CD45 (PTPRC)
Synonyms
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
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
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
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
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).
Summary
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.
References
- Alexander D. The CD45 tyrosine phosphatase: a positive and negative regulator of immune cell function. Semin Immunol. 2000;12:349–59.CrossRefPubMedGoogle Scholar
- Dustin ML, Chakraborty AK, Shaw AS. Understanding the structure and function of the immunological synapse. Cold Spring Harb Perspect Biol. 2010;2:a002311.CrossRefPubMedPubMedCentralGoogle Scholar
- Hermiston M, Xu Z, Weiss A. CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol. 2003;21:107–37.CrossRefPubMedGoogle Scholar
- Hermiston ML, Tan AL, Gupta VA, Majeti R, Weiss A. The juxtamembrane wedge negatively regulates CD45 function in B cells. Immunity. 2005;23:635–47.CrossRefPubMedGoogle Scholar
- Hermiston M, Zikherman J, Zhu JW. CD45, CD148, and Lyp/Pep: critical phosphatases regulating Src family kinase signaling networks in immune cells. Immunol Rev. 2009;228:288–311.CrossRefPubMedPubMedCentralGoogle Scholar
- Hibbs ML, Harder KW. The duplicitous nature of the Lyn tyrosine kinase in growth factor signaling. Growth Factors. 2006;24:137–49.CrossRefPubMedGoogle Scholar
- Huntington ND, Tarlinton DM. CD45: direct and indirect government of immune regulation. Immunol Lett. 2004;94:167–74.CrossRefPubMedGoogle Scholar
- Johnson P, Cross JL. Tyrosine phosphorylation in immune cells: direct and indirect effects on toll-like receptor-induced proinflammatory cytokine production. Crit Rev Immunol. 2009;29:347–67.CrossRefPubMedGoogle Scholar
- Johnson P, Felberg J. CD45: a key regulator of Lck and T cell activation. Mod Asp Immunobiol. 2000;1:156–9.Google Scholar
- Johnson P, Maiti A, DHW N. CD45: a family of leukocyte specific cell surface glycoproteins. In: Herzenberg LA, Weir DM, Herzenberg LA, Blackwell C, editors. Weir’s Handbook of experimental immunology. 5th ed. Malden: Blackwell; 1997. p. 62.1–62.16.Google Scholar
- Penninger JM, Irie-Sasaki J, Sasaki T, Oliveira-dos-Santos AJ. CD45: new jobs for an old acquaintance. Nat Immunol. 2001;2:389–96.PubMedCrossRefGoogle Scholar
- Saunders AE, Johnson P. Modulation of immune cell signalling by the leukocyte common tyrosine phosphatase, CD45. Cell Signal. 2010;22:339–48.CrossRefPubMedGoogle Scholar
- Scapini P, Pereira S, Zhang H, Lowell CA. Multiple roles of Lyn kinase in myeloid cell signaling and function. Immunol Rev. 2009;228:23–40.CrossRefPubMedPubMedCentralGoogle Scholar
- Thomas ML. The leukocyte common antigen family. Annu Rev Immunol. 1989;7:339–69.CrossRefPubMedGoogle Scholar
- Tomlinson MG, Lin J, Weiss A. Lymphocytes with a complex: adapter proteins in antigen receptor signaling. Immunol Today. 2000;21:584–91.CrossRefPubMedGoogle Scholar
- Trowbridge IS, Thomas ML. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu Rev Immunol. 1994;12:85–116.CrossRefPubMedGoogle Scholar
- van der Merwe PA, Dushek O. Mechanisms for T cell receptor triggering. Nat Rev Immunol. 2011;11:47–55.CrossRefPubMedGoogle Scholar
- Williams JC, Wierenga RK, Saraste M. Insights into Src kinase functions: structural comparisons. Trends Biochem Sci. 1998;23:179–84.CrossRefPubMedGoogle Scholar
- Xu Y, Harder KW, Huntington ND, Hibbs ML, Tarlinton DM. Lyn tyrosine kinase: accentuating the positive and the negative. Immunity. 2005;22:9–18.PubMedGoogle Scholar