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

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 recog

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 2 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 anti-diphtheria 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 1 (T1) and transitional 2 (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 protein 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 pathogen-associated 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

non-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) 2 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 containing leukocyte-specific phosphoprotein of 65 kDa (SLP-65) BLNK lacks catalytic

domain-or enzymatic activity, but instead, functions as an adapter between the receptdomain-or 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,

phosphoinositide 3-kinase (PI3-K)/protein kinase B (PKB), phospholipase C-γ2 γ2), and Rho GTPase pathways

(PLC-Diagram 1 B cell receptor signaling pathways (obtain from Cell Signaling Technology website)

Receptor aggregation rapidly activates Src family kinases, including Lyn, Blk and Fyn, Syk and Btk tyrosine kinases, initiating complex signaling cascades involving multiple adaptors, kinases, phosphatases, G-proteins and transcription factors The complexity of BCR signaling permits many distinct outcomes, including proliferation, differentiation, apoptosis, survival and tolerance Many other transmembrane proteins, some of which are receptors, are known to modulate specific elements of BCR signaling A few of these, including CD45, CD19, CD22, and FcγRIIB1 (CD32), are indicated above in yellow

1.3.1 The PLCγ2 pathway

One of the signaling pathways shown to be activated by the BCR is the phosphoinositide pathway which involves the hydrolysis of the phosphatidylinositol 4,5-bisphosphate (PI4,5-P2) by PLCγ2 to form diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) (Kurosaki et al., 2000) Both molecules act as second messengers, with the former critical for activating protein kinase C (PKC) family members, and the latter inducing the release of calcium from intracellular stores by binding to IP3 receptors on the endoplasmic reticulum (ER) One important substrate of PKC appears to be the cAMP response element binding protein (CREB) transcription factor, which is regulated

by cAMP in some cell types but by PKC in B cells (Xie and Rothstein, 1995; Xie et al., 1996) Calcium elevation in B cells leads to the activation of both calcium-calmodulin-dependent protein kinase II and the calmodulin-activated protein serine/threonine phosphatase, calcineurin Among the events regulated by calcium elevation are the phosphorylation of the transcription factor Ets-1 and the calcineurin-regulated dephosphorylation of the cytosolic component of the nuclear factor of activated T cells

(NFATc), which causes NFATc translocation from the cytoplasm to the nucleus where it can participate in transcriptional regulation

1.3.2 The Ras signaling pathway

BCR stimulation also leads to the activation of Ras, which in turn activates the classical mitogen-activated protein kinase (MAPK) pathway The MAPK cascades constitute a group of signal transduction pathways characterized by successive phosphorylation of coupled serine/threonine or dual specificity kinases The conserved signaling modules consist of a MAPK, a MAPK kinase (MAPKK), normally a dual-specificity kinase that phosphorylates MAPK on threonine and tyrosine residues, and a MAPKK kinase (MAPKKK), which phosphorylates MAPKK on serine and activates it

The best characterized MAPK pathway is the extracellular-signal regulated kinase (ERK) pathway The classical MAP kinases, ERK-1 and ERK-2 are important for the ability of the BCR to stimulate proliferation of resting splenic B cells BCR ITAM phosphorylation recruits Shc that forms a complex with Grb2 and SOS resulting in Ras activation and initiation of the Ras/Raf/MEK cascade This pathway receives a primary signal from Ras-GTP, which binds directly to Raf-1, the MAPKKK in this cascade Activated Raf-1 activates MEK-1 and MEK-2 (the MAPKK), which both in turn phosphorylate ERK-1 and ERK-2 Phosphorylated ERKs form dimers, a step required for nuclear translocation and subsequent phosphorylation of transcriptional regulatory proteins, including Fos, Jun and members of the Ets family (Cohen, 1997)

1.3.3 The Rho GTPase pathway

Rho GTPases are instrumental in the organization of actin cytoskeleton, but also for the control of gene expression Although these proteins have been classically

implicated in chemotaxis, there are now clear indications on how differential signaling toward other, more specific functions, such as phagocytosis or the production of reactive oxygen species (Van Hennik and Hordijk, 2005) Unlike the ERK pathway, the c-Jun N-terminal kinase (JNK)/ stress-activated protein kinase (SAPK) pathway is delivered by another family of small GTP-binding proteins, the Rho GTPases Rac1 and Cdc42 (Coso et al., 1995; Teramoto et al., 1996a; Teramoto et al., 1996b) Rac1 and Cdc42 activate NF-κB (Perona et al., 1997) and the serum response factor transcription factors (SRF) (Hill et al., 1995) The JNK cascade elements are positioned in a conventional signaling cascade involving MKK1-4 (the MAPKKK), SEK1 (the MAPKK) and JNK (Tibbles et al., 1996) JNK translocates to the nucleus where it can regulate the activity of multiple transcription factors Among the targets of this cascade are the transcriptional factors c-Jun and ATF-2 JNK phosphorylates c-Jun in its N-terminal region, greatly promoting its transcriptional activating ability (Treisman, 1996)

