Elucidating the role of dok 3 in b cell receptor signaling using gene knockout mice 2

1 1.1 Innate and adaptive immunity The human body encounters numerous foreign substances, of which, some are microbes that can elicit an immune response. The physiological function of the human immune system is to recognize and eliminate invading microbes. Defense against microbes is mediated by the early reactions of innate immunity and later responses of adaptive immunity. Innate immunity consists of mechanisms that exist prior to an encounter with microbes, are rapidly activated, and react similarly to repeated infection. In contrast to innate immunity, more highly evolved defense mechanisms are stimulated by exposure to infectious agents and increase in magnitude and defensive capabilities with each successive exposure to a particular microbe. Because this form of immunity develops as a response to infection and adapts to the infection, it is called adaptive immunity. Innate immunity provides the early lines of defense against microbes. This system uses pattern recognition receptors to recognize invariant structures that are shared by microbes. Most of them are often essential for survival of the microbes, such as lipopolysaccharides (LPS) that constitute the cell wall in gram-negative bacteria or unmethylated microbial DNA, thus limiting the capacity of microbes to evade detection (Akira, 2006). The principle components of innate immunity includes physical and chemical barriers such as epithelia and antimicrobial substances produced at epithelial surfaces; phagocytic cells and natural killer cells; and blood proteins such as complements factors and cytokines. In contrast to innate immunity, there are two types of adaptive immune responses, called the humoral immunity and cell-mediated immunity, which are mediated by different components of the immune system and function to eliminate different types of microbes. Neutralization of foreign extracellular microbes and soluble microbial toxins is mediated by humoral immunity by means of secretory molecules such as antibodies produced by B lymphocytes. Eradication of intracellular bacteria and viruses requires cell-mediated immunity, also called cellular immunity. Intracellular microbes could evade circulating antibodies, survive and proliferate inside host cells. Cell-mediated immunity is mediated by circulating lymphocytes, mainly T lymphocytes and macrophages, which promotes the lysis of infected host cells or destruction of microbes by phagocytosis, respectively. 1.2 B lymphocytes The human body contains approximately X 1012 lymphocytes, of which 5-15% are B cells. B cells are small (6-10 µm) and have a dense nucleus and little cytoplasm. They were discovered together with T cells in the early 1960s and can be distinguish phenotypically only by analysis of cell surface markers. B lymphocytes were so called because in birds they were found to mature in an organ called the bursa of Fabricius (Glick, 1991). In mammals, no anatomic equivalent of the bursa exists, and the early stages of B cell maturation occur in the bone marrow and in the fetus liver. Thus, “B” lymphocytes refer to bursa-derived lymphocytes or bone marrow-derived lymphocytes. The humoral immune response is the aspect of immunity that is mediated by antibodies produced by B cells and is so called because it involves substances found in the humours, or body fluids. The concept of humoral immunity developed based on analysizing antibacterial components in the serum. Hans Buchner is credited with the development of the humoral theory. In 1890 he described alexins, or “protective substances”, which exist in the serum and other bodily fluids and are capable of killing microorganisms. Alexins, later redefined as "complement" by Paul Ehrlich, were shown to be the soluble components of the innate response. In the same year, Emil von Behring and Shibasabo Kitasato in Koch’s laboratory discovered that injecting diphtheria toxin into animals produces a serum containing an antitoxin that provided passive antidiphtheria immunity to people (Behring and Kitasato, 1965). This component in serum is later termed antibodies or immunoglobulins (Igs). B lymphocytes constitute an unique and vital element of both innate and adaptive immune system. They are the sole antibody production factories of our immune system. Naturally occurring or secreting antibodies are distributed throughout our body, in the blood streams, in