hemopoietic cells (5, 6). In T-cells it constitutes ϳ10% of all cell surface glycoproteins. Previous studies have shown that CD45 is necessary for T-cell activation because of its ability to promot...

Section: Discussionsupporting

confidence: 73%

Section: Discussionsupporting

confidence: 70%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Veillette¹,

Soussou²,

Latour³

et al. 1999

“…2 Since p56 lck has not been co-precipitated in the absence of the 30-kDa protein (CD45AP or LPAP), it is not known whether p56 lck can directly associate with CD45 in T cells. In contrast, p30 has been shown to co-precipitate with CD45 in the absence of p56 lck (20), and recently, the transmembrane region of CD45 has been shown to be required for the association of p30 (42). However, as only a small percentage of p56 lck is co-precipitated with CD45, 2 the physiological significance of this association remains to be established.…”

Section: Fig 6 Binding Of Recombinant Gst Sh2 Domain Fusion Proteinmentioning

confidence: 93%

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

Watts

Aebersold

et al. 1996

Self Cite

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. ...

“…For example, the extracellular domain of CD45, primarily CD45R0, has been reported to associate with CD4, which in turn binds Lck (41,42). Although the transmembrane region of CD45 is not required for its association with Lck, it does mediate an interaction with CD45AP (43), which also binds to Lck (44). Although CD45RABC-D2 can associate with Lck in the absence of CD45, CD45RABC-D2 may also bind to CD45 because a small amount co-immunoprecipitated with endogenous CD45 (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Expression of CD45 Lacking the Catalytic Protein Tyrosine Phosphatase Domain Modulates Lck Phosphorylation and T Cell Activation

Wang

Johnson

2005

Self Cite

The function of the second protein tyrosine phosphatase domain (D2) in two-domain protein tyrosine phosphatases (PTP) is not well understood. In CD45, D2 can interact with the catalytic domain (D1) and stabilize its activity. Although D2 itself has no detectable catalytic activity, it can bind substrate and may influence the substrate specificity of CD45. To further explore the function of D2 in T cells, a full-length construct of CD45 lacking the D1 catalytic domain (CD45RABC-D2) was expressed in CD45؉ and CD45 ؊ Jurkat T cells. In CD45؊ Jurkat T cells, CD45RABC-D2 associated with Lck but, unlike its active counterpart CD45RABC, did not restore the induction of tyrosine phosphorylation or CD69 expression upon T cell receptor (TCR) stimulation. Expression of CD45RABC-D2 in CD45؉ Jurkat T cells resulted in its association with Lck, increased the phosphorylation state of Lck, and reduced T cell activation. TCR-induced tyrosine phosphorylation was delayed, and although MAPK phosphorylation and CD69 expression were not significantly affected, the calcium signal and IL2 production were severely reduced. This indicates that the non-catalytic domains of CD45 can interact with Lck in T cells. CD45RABC-D2 acts as a dominant negative resulting in an increase in Lck phosphorylation and a preferential loss of the calcium signaling pathway, but not the MAPK pathway, upon TCR signaling. This finding suggests that, in addition to their established roles in the initiation of TCR signaling, CD45 and Lck may also influence the type of TCR signal generated. CD45 is a transmembrane two-domain protein tyrosine phosphatase (PTP)1 expressed exclusively in leukocytes (reviewed in Refs. 1-4). There are 21 transmembrane PTP in the human genome, and 12 have two domains (5). The catalytic activity of CD45 and other transmembrane two-domain PTP, such as PTP␣ and LAR, resides primarily in the first membrane proximal PTP with little or no activity attributed to the second PTP domain, D2 (6). In the case of CD45, the majority of data indicate that D2 is inactive (7-9). It lacks critical catalytic residues and cannot readily bind phosphotyrosine (10). Two functions have been proposed for CD45-D2: 1) to interact with and stabilize the catalytic D1 domain of CD45 (7, 11) and 2) to bind substrate and facilitate substrate recruitment (10, 12).CD45 is required for T cell activation by constitutively dephosphorylating the negative regulatory tyrosine of Lck, Tyr 505 (reviewed in Refs. 4 and 13-15). The restoration of T cell signaling by activated Lck in CD45 Ϫ T cells (16) and restoration of T cell development in the CD45 null mouse by expression of Lck Y505F (17) support this finding. Dephosphorylation at Tyr 505 creates a "primed" Lck molecule that can become activated upon TCR encounter with antigen, which initiates the signal transduction cascade by phosphorylating downstream substrates such as CD3 and ZAP-70 and leads to the activation of PLC␥-1, inositol 1,4,5-trisphosphate (IP 3 ) generation, an increase in intracellular calcium, CD69 expressi...