Regulation of the p38 MAPK is also achieved through a serine kinase cascade and Rac1 and Cdc42 Unlike the requirement for both PKC and calcium in JNK activation, maximum p38 activation appears to require only PKC, not calcium As with other MAPK cascades, the membrane-proximal component is a MAPKKK, typically a MEKK or a mixed lineage kinase (MLK) The MAPKKK phosphorylates and activates MKK3/6, the p38 MAPK kinases MKK3/6 can also be activated directly by ASK1, which is stimulated by apoptotic stimuli p38 MAPK is involved in regulation of HSP27 and MK2 (MAPKAPK-2), MK3 (MAPKAPK-3) and several transcription factors including ATF-2,

Stat1, the Max/Myc complex, MEF-2, Elk-1 and indirectly CREB via activation of MSK1

1.3.4 The PI3-K pathway

A fourth signaling pathway important for BCR signaling involves the activation

of phosphatidylinositol 3-kinase (PI3-K), which phosphorylates the inositol ring of phosphatidylinositol-4,5-biphosphate at the 3 position The product of this reaction, phosphatidylinositol-3,4,5-triphosphate (PIP3), is known to recruit PLCγ (Falasca et al., 1998), as well as the kinases Btk (Buhl et al., 1999) and Akt (Gold et al., 1999) These and other effectors bind via pleckstrin homology (PH) domain interactions with PIP3 to the plasma membrane where, in the context of additional modifications, they are activated While Akt activation generates cell survival signals, PLCγ and Btk activation leads to phosphoinositide hydrolysis yielding inositotl-1,3,4-triphosphate, which mediates mobilization of calcium (Fluckiger et al., 1998) PIP3 is also essential for full activation

of MAP kinases and regulates the activity of NF-κB (Kane et al., 1999) Loss of PIP3

generation, through genetic ablation of the regulatory subunit of PI-3K p85α, results in

an absence of mature B cells in the periphery (Fruman et al., 1999; Suzuki et al., 1999) Thus signaling cascades that mediate production of PIP3 are critical not only for active BCR-mediated responses but also for B cell development and survival

1.4 Negative regulators of B cell receptor signaling

Although early studies focused principally on the role of positive signals on lymphocyte activation, increasing evidence suggests that each mode of cellular activation

is finely regulated by an integrated series of both positive and negative regulators Upon the complete elimination of invaders, auto-regulatory and negative feedback mechanisms

step in to maintain homeostasis and returning the immune system to basal resting state Unchecked or prolonged activation of immune responses are detrimental to the host Several mechanisms exist to prevent inappropriate B-cell activation and to avoid generation of autoreactive antibodies and autoimmune diseases One type of negative regulation involves the direct or indirect recruitment of inhibitory signals by a large group of receptors carrying immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains Example of such inhibitory receptors are PD-1, which recruits Src homology 2 (SH2) domain-containing protein tyrosine phosphatases (SHPs) (Okazaki et al., 2001), as well as FcγRIIB, which binds the SH2 domain-containing inositol-5 phosphatase-1 (SHIP-1) (Coggeshall, 1998; Ono et al., 1996) Ligation-induced phosphorylation of ITIM motifs in the cytoplasmic tails of most of these receptors results

in the recruitment of phosphatases These two classes of phosphatases prevent B-cell activation by inhibiting critical steps in the BCR signaling cascade For example, the phosphatidylinositol-3 kinase (PI-3K) pathway plays a central role in regulating numerous biologic processes, including survival, adhesion, migration, metabolic activity, proliferation, differentiation, and cell activation through the generation of the potent second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P(3) or PIP3) To ensure that activation of this pathway is appropriately suppressed/terminated, the ubiquitously expressed tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) hydrolyzes PI-3,4,5-P(3) to PI-4,5-P(2), whereas SHIP-1 and SHIP-2 break it down to PI-3,4-P(2)