the mucosal tissues, and to a lesser extent, the interstitial fluid of the tissues. These Y-shaped secreted antibodies, of which there are four classes (IgM, IgG, IgA and IgE), are composed of two heavy and two light chains. Each of the millions of different antibody molecules synthesized is capable of recognizing a different antigen. B cells achieve this very large repertoire of antibodies by complex mechanisms such as V(D)J recombination Ig genes as well as other processes including somatic hypermutation and gene conversion. Apart from its pivotal role in antibody production, B lymphocytes also participate in adaptive immune responses by functioning as antigen presenting cells. The ability of B lymphocytes to capture, process and present antigens to T cells is requisite for normal humoral immune responses. B lymphocytes are remarkably efficient at presenting protein antigens that bind to their Ig receptors to class II MHC-restricted CD4+ helper T cells. Ig molecules function as high-affinity binding sites for capturing antigens at limiting concentrations. In addition, Ig receptor-mediated endocytosis leads to an intracellular pathway of protein traffic that favors recycling of antigens and optimizes the processing of these antigens. As a result, small amounts of endocytosed antigens are capable of associating with MHC molecules and being presented in a form that can be specifically recognized by T lymphocytes. The antigen presenting function of B cells is particularly important in secondary antibody responses, which require low concentrations of antigens and MHC-restricted T-B cell cooperation. 1.2.1 Subpopulations of B lymphocytes B cells are divided into different types according to the expression of surface molecules and anatomical location. Two major B cell subsets, designated B-1 and B-2 (B-0 or follicular B), exist in human and mice. B-1 cells are located in the peritoneal and pleural cavities (Hardy, 2006). They expressed high levels of surface IgM, low levels of B220 and IgD and moderate levels of CD5, and absence of CD23. In contrast, conventional, or B-2, cells are the predominant B cells found in secondary lymphoid organs, such as spleen and lymph node, and in the circulation. B-2 cells express high levels of B220, IgD and CD23 and moderate levels of IgM and lack surface CD5 expression. The B-1 population can be further subdivided into B-1a and B-1b subpopulations. The B-1b “sister population” lacks surface CD5 but shares the other attributes of B-1a cells, such as natural antibody production and low in B220 expression. Another subset of B cells is the marginal zone (MZ) B cells and is normally found in the spleen (Martin and Kearney, 2000). Unlike follicular B-2 cells that express high levels of IgD and CD23, with either high or low levels of IgM, MZ B cells express high levels of IgM and very low levels of IgD and CD23. 1.2.2 B cell development B cell development is a highly regulated multi-step process that proceeds through the ordered maturation of a B cell from a committed precursor and ends with the generation of an immunocompetent mature B cell (Hardy and Hayakawa, 2001). Mammalian B cells develop from lymphoid progenitors in the bone marrow (Osmond, 1990). In the bone marrow, hematopoietic stem cells differentiate into common lymphocyte progenitors (CLP) that can develop into mature B cells. The main goal of this developmental process is to generate a population of cells expressing a diverse repertoire of B-cell antigen receptors (BCRs), with different specificities that are capable of recognizing and responding to new and recurring pathogens (Kurosaki, 2000). In contrast to other cell lineages, developmental progression of B cells relies on a mechanism of selection that ensures the survival of B cells expressing BCRs with certain characteristics and specificities (Seagal and Melamed, 2003). This selection process not only serves to promote the development of B cells with functional antigen receptors (positive selection) but also provides a mechanism for the elimination of clones that are able to respond to endogenous or self-antigens (negative selection). Importantly, the ordered assembly and expression of individual components of the BCR drives the maturation process; development is arrested unless a specific component of the BCR has been successfully expressed (Benschop and Cambier, 1999). These points of developmental arrest have defined checkpoints where the B cell interrogates