Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


  • Pauline Johnson
  • Asanga Samarakoon
  • Amy E. Saunders
  • Kenneth W. Harder
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_34


Historical Background

CD45 was first identified by antisera and then by monoclonal antibodies (mAbs) as a major lymphocyte cell surface glycoprotein. CD45 is leukocyte specific, expressed on all hematopoietic cells except red blood cells and this has led to the clinical use of pan-specific CD45 mAbs to identify leukocytes and cells of hematopoietic origin. Some mAbs identify specific isoforms of CD45 that are expressed in a cell type and developmentally regulated manner and are referred to as CD45R mAbs. CD45 isoforms range from 180 to 220 kDa and cloning of the CD45 gene revealed that these isoforms arise by alternative splicing of at least three exons (ABC) that encode a region near the amino terminus of the extracellular domain of CD45 (Thomas 1989; Johnson et al. 1997). The B220 (CD45RABC) isoform of CD45 is expressed primarily on B cells and the CD45RA and RB isoforms are expressed on T cells and downregulated on T cell activation whereas the CD45RO isoform, which does not include any of the alternatively spliced exons, is upregulated on T cell activation. Thus CD45 isoform expression is a useful parameter to monitor the activation state of the T cell and has been particularly useful for human cells. The PTPRC gene, which encodes CD45, was cloned in the 1980s, revealing significant sequence identity of the cytoplasmic domain of CD45 with the protein tyrosine phosphatase, PTP1B. This led to the identification of CD45 as a receptor-like protein tyrosine phosphatase (Trowbridge and Thomas 1994). The phosphatase activity of CD45 was subsequently shown to be important for antigen receptor signaling in T and B lymphocytes where it regulates the phosphorylation state and activity of specific Src family kinases (Alexander 2000). In addition, work with CD45-deficient cells and mice has implicated CD45 in regulating leukocyte adhesion, cytokine signaling, and immune receptor signaling involving Fc, NK, and Toll-like receptors.

CD45 is a Transmembrane Protein Tyrosine Phosphatase

The PTPRC gene is conserved across several vertebrate species, from zebra fish, lamprey, and shark to chickens, cows, and humans. It spans over 50 MB and is located on chromosome 1q31–32 in humans and chromosome 1 at 74 cM in mice. Further information on the PTPRC gene, the protein sequences from the different species, and the crystallographic structure of the cytoplasmic domain of CD45 can be found at the National Center for Biotechnology Information, NCBI (http://www.ncbi.nlm.nih.gov). CD45 is a type I transmembrane protein, which is abundantly expressed on leukocytes. By electron microscopy, CD45 has a globular cytoplasmic domain of 12 nm and a rod-like extracellular domain of 28 nm, which extends to 51 nm when the alternatively spliced exons are included (Johnson and Felberg 2000). This makes CD45 one of the largest molecules on the lymphocyte membrane, being considerably larger than the cell surface molecules involved in antigen recognition that are estimated to extend approximately 8 nm from the cell surface. CD45 is heavily glycosylated with 25% of its mass attributed to carbohydrate. N-linked glycosylation sites are interspersed throughout the extracellular domain whereas O-linked sites are enriched in the amino terminal and alternatively spliced regions. The rest of the extracellular domain is relatively cysteine rich and may contain up to three fibronectin type III-like domains (Fig. 1).
CD45 (PTPRC), Fig. 1

Schematic of CD45 structure. CD45 is a type I transmembrane glycoprotein. The extracellular domain is relatively large and heavily glycosylated with several N-linked carbohydrate sites interspersed throughout the region whereas the majority of O-linked glycosylation sites are localized in the amino terminal region and the alternatively spliced regions (designated A, B, and C) of CD45. The cytoplasmic region of CD45 consists of a membrane proximal wedge region followed by two protein tyrosine phosphatases (D1 and D2), with the catalytic cysteine residing in D1, and then a short C-terminal region

CD45 has a single transmembrane region and a large intracellular domain of over 700 amino acids that has been shown by crystallographic studies to contain two protein tyrosine phosphatase domains. Only the membrane proximal phosphatase domain (D1) has catalytic activity but the second domain is required for optimal activity (Johnson and Felberg 2000). Since basal phosphotyrosine levels in naive lymphocytes are low, CD45 is thought to be a constitutively active phosphatase, whose activity can be regulated by its level of expression and by access to substrate. No specific ligands have been identified for CD45 but the carbohydrates of CD45 can bind lectin molecules such as galectin 1, which cluster CD45 and modulate its activity. Serine phosphorylation and the interaction of CD45 with other proteins (such as CD45AP, the CD45 associated protein) may also regulate its ability to dephosphorylate substrates, but these mechanisms are not well understood.