In addition to cell-surface receptors, it is now clear that adaptor molecules are also involved in inhibiting signal transduction by mediating protein-protein or protein-lipid

interactions Adaptor or docking proteins are non-enzymatic but possess multiple modular domains responsible for recruiting signaling proteins to activated receptors, nucleating intermolecular complexes and positively or negatively modulating effector protein activity by inducing conformational changes or phosphorylation/ dephosphorylation Adaptor molecules are commonly defined as proteins that possess protein-protein or protein-lipid interaction domains However, many enzymes can be considered to function as adaptors proteins, because they additionally contain protein- or lipid-binding modules For example, the Src-family PTK, Lyn has one SH2 domain and one SH3 domain, as well as an enzymatic domain The interaction modules in adaptors and enzymes act to localize proteins to specific subcellular sites, control enzymatic activities and direct the formation of multiprotein complexes – all of which, in turn, contribute to the qualitative and quantitative 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 immunoreceptor tyrosine-based inhibitory motif (ITIM), a 13-amino acid sequence required for inhibitory function (Muta et al., 1994) The comparison of sequences of several inhibitory receptors revealed the conservation of a valine or an isoleucine residue at position Tyr-2, resulting in the characteristic ITIM sequence V/IxYxxL FcγRIIB receptor is expressed on B cells, macrophages, neutrophils and mast cells FcγRIIB receptors suppress cellular activation

by promoting dephosphorylation reactions, resulting from the recruitment of SHIP-1 to the ITIM SHIP-1 decreases the cellular levels of phosphatidylinositol PIP3, ultimately preventing the influx of extracellular calcium FcγRIIB inhibition of cell activation was

also proposed to be mediated by the adaptor protein Dok-1 through suppressing of ERK activation (Yamanashi et al., 2000; Ott et al., 2002)

FcγRIIB1 receptor isoforms are preferentially expressed in B lymphocytes, and are involved in the negative regulation of antibody production and B cell proliferation (Van den Herik-Oudijk IE et al., 1994) FcγRIIB1 receptors are important factors in controlling the amplitude of B cell activation in response to antigen Specific IgG antibodies that bind to the BCR through their Fab region can interact through their Fc portion with inhibitory FcγRIIB1 on B cells and deliver an inhibitory signal Coaggregation of FcγRIIB1 with the BCR inhibited independently ligated receptors whose signaling required PIP3 Negative signaling by FcγRIIB1 represents a feedback suppression system that functions to inhibit B cell activation, proliferation and antibody production (Daeron et al., 1995)

FcγRIIB2 isoforms are coexpressed with activating FcγR in primary monocytes, neutrophils and monocyte-derived dendritic cells In mast cells and basophils, coclustering of FcγRIIB1 and FcγRIIB2 isoforms with activating FcγR caused inhibition

of degranulation Follicular dendritic cells (FDC) express high levels of FcγRIIB FcγRIIB receptors expressed on FDC in germinal centers are involved in the retention of immune complexes and in the generation of recall responses A defect in the maturation

of FDC in the germinal centers was noticed in FcγRIIB-deficient animals Because of their ability to inhibit cell activation, FcγRIIB receptors are believed to promote noninflammatory clearance of immune complexes The inhibitory capacity of FcγRIIB2

in cells of the mononuclear phagocyte system is exerted upon coaggregation with bearing FcγRs, leading to inhibition of effector functions In monocytes and macrophages,

ITAM-FcγRIIB2 receptors regulate the production of cytokines and the amplitude of inflammation in response to immune complexes

1.4.2 SH2-containing inositol 5’-phosphatase-1 (SHIP-1)

An unknown tyrosine-phosphorylated p145 protein was isolated, together with Shc, while using a Grb2 C-terminus SH3 domain-based affinity chromatography to identify potential binding partners of Grb2 upon IL-3 stimulation (Damen et al., 1996) Its predicted amino acid sequence revealed an amino-terminal SH2 domain, a central 5’-phosphatase domain, two NPXY sequences and a proline rich C-terminal tail This protein was named SH2-containing inositol phosphatase-1 or SHIP-1 Ware et al described cloning of human SHIP from a human megakaryocytic cell line cDNA library using 2 nonoverlapping mouse SHIP cDNA fragments as probes (Ware et al., 1996) This interesting novel gene was also independently cloned by three groups using different approaches (Drayer et al., 1996; Kavanaugh et al., 1996; Lioubin et al., 1996) Northern blot analysis suggested that human SHIP-1 is expressed as a 5.3-kb mRNA in bone marrow and a wide variety of other tissues Sequence analysis of the cDNA predicted a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mouse