the functionality of the BCR, and only those B cells in which proper assembly has occurred are selected for continued developmental progression. The mature form of the BCR consists of two functional units, the antigen recognition unit formed by the immunoglobulin (Ig) heavy (H) and light (L) chains, and the signaling unit formed by the proteins Igα (CD79a) and Igβ (CD79b). The process of B-cell development is highly dependent on assembly of a competent BCR. The signaling proteins Igα and Igβ are expressed first, beginning at the earliest defined cell stage committed to the B lineage, the pro-B cell. The genes encoding the Ig heavy and light chains are comprised of a series of segments termed variable (V), diversity (D), and joining (J), which are brought together by a site specific recombination process termed V(D)J recombination (Chen and Alt, 1993). During the pro-B stage, the Ig heavy-chain locus is in the process of rearrangement. Despite the absence of heavy-chain protein, there is some indication that Igα and Igβ may be expressed at the pro-B-cell surface. Although Igα and Igβ function at the pro-B stage is unclear, it has been proposed that this complex constitutes a pro-B-cell receptor that may signal continued development to the pre-B cell stage. Following successful recombination of the heavy chain, this protein is assembled into the pre-BCR complex together with the surrogate light chain (SLC) proteins, VpreB, λ5, Igα and Igβ (Burrows et al., 2002). The pre-BCR then provides the context where the functionality of the newly synthesized heavy chain is tested, and signals generated by this receptor allow the transition to the pre-B stage. Pre-BCR signaling is also necessary for allelic exclusion of the un-rearranged heavy-chain locus, light-chain recombination, and the proliferative expansion of pre-B cells that occurs prior to light-chain recombination. Successful light-chain recombination at the pre-B stage is followed by displacement of the SLC proteins in the receptor complex. Assembly and surface expression of the mature BCR containing light and heavy chains marks the transition to the immature stage. It is at the immature stage that the BCR is first able to interact with conventional polymorphic ligands. In contrast to previous selection steps that tested the signaling capacity of the receptor complex, selection at the immature stage is designed to test the receptor–ligand interaction (Benschop and Cambier, 1999; Seagal and Melamed, 2003). Intimate contact between the immature B cell and bone marrow stromal cells allows receptors capable of recognizing self-antigens to be identified and eliminated through a variety of mechanisms collectively termed ‘negative selection’. Non-self-reactive B cells exit to the periphery and reach the spleen, where they may still be tested for reactivity against self-antigens before transiting to the mature stage. Immature B cells in the spleen are further divided into transitional (T1) and transitional (T2) cells by virtue of their cell-surface phenotype (Rudin and Thompson, 1998). The phenotype of T1 cells most closely resembles that of bone marrow immature B cells. T1 cells inhabit the spleen’s red pulp and give rise to T2 and mature naive B cells, when entering into the spleen follicles. In contrast to immature B cells that undergo negative selection following ligand binding, mature B cells initiate pathways that lead to proliferation and further differentiation into antibody producing B cells (plasma cells) (Manz et al., 2002) or memory B cells (Tsiagbe et al., 1992). 1.3 B cell receptor signal transduction Most lymphocytes such as T cells, B cells, natural killer cells and macrophages express specific cell surface receptors that enable them to recognize foreign microbial antigens. T cells, via T cell receptors (TCR), recognize foreign protein antigens presented to them on a major histocompatibility complex (MHC) molecule by our own antigen presenting cells; B cells utilize B cell receptors (BCR) to distinguish protein and nonprotein antigens. On the other hand, Toll-like receptors (TLR), known to be expressed by many cell types, are capable of identifying diverse classes of non-protein pathogenassociated molecular patterns (PAMPS) present in microbes such as unmethylated bacterial DNA, double-stranded and single-stranded viral RNA and bacterial cell membrane constituents such as lipopolysaccharides (LPS) (Akira and Hemmi, 2003; Benschop and Cambier, 