CD45 is a Major Regulator of the Src Family Kinases, Lck and Lyn

The  Src family tyrosine kinase, Lck, was identified as a key substrate for CD45 in T cells and Lyn was identified as a major substrate in B cells. Fyn may also be a CD45 substrate in T cells, but is less affected by the absence of CD45. In macrophages, the Src family tyrosine kinases Lyn and Hck have been identified as substrates for CD45 (Alexander 2000; Hermiston et al. 2003; Saunders and Johnson 2010). The Src family tyrosine kinases are regulated by two major tyrosine phosphorylation sites: a negative regulatory site close to the C-terminus of the protein and a positive regulatory site in a loop close to the active site of the kinase which, when phosphorylated, moves the loop away from the catalytic site thereby allowing substrate access (Williams et al. 1998). CD45 was shown to dephosphorylate Lck at the negative regulatory site in T cells, thus priming Lck for activation. However, subsequent work showed that CD45 could also dephosphorylate the positive regulatory site that is normally autophosphorylated in the active kinase. Thus CD45 has the ability to both upregulate and downregulate Lck activity in T cells and has the ability to maintain Lck in the primed, dephosphorylated state (Fig. 2). Although CD45 has been identified as a significant regulator of Src family kinase phosphorylation in hematopoietic cells, it is not the only regulator, as other tyrosine phosphatases such as CD148 can dephosphorylate the negative regulatory site and SHP-1 and Lyp/PEP can dephosphorylate the positive regulatory site (Hermiston et al. 2009).
CD45 (PTPRC), Fig. 2

Regulation of Src family kinase phosphorylation by CD45. CD45 can dephosphorylate both the negative (Y505) and positive (Y394) regulatory tyrosines of the Src family kinase, Lck in T cells. In its dephosphorylated state, Lck is maintained in a primed state. CD45 acts reciprocally to the  Csk kinase to dephosphorylate Lck at Y505. This releases the intracellular binding of phosphorylated Y505 to the SH2 domain to create an open, primed Lck. Lck either autophosphorylates or transphosphorylates Y394 which displaces the loop from the catalytic site, and creates an active kinase. CD45, as well as other phosphatases such as PEP, downregulate Lck activity by dephosphorylating Y394

CD45 and T Cell Antigen Receptor Signaling

The distribution of CD45 in the membrane, as well as the recruitment of Lck to the membrane either by myristoylation or by association with the CD4 or CD8 transmembrane proteins influences the ability of CD45 to access and dephosphorylate Lck. The basal activity of CD45 in naïve T cells primes Lck so it is ready to effectively participate in T cell receptor (TCR) signaling. Recognition by the TCR of peptide antigen presented by MHC class I or II molecules together with the binding of CD4 or CD8 co-receptor molecules to MHC molecules brings Lck into close proximity with the TCR associated  CD3 chains. Antigen recognition is thought to induce clustering of these TCR complexes, which excludes CD45 and allows Lck to phosphorylate the  CD3 chains at specific immunoregulatory tyrosine activation motifs (ITAMs) (van der Merwe and Dushek 2011). This results in the recruitment of the SH2 domain–containing tyrosine kinase  Zap-70 that further propagates the phosphorylation cascade and the recruitment of signaling proteins (Tomlinson et al. 2000 and Fig. 3). Signaling from these microclusters leads to T cell activation and proliferation. These microclusters also aggregate to form the center of an immune synapse (cSMAC), which is surrounded by a ring of adhesion molecules referred to as the pSMAC (Dustin et al. 2010). CD45 is dynamically regulated during this process; it is initially excluded from the microclusters and the initial cSMAC then accumulates at the cSMAC at later time points, where it may play a role in downregulating Lck kinase activity and terminating the phosphorylation-induced signals. In the absence of CD45, Lck is constitutively phosphorylated in T cells, which leads to inefficient TCR signaling and an increased threshold for T cell activation. This disrupts both positive and negative selection in the thymus, leading to reduced numbers of peripheral T cells in CD45-deficient mice that do not respond appropriately on antigen recognition (Alexander 2000; Hermiston et al. 2003; Saunders and Johnson 2010).
CD45 (PTPRC), Fig. 3