SHIP-1 Liu et al studied the expression of the ship gene during mouse development (Liu

et al., 1998) They found that the gene is expressed in late primitive-streak stage embryos (7.5 days postcoitum), when hematopoiesis is thought to begin, and the expression is restricted to the hematopoietic lineage In adult mice, SHIP-1 expression continues in most cells of hematopoietic origin, including granulocytes, monocytes, and lymphocytes, and is also found in the spermatids of the testis Furthermore, the level of SHIP-1 expression is developmentally regulated during T-cell maturation These results

suggested a possible role for SHIP-1 in the differentiation and maintenance of the hematopoietic lineages and in spermatogenesis

SHIP-1 acts by hydrolyzing inositol metabolites phosphorylated at the 5’ position

of the inositol ring, namely, phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] and 1,3,4,5-tetrakisphosphates [I(1,3,4,5)P4] The membrane-bound PI(3,4,5)P3 is critical for binding and membrane recruitment of pleckstrin homology (PH) domain containing molecules like the PTK Btk, a pivotal effector of B-cell activation (Bolland et al., 1998), and the serine-threonine-specific protein kinase Akt/PKB, a prosurvival factor (Carver et al., 2000; Baran et al., 2003) By converting PI(3,4,5)P3 to PI(3,4)P2, SHIP-1 precludes activation of these PH domain bearing effectors and can prevent B-cell activation (Helgason et al., 2000; Liu et al., 1998; Brauweiler et al., 2000) In support of this idea, it has been reported that B cells freshly isolated from SHIP-1-/- mice exhibited augmented BCR-induced proliferation Moreover, in vivo B-cell maturation is accelerated in SHIP-1-

/- mice (Helgason et al., 2000; Liu et al., 1998; Brauweiler et al., 2000)

Diagram 2 SHIP-1-mediated inhibition of cellular activation (Ravetch and Lanier, 2000)

Although viable and fertile, SHIP-1-/- mice failed to thrive, and survival was only 40% by 14 weeks of age (Helgason et al., 1998) The mice exhibited a myeloproliferative-like syndrome with consolidation of the lungs caused by infiltration of macrophages (Helgason et al., 1998; Oh et al., 2007) They concluded that SHIP-1 plays

a crucial role in modulating cytokine signaling within the hematopoietic system

The primary mode of recruitment of SHIP-1 in activated B cells is believed to involve FcγRIIB Engagement of FcγRIIB by the Fc portion of immunoglobulin G (IgG) present in immune complexes (which are generated as a consequence of productive B-cell activation) results in tyrosine phosphorylation of the ITIM of FcγRIIB, thus triggering binding of the SHIP-1 SH2 domain and membrane translocation of SHIP-1 Analyses of ex vivo B cells or B-cell lines lacking SHIP-1 have provided evidence that FcγRIIB-associated SHIP-1 inhibits B-cell activation by preventing BCR-induced

PI(3,4,5)P3 accumulation, activation of Btk (Bolland et al., 1998) and Akt/PKB (Aman et al., 1998), calcium fluxes (Okada et al., 1998; Hashimoto et al., 1999), and ERK activation (Brauweiler et al., 2001; Ganesan et al., 2006; Ono et al., 1997; Pearse et al., 1999) There are also FcγRIIB-independent mechanisms for recruiting SHIP-1 in B cells

In agreement with this, it has been reported that SHIP-1-deficient B cells display enhanced BCR-elicited PI(3,4,5)P3 generation and Akt activation even in the absence of FcγRIIB coligation While the exact mechanism of recruitment of SHIP-1 in this setting

is not known, it likely involves interactions with other molecules This view is also consistent with the finding that SHIP-1 can associate with intracellular adaptor molecules like Shc (Tridandapani et al., 1999; Tridandapani et al., 1997), Grb2 (Poe et al., 2000) and Dok-1 (Kepley et al., 2004; Abramson and Pecht, 2002), -2 (Dong et al., 2006) and -

3 (Robson et al., 2004; Lemay et al., 2000)