1999; Seagal and Melamed, 2003). Binding of antigens to these membrane-bound receptors trigger a wide variety of cellular responses. The distinct intracellular signaling cascades downstream of each receptor relays messages to the nucleus, transcribing new gene products that lead to cell proliferation, cell differentiation, cell migration, cytokine production and even cell death, just to name a few. The B cell receptor (BCR) is an integral membrane protein complex that is composed of an antigen binding subunit (two Ig H chains and two Ig L chains) and a signaling subunit (a disulfide-linked heterodimers of Igα and Igβ). Igα and Igβ each contain a sequences motif of approximately 26 amino acids residues within their cytoplasmic regions called immunoreceptor tyrosine activation motif (ITAM). The ITAM consists of two YXXL motifs separated by 6–8 amino acids, where Y stands for tyrosine, L is for Leucine and X stands for any amino acid. Both tyrosine residues in the ITAM become phosphorylated upon receptor aggregation and initiates signal transduction by providing specific binding site for Src-homology (SH) domain containing effectors, including several classes of cytoplasmic protein tyrosine kinases (PTKs), which phosphorylate intracellular enzymes and adaptor molecules. Such phosphorylation events cause increased levels of intracellular calcium, activation of phosphatidylinositol 3-kinase (PI3-K), cytoskeletal reorganization, transcriptional activation, and, finally, B cell maturation, proliferation, and antibody secretion. A temporal analysis of the activity of the PTKs after BCR aggregation has shown that the Src family kinases such as Lyn or Blk is activated first, mediating the phosphorylation of the ITAM tyrosines. The doubly phosphorylated ITAM provides a binding site for the PTK, Spleen-associated tyrosine kinase (Syk), which binds via its tandem SH2 domains. Once bound, Syk becomes phosphorylated on tyrosine residue 519, and its activity is greatly increased. In addition, Bruton’s tyrosine kinase (Btk) is activated. These receptor-associated proteins undergo transphosphorylation, which then initiates SH2-mediated recruitment of other signal transduction molecules. Among the proteins recruited is B-cell linker protein (BLNK)/SH2 domaincontaining leukocyte-specific phosphoprotein of 65 kDa (SLP-65). BLNK lacks catalytic or enzymatic activity, but instead, functions as an adapter between the receptor complex and downstream signaling proteins. BLNK contains several modules that facilitate protein-protein and protein-lipid interactions between members of the signaling cascade. Tyrosine phosphorylation of BLNK is mediated by Syk and occurs in the cytoplasm. Once phosphorylated, BLNK associates with many other cytoplasmic signaling proteins, such as phospholipase C-γ2 (PLC-γ2), Vav (a guanine nucleotide exchange factor for the Rho/Rac family of GTPases), and the Shc/Grb2/son of sevenless (SOS) complex and promotes their translocation to the membrane compartment, thus, promoting the formation of the BCR signalosome. The four most intensively studied biochemical signaling pathways associated with the activated BCR are the Ras GTPase, 10 phosphoinositide 3-kinase (PI3-K)/protein kinase B (PKB), phospholipase C-γ2 (PLCγ2), and Rho GTPase pathways. Diagram B cell receptor signaling pathways (obtain from Cell Signaling Technology website) 117 full-length Dok-3 as a bait for yeast-two-hybrid screening (Tan J, 2004, unpublished data). She demonstrated that DIP also interacts only with Dok-1 and but not Dok-2, and 5. It is well-documented that Dok-1 and are co-expressed together and even function in common pathways or independently binds SHIP-1, Csk, Grb2 and Abl. Thus it is not unusual for Dok-1 and to share other binding partners such as DIP, G3BP-1 and 2. To dissect the mechanism of interaction between them, we had generated domain mutants of Dok-1, Dok-3, G3BP-1 and G3BP-2 for coimmunoprecipitation experiments. FLAG-tagged Dok-1 and mutants expressing only PH alone, PTB alone, PH and PTB, or C-terminus regions will resolve which domain(s) are responsible to bind G3BPs. Vice verse, HA-tagged G3BPs mutants expressing only NTF2 alone, acidic domain plus proline rich regions, or C-terminus RNP1, and RG-rich domains will demonstrate which domain(s) bind Doks. Since Dok-1 and are co-expressed in B cells, we hypothesized that G3BP-1 may act as a positive regulator in B cells activation and be inhibited via interaction with Dok-1 and/or 3. 