Function of CD45 in TCR signaling. In the unactivated T cell, CD45 dephosphorylates Lck on the negative regulatory site and maintains Lck in a primed, dephosphorylated state. On engagement of peptide-MHC on the antigen-presenting cell (APC) by the TCR, costimulatory molecules (CD4 or CD8) are recruited along with Lck and signaling clusters are formed that exclude CD45. Active Lck then phosphorylates tyrosine residues in the ITAMs on CD3 molecules (δ, ε and ζ), which leads to the recruitment and phosphorylation of the kinase ZAP-70. Activated ZAP-70 then phosphorylates signaling molecules and initiates a downstream signaling cascade that leads to T cell activation. Later in the signaling process, TCR clusters form an immune synapse and CD45 moves into the synapse where it is thought to inactivate Lck and terminate TCR signaling

CD45 and B Cell Antigen Receptor Signaling

B cell antigen receptor (BCR) signaling, like TCR signaling, requires protein tyrosine kinase cascades to propagate signals required for B cell development and function. However the BCR is less dependent on CD45 and Src family kinases and is instead more dependent on the Syk tyrosine kinase. Syk is not as dependent on CD45 for activation as some BCR signaling events do occur in the absence of CD45. As a result, B cell development occurs in CD45-deficient mice but with increased numbers of immature IgMhi cells and marginal zone B cells in the spleen and decreased numbers of circulating mature B cells (Hermiston et al. 2005). In CD45-deficient B cells, Syk and Btk are activated to some extent, Lyn is hyperphosphorylated and Fyn and Blk are not activated upon BCR engagement. This results in Igα and Igβ tyrosine phosphorylation but reduced ERK phosphorylation and diminished calcium signaling, which is insufficient to induce B cell proliferation in response to IgM cross-linking and points to a role for CD45 in enhancing BCR signaling strength. This reduced BCR signaling, as well as reduced phosphorylation of the costimulatory molecule, CD19, implicates CD45 in the positive regulation of Lyn, Fyn, and Blk (Fig. 4). However, CD22 was hyperphosphorylated in a CD45-deficient B cell line and Lyn was hyperphosphorylated at both the negative and positive regulatory sites and was more active in an in vitro assay, suggesting that CD45 may also negatively regulate Lyn (Huntington and Tarlinton 2004; Saunders and Johnson 2010). In B cells and myeloid cells, Lyn has the unique ability to phosphorylate immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in key negative regulatory molecules such as CD22, PIR-B, and FcγRIIB1 (Xu et al. 2005; Scapini et al. 2009). This non-redundant role for Lyn became evident through studies of Lyn-deficient mice where B cells are hyperresponsive to BCR ligation and resistant to signal inhibition induced by inhibitory receptor BCR co-ligation. Lyn-deficient mice also showed diminished tyrosine phosphorylation of key inhibitory phosphatases such as SHP-1 and SHIP-1. Many of these effects were opposite to those observed in CD45-deficient B cells, consistent with the loss of CD45 having an activating effect on Lyn. This view is strengthened by analysis of Lyn gain-of-function (Lynup) mice, which have enhanced BCR-induced calcium mobilization and tyrosine phosphorylation of both stimulatory (PLCγ, Syk) and inhibitory proteins, impaired B cell proliferation, and reduced circulating B cells in vivo. Thus there are many similarities between the CD45-deficient and Lynup B cells, with the minor differences perhaps being explained by the effect of CD45 on Blk and Fyn. It is also possible that CD45 has a dual effect on Lyn in B cells, downregulating its unique role activating inhibitory receptors and promoting Lyn’s role in BCR signaling (Fig. 4), but exactly how this occurs is not known. Overall, the data indicate a clear role for CD45 in regulating BCR-induced signaling by regulating Lyn kinase.
CD45 (PTPRC), Fig. 4

Function of CD45 in BCR and inhibitory protein (CD22) signaling. Antigen binding cross-links the BCR, which activates the Src family kinases (Lyn, Fyn, and Blk) to phosphorylate ITAMs on Igα and Igβ. This leads to the recruitment and activation of Syk, which can then phosphorylate signaling molecules propagating a downstream signal that leads to B cell activation and proliferation. CD45 dephosphorylates and positively regulates these Src family kinases involved in BCR signal transduction. However, Lyn also negatively regulates BCR signaling by phosphorylating ITIMs present on inhibitory receptors such as CD22. This recruits the SHP-1 tyrosine phosphatase which downregulates BCR signaling. The data suggest that in this situation, CD45 negatively regulates Lyn activity, thereby promoting BCR signaling. Thus the net effect of CD45 is a partial inhibitory effect on BCR signaling

Regulation of Additional Signaling Pathways by CD45 in Leukocytes

CD45 regulates other signals that are associated with ITIM or ITAM containing signaling molecules such as the Fc and NK receptors (Hermiston et al. 2009; Saunders and Johnson 2010). These receptors associate with ITAM-containing signaling proteins such as DAP12, FcRγ and  CD3ζ chains, which are phosphorylated by Src family kinases and recruit Syk or  Zap-70; thus, a similar pattern to antigen receptor regulation by CD45 is thought to exist. CD45 is required for IgE-mediated degranulation and IgE-mediated anaphylaxis in mast cells; however, FcRγ-mediated events such as IgG-mediated phagocytosis and antibody-dependent cytotoxicity in NK cells are not CD45 dependent. Interestingly, cytokine production induced by cross-linking NK cell receptors is CD45 dependent and correlates with impaired calcium mobilization and Syk and ERK activation. Thus the effect of CD45 may depend on the relative importance of Src family kinases in the response.

CD45 can also regulate cytokine signaling in hematopoietic cells (Saunders and Johnson 2010). CD45 can either upregulate or downregulate IFNα signaling in T cells and downregulates IL-3-induced proliferation in bone marrow–derived mast cells. CD45 can also negatively regulate erythropoietin-stimulated bone marrow progenitors to produce erythroid colony-forming units (CFU), although others see no difference in CFU after IL-3 stimulation. Penninger’s group showed that the Janus kinase, JAK2, was hyperphosphorylated in IFNα-stimulated CD45-deficient thymocytes, Jurkat T cells, and in IL-3-induced bone marrow–derived mast cells. This led the authors to conclude that CD45 is a JAK phosphatase (Penninger et al. 2001). Interestingly, Lyn-deficient mice have increased splenic CFUs in response to IL-3, GM-CSF, and CSF-1, illustrating that Lyn may also negatively regulate cytokine signaling (Hibbs and Harder 2006). Thus it is possible that CD45 may activate Lyn to downregulate cytokine signaling by phosphorylating inhibitory receptors. The receptors recruit inositol (SHIP-1) and tyrosine (SHP-1) phosphatases that inhibit cytokine signaling through the attenuation of the PI3K pathway or by dephosphorylation of Janus kinases, respectively.

In CD45-deficient macrophages, autophosphorylation and activation of Hck and Lyn kinases leads to dysregulated αMβ2-mediated adhesion. In CD45-deficient T cells, enhanced α5β1 integrin- and CD44-mediated adhesion leading to enhanced signaling and sustained Src family kinase activity is observed. Thus the negative regulation of Src family kinases by CD45 also impacts leukocyte adhesion.

In dendritic cells, CD45 can modulate pro-inflammatory cytokine production in response to Toll-like receptor (TLR) stimulation. The effect depends on the type of TLR activated and may be explained by a differential effect of CD45 on the  MyD88 dependent and independent TLR signaling pathways. Although Btk as well as Hck and Lyn have been implicated in TLR signaling, tyrosine phosphorylation is not considered a major component of the TLR signaling pathway and exactly how signals from these kinases are integrated into the TLR signaling pathway is not well understood. TLR signals can also be modulated by signals derived from other receptors such as integrins, cytokine, and inhibitory receptors, raising the possibility that CD45 may also impact TLR signaling by modulating this cross talk (Johnson and Cross 2009).


CD45 is a protein tyrosine phosphatase, conserved throughout the evolution of vertebrates. CD45 is leukocyte specific and dephosphorylates specific Src family kinases, namely, Lck and Fyn in T cells and Lyn and Hck in B cells and myeloid cells. As CD45 can both positively and negatively regulate Src family kinases, it is challenging to determine whether Src kinase substrates are also direct CD45 substrates or whether their phosphorylation state is indirectly determined by the effect of CD45 on the kinase. Although the lack of CD45 in leukocytes significantly affects the phosphorylation state and activity of these Src family kinases, it is not the only regulator of these kinases. Specific regulators of Src family kinases may operate in specific locations under specific circumstances or some may have overlapping roles. Understanding when and where CD45 regulates Src family kinases will also provide a better understanding of immune cell activation. One of the main functions of CD45 is to help maintain specific Src family kinases in a primed, dephosphorylated state, preventing both hyperactivation and inactivation, which both lead to severe immune dysfunction. Indeed, the loss of CD45 in humans and mice results in severe combined immunodeficiency, illustrating the importance of CD45 in leukocyte function.


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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Pauline Johnson
    • 1
  • Asanga Samarakoon
    • 1
  • Amy E. Saunders
    • 1
  • Kenneth W. Harder
    • 1
  1. 1.Department of Microbiology and Immunology, Life Sciences InstituteUniversity of British ColumbiaVancouverCanada