SHIP-1-/- mast cells were found to be far more prone to degranulation, after the crosslinking of IgE preloaded cells (Huber et al., 1998) IgE alone also stimulated massive degranulation in SHIP-1-/- but not wildtype mast cells This degranulation with IgE alone, which may be due to low levels of IgE aggregates, correlated with a higher and more sustained intracellular calcium level than that observed with wildtype cells and was dependent on the entry of extracellular calcium The results showed the critical role that SHIP plays in setting the threshold for degranulation and demonstrated that SHIP directly modulates a 'positive-acting' receptor

1.4.3 Lyn tyrosine kinase

The Src family kinase Lyn might support BCR activation by phosphorylating the first tyrosine of the ITAM sequence and other BCR proximal signaling elements and

presumably by stimulating oxidase activity (Hibbs and Dunn, 1997) Apart from its positive effect on BCR signaling, however, Lyn participates in a negative feedback loop that terminates BCR signaling (Chan et al., 1998) Upon activation, Lyn phosphorylates inhibitory receptors like CD22 and CD72, which then bind phosphatases that dephosphorylate the ITAMs and inhibit signal transduction Indeed, B cells from Lyn-deficient mice do not show drastic developmental defects and are hyperreactive rather than hyporeactive Furthermore, Lyn-deficient mice show a high susceptibility to the development of autoimmune diseases, characterized by circulating autoreactive antibodies and the deposition of IgG immune complexes in the kidney (Silver et al., 2006; Hibbs et al., 1995b) These results demonstrate the importance of Lyn as an inhibitory element of BCR signaling, whereas Lyn’s role as a positive signaling element

of BCR signaling is only seen if Lyn deficiency is combined with deficiencies of other signaling elements, such as Btk In the chicken B cell line DT40, Lyn deficiency results

in the reduction of both calcium mobilization and tyrosine phosphorylation of substrate proteins (Takata et al., 1994)

B cells from Lyn-/- mice exhibit hyperproliferation to BCR and BCR-FcγRIIB stimulation, along with enhanced MAP kinase activation and Ca2+ influx (Wang et al., 1996; Chan et al 1997a,b; Nishizumi et al., 1998) This hyper-responsiveness is thought

to be a cause of the autoimmune disease that develops in Lyn-/- mice (Hibbs et al., 1995; Nishizumi et al., 1995) The phosphorylation of Dok-1 under both conditions are Lyn-dependent as shown using Lyn-/- B cells, there is loss of Dok-1 phosphorylation This data thus suggest that Dok-1 could act in pathways downstream of Lyn

1.4.4 C-terminal Src tyrosine kinase (Csk)

Csk was originally purified as a kinase which can phosphorylate the negative regulatory tyrosine residue (Tyr-527) of c-Src, thereby suppressing their activity, and was subsequently shown to phosphorylate other members of the Src PTKs, such as Lyn, Fyn, Yes and Lck, at their C-terminal tyrosine residues in vitro (Nada et al., 1991) The amino-terminus of Csk contains SH3 and SH2 domains and a kinase domain at its carboxyl-terminus Csk-/- embryos exhibit defects in the neural tube and die between day 9 and day 10 of gestation (Imamoto and Soriano, 1993; Nada et al., 1993) 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 and macrophages, but not in B cells and mast cells In Csk-/- DT40 B cells, Lyn and Syk became constitutively phosphorylated but cells still required additional signals from BCR to elicit other biochemical events such as calcium mobilization (Hata et al., 1994)

1.4.5 Downstream of tyrosine kinases (Dok)

Dok (downstream of tyrosine kinases) adaptor proteins constitute a family of seven members, consisting Dok-1 (p62dok), Dok-2 (p56dok-2, Dok-R, FRIP), Dok-3 (Dok-L), 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-2are 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, kidneys and lungs Dok-5 is expressed most exclusively in the central nervous system Dok-6 is highly expressed in developing central nervous system Dok-7 is preferentially expressed in skeletal muscles and heart These proteins though

having diverse expression profiles, are structurally similar, containing an N-terminalmodule composed of tandem pleckstrin homology (PH)-phosphotyrosine binding (PTB) domainsfollowed 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 by binding phospholipids in the plasma membrane

Potential tyrosine kinases sites, association with effector molecules containing SH2, SH3

Potential tyrosine kinases sites, association with effector molecules containing SH2, SH3

Diagram 3 Structure and domains of Dok family members

Dok family members share conserved structure, consisting of a PH domain (green), PTB domain (yellow) and multiple consensus tyrosine residues that can be phosphorylated upon activation

Unknown Neuronal

differentiation Unknown

c-Ret Brain,

cerebellum, spinal cord

38

Dok-6

Unknown Neuronal

differentiation, insulin signaling

Unknown c-Ret, IR

Brain, activated T Cells

36

Dok-5/

IRS-6

Lack neuromuscular synapses, immobile at birth

Neuromuscular synaptogenesis MuSK

MuSK Cardiac/

thigh/

diaphragm muscle

55

Dok-7

lymphoid tissues, activated T cells

Non-B cells, macrophage, mast cells, myeloid cells

T cells, macrophage, mast cells, myeloid cells

Ubiquitous

Expression profile

Unknown Neuronal

differentiation, insulin signaling

Crk, Src, Fyn c-Ret, Tie2,

IR 41

Dok-4/

IRS-5

Unknown BCR signaling

c-Abl, Csk, SHIP-1, Grb2

BCR, FcγRIIb

58/62

Dok-3/

DokL

-ve regulator of TLR-4 and LPS hypersensitivity

Cell migration,

T cell development, cell

proliferation, TLR-4 signaling

RasGAP, Nck, SHIP-1, c-Abl, CrkL, BCR-ABL, c- Src, Grb2

IL-4R, CD2, Tie2/Tek, FcεR, EGFR

ve regulator of TLR-4 and LPS hypersensitivity

Cell migration,

B cell apoptosis, cell proliferation, TLR-4 signaling

RasGAP, Nck, SAP, Smad3, Smad4, SHIP-

1, Csk, CrkL, Tec, Lyn, BCR-ABL, Grb2

IR, CD2, EGFR, FcεR, FcγRIIb, c-kit, activin receptor Type I/II

62

p62 dok /

Dok-1

Knockout mice studies Functions

Intracellular interacting proteins

Receptor pathways MW

Dok

members

Unknown Neuronal

differentiation Unknown

c-Ret Brain,

cerebellum, spinal cord

38

Dok-6

Unknown Neuronal

differentiation, insulin signaling

Unknown c-Ret, IR

Brain, activated T Cells

36

Dok-5/

IRS-6

Lack neuromuscular synapses, immobile at birth

Neuromuscular synaptogenesis MuSK

MuSK Cardiac/

thigh/

diaphragm muscle

55

Dok-7

lymphoid tissues, activated T cells

Non-B cells, macrophage, mast cells, myeloid cells

T cells, macrophage, mast cells, myeloid cells

Ubiquitous

Expression profile

Unknown Neuronal

differentiation, insulin signaling

Crk, Src, Fyn c-Ret, Tie2,

IR 41

Dok-4/

IRS-5

Unknown BCR signaling

c-Abl, Csk, SHIP-1, Grb2

BCR, FcγRIIb

58/62

Dok-3/

DokL

-ve regulator of TLR-4 and LPS hypersensitivity

Cell migration,

T cell development, cell

proliferation, TLR-4 signaling

RasGAP, Nck, SHIP-1, c-Abl, CrkL, BCR-ABL, c- Src, Grb2

IL-4R, CD2, Tie2/Tek, FcεR, EGFR

ve regulator of TLR-4 and LPS hypersensitivity

Cell migration,

B cell apoptosis, cell proliferation, TLR-4 signaling

RasGAP, Nck, SAP, Smad3, Smad4, SHIP-

1, Csk, CrkL, Tec, Lyn, BCR-ABL, Grb2

IR, CD2, EGFR, FcεR, FcγRIIb, c-kit, activin receptor Type I/II

62

p62 dok /

Dok-1

Knockout mice studies Functions

Intracellular interacting proteins

Receptor pathways MW

Dok

members

Table 1 Summary of Dok family members

The known pathways, interacting proteins and functions of the various Dok members are summarized as above Phenotypes of mice deficient in Dok-1, 2 and 7 have been reported but the rest are to be determined

1.4.5.1 Dok-1 and Dok-2

Chronic Myelogenous Leukemia (CML) affects 1 to 2 people per 100,000 and accounts for 7 - 20% cases of leukemia This myeloproliferative disease is characterised

by chronic unregulated expansion of bone marrow stem cell that forms the myeloid lineage such as granulocytes and macrophages These cells contain a chromosomal

translocation in which the 5’ exons of the bcr (breakpoint cluster) gene on chromosome

22 are fused to the c-abl tyrosine kinase on chromosome 9 (de Klein et al., 1982) The translocation places the three exons which encode for tyrosine kinase domain of c-abl, downstream of either the first or second exon of bcr The resulting chromosome is also

known as the Philadelphia chromosome (Ph) This chromosomal abnormality is so named because it was first discovered and described in 1960 by two scientists from Philadelphia, Pennsylvania: Peter Nowell of the University of Pennsylvania and David Hungerford of

the Fox Chase Cancer Center The products of the chimera bcr-abl gene are two fusion

proteins, p185bcr-abl or p210bcr-abl Relative to normal c-Abl, Bcr-Abl is constitutively active, exhibits increased tyrosine kinase activity and activates a cascade of proteins which control cell cycle Moreover, Bcr-Abl inhibits cellular DNA repair, therefore causing genomic instability and increase in cell susceptibility to developing further genetic abnormalities Indeed, Bcr-Abl has been shown transforming a variety of fibroblastic and hematopoietic cell lines in culture (Konopka et al., 1984; Lugo et al., 1990)

Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and coimmunoprecipitated with p120 Ras GTPase-activating protein (GAP), a negative regulator of p21ras (Ellis et al., 1990) A p62 is also rapidly phosphorylated upon platelet-derived growth factor- (PDGF), colony-stimulatory factor-1 (CSF-1) (Heidaran et al., 1992), insulin (Hosomi et al., 1994), insulin growth factor (IGF) (Sanchez-Margalet et al., 1995) and vascular-endothelial growth factor (VEGF) stimulation of cells Stimulation of B cell receptors or Fcγ receptors also induced tyrosine phosphorylation of a p62 Assuming that all of these are the same p62, these data strongly suggest that it plays a central role in signaling mediated by a wide range of tyrosine kinases This p62 protein which was simultaneously identified, sequenced and cloned by two groups of researchers, was henceforth termed p62dok for p62 protein downstream of tyrosine kinases, but subsequently renamed Dok-1 as more family members were

identified (Carpino et al., 1997; Yamanashi and Baltimore, 1997) Apart from Bcr-Abl, stimulation of c-Kit receptor tyrosine kinase also induced phosphorylation of Dok-1, implicating Dok-1 in facilitating signal transduction events downstream of the c-Kit receptor, a receptor for stem cell factor (Carpino et al., 1997) Indeed using in vitro assay, Dok-1 was shown to be phosphorylated by multiple hematopoietic cell-specific tyrosine kinases such as Tec, Lyn, Syk and Pyk2 when co-expressed in 293 cells (Yoshida et al., 2000)

A search through NCBI’s Entrez Gene reveals that Dok-1 is expressed across many species, including human, mouse, dog, monkey, rat, cow and zebrafish Northern blot analysis indicated a human Dok-1 message of approximately 1.9 – 2.0 kb, encoding

for a 481 amino acid protein (Carpino et al., 1997) Overall amino acid identity between human and mouse Dok-1 is 83% (Yamanashi and Baltimore, 1997) Dok-1 has overall structural similarity to insulin receptor substrate-1 (IRS-1), having both pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in its amino-terminal portion, followed by many binding sites for SH2 domains The N-terminal region of Dok-

1 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 and Crk (Songyang et al., 1993) Dok-1 is also relatively proline-rich, with ten PXXP motifs The PXXP motif has been demonstrated to be the most conserved motif within known SH3 domain ligands, suggesting that Dok-1 may form signaling complexes with other molecules bearing SH3 domains IRS-1 binds to activated and autophosphorylated insulin receptor through its amino-terminal PH and PTB domains and is subsequently tyrosine phosphorylated, allowing its carboxy-terminal region to act as docking sites for SH2-containing signaling molecules such as PI-3 kinase (Yenush et al., 1998) and SHP-2 (Myers, Jr et al., 1998) Given that Dok-1 structurally contains multiple signaling modules, it may function as a docking/adaptor protein

A year following the discovery of Dok-1, Dok-2 was cloned, characterized and separately named by three research groups (Di Cristofano et al., 1998; Jones and Dumont, 1998; Nelms et al., 1998) Di Cristano et al sequenced a 56 kDa RasGAP binding phospho-protein isolated from lysates from Mo7p210 cells, a megakaryoblastic cell line that expresses p210bcr-abl, naming this novel protein p56dok-2 An extensive analysis of an

expressed sequence tag database revealed the presence of four more potential members of Dok family They proposed the following nomenclature for the Dok family, denoting them Dok-1 to Dok-6 True enough, more Dok family members were cloned in subsequent years, all through database searches