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Dok- L in NIH3T3 cells potently inhibited the transforming activity of v-Abl Dok- 3 becomes rapidly tyrosine phosphorylated in response to B- cell activation and associates with the SH2 domains of SHIP-1 and Csk (Lemay et al., 20 00) Overexpression of Dok- 3 in the A20 B- cell line caused an inhibition of BCR-induced release of IL -2 (Robson et al., 20 04) Mutating four critical carboxyl-terminal tyrosines into... inhibits the Ras-ERK pathway 36 Astoundingly, while analyzing the signaling role of Grb2 in B cells using Grb2-/DT40 cells, a group of researchers reveal a 50 kDa protein that remains almost unphosphorylated upon BCR-stimulation in the absence of Grb2 (Stork et al., 20 07) They isolated this unknown protein, sequenced and determined it to be an avian ortholog of Dok- 3 Previously Grb2 was shown to be... transmembrane proteins, some of which are receptors, are known to modulate specific elements of BCR signaling A few of these, including CD45, CD19, CD 22, and FcγRIIB1 (CD 32) , are indicated above in yellow 1 .3. 1 The PLC 2 pathway One of the signaling pathways shown to be activated by the BCR is the phosphoinositide pathway which involves the hydrolysis of the phosphatidylinositol 4,5bisphosphate (PI4,5-P2)... binding sites for SH2 domains The N-terminal region of Dok1 consists of a PH domain, that may be involved in membrane localization and a PTB domain, which binds phosphotyrosine-containing motifs of the form NPXpY Dok- 1 has fifteen tyrosines, ten of which are located within the C-terminal end Six of these have the requisite proline at +3 (pYXXP), a preferential target site for the SH2 domains of Abl... an inhibitor of Ca2+ mobilization (Stork et al., 20 04) They generated a Dok- 3 deficient DT40 variant by gene targeting Dok- 3- /cells showed a biphastic Ca2+ profile, which is almost identical to that of Grb2-/- cells This result suggests that both adaptor proteins function in a common pathway that regulates Ca2+ mobilization Mutation of Dok- 3 at Tyr -33 1, a residue critical for binding to SH2 domain of. .. followed by a region rich in binding motifs to Src homology (SH )2 and SH3 domains at their C-terminus These multiple modular domains are responsible for recruiting signaling proteins, PTB domain is known to bind phosphotyrosine-containing motif and phosphorylated tyrosines in carboxy-terminal region can act as docking sites for SH2-containing signaling molecules PH domain can be involved in membrane localization... phenylalanines abolishes this inhibition and the binding to SHIP-1, thus demonstrating Dok- 3 as an inhibitor of B- cell activation on the basis of its capacity to recruit SHIP-1 (Robson et al., 20 04) The Dok- 3 SHIP-1 complex functions by selectively suppressing JNK signaling cascade without affecting the activation of known targets of SHIP-1 like Akt/PKB BCR-triggered activation of JNK is enhanced in B cells... 1991) The aminoterminus of Csk contains SH3 and SH2 domains and a kinase domain at its carboxylterminus Csk-/- embryos exhibit defects in the neural tube and die between day 9 and day 10 of gestation (Imamoto and Soriano, 19 93; Nada et al., 19 93) Cells derived from these embryos exhibit an increase in activity of Src and the related Fyn kinase Csk is a potent inhibitor of immunoreceptor signaling in T cells... (p6 2dok) , Dok- 2 (p5 6dok- 2, Dok- R, FRIP), Dok- 3 (DokL), Dok- 4 (IRS-5), Dok- 5 (IRS-6), Dok- 6 and Dok- 7 (Diagram) Dok- 1 and Dok- 2 are highly related in structure and are preferentially expressed, together with Dok- 3, in hematopoietic cells Dok- 1 and Dok- 2 are expressed in the T cell lineage, whereas B cells express Dok- 1 and Dok- 3 Dok- 4 is strongly expressed in non-hematopoietic organs, particularly intestines, . control of B cell signaling. 1.4.1 FcγRIIB receptor FcγRIIB receptors are single-chain molecules bearing IgG-binding sites in their extracellular domains and cytoplasmic domains containing an. subpopulations. The B- 1b “sister population” lacks surface CD5 but shares the other attributes of B- 1a cells, such as natural antibody production and low in B2 20 expression. Another subset of B cells is the. beginning at the earliest defined cell stage committed to the B lineage, the pro -B cell. The genes encoding the Ig heavy and light chains are comprised of a series of segments termed variable

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