Independently, Nelms et al utilised the yeast two-hybrid approach to screen a mouse thymus cDNA library for interacting proteins using the interleukin-4 (IL-4) receptor I4R motif as bait They discovered a new protein which was designated FRIP in

short for IL-Four Receptor Interacting Protein Finally, Jones and Dumont also cloned a

novel docking molecule which has sequence homology to 1 which was called

Dok-R (for Dok-Dok-Related) when employing the yeast two-hybrid system to identify molecules that can interact with murine Tek receptor intracellular domain in a phospho-dependent manner

Both human and mouse full length Dok-2 mRNA encodes for a 412 amino acid protein Structurally similar to Dok-1, it has a PH and PTB domain at the N-terminus, thirteen potential tyrosine phosphorylation site and six PXXP motifs Overall there is 34.8% identity between the two proteins Although Dok-1 is more widely expressed, the expression profiles of Dok-1 and 2 were coincident; both transcripts were highly expressed in cells and tissues of hematopoietic origin Dok-2 is in particular specific, being only expressed in thymus, spleen and lymph nodes (Figure 1.1)

Given that Dok-1 and 2 are rapidly phosphorylated upon activation of receptors, they are likely to play an important role in mediating signaling cascades Reports pertaining to the studies of Dok-1 and 2 were mainly explored in cells of hematopoietic origin for the following reasons Hematopoietic cells, in particular T cells, mast cells,

macrophages and myeloid cells simultaneously express Dok-1 and 2 (Lemay et al., 2000) Various reports had also pointed towards the importance of Dok-1 and 2 in immune cell signal transduction such as B cell receptor, CD2 receptor, Fc receptor and Toll-like receptor 4, all of which are exclusively expressed by hematopoietic cells (Berg

et al., 1999; Kepley et al., 2004; Lock et al., 1999; Nemorin and Duplay, 2000; Nemorin

et al., 2001; Okabe et al., 2005; Shinohara et al., 2005; van Dijk et al., 2000; Sattler et al., 2001a) Finally, evidences obtained from physiological studies of Dok-1 and 2 single and double knockout mice strongly indicate the importance of these adaptors in regulating the homeostasis of myeloid cells, leukemogenesis, LPS endotoxin shock and T cell receptor signaling (Di Cristofano et al., 2001; Niki et al., 2004; Shinohara et al., 2005; Yamanashi

et al., 2000; Yasuda et al., 2004; Yasuda et al., 2007) Due to all the above reasons, experiments conducted using Dok-1 and Dok-2 deficient mice were the most relevant and reliable

Dok-1 or Dok-2 deficiency alone does not affect lymphopoiesis in the mouse (Di Cristofano et al., 2001; Niki et al., 2004; Yamanashi et al., 2000) These mice were viable, fertile and born at the Mendelian frequency Dok-1 is phosphorylated upon treatment with F(ab’)2 form of IgM, which crosslink only BCR but is more strongly phosphorylated after crosslinking with intact whole IgG, which crosslink both BCR and FcγRIIB Proliferation of Dok-1-/- B cells upon stimulation through BCR is normal although hyperphosphorylation of ERK2 is detected (Yamanashi et al., 2000) Interestingly, Dok-1 is dispensable for FcγRIIB phosphorylation and its subsequent binding to SHIP-1 upon BCR-FcγRIIB stimulation (Yamanashi et al., 2000)

The loss of Dok-1 or Dok-2 alone did not exacerbate their health, although these single deficient mice were shown to be more susceptible to endotoxin shock due to hyper-sensitivity to bacterial LPS challenge (Shinohara et al., 2005; Yamanashi et al., 2000) However when both gene are lost, mice succumbed to myelomonocytic leukemia

at about 1 year of age (Yasuda et al., 2004) These double knockout (DKO) mice have enlarged spleen and kidney due to infiltration of granulocytes and lymphocytes, and expansion of lymphocytes population in the peripheral blood Further studies showed that this was in part due to hyper-responsiveness and hypo-apoptosis of myeloid cells upon treatment with or deprivation from cytokines such as stem cell factor (SCF) and IL-3 Thus Dok-1 and 2 function as negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia

More recently, DKO mice were analysized further to examine for their role in thymocytes development and function (Yasuda et al., 2007) Results suggest that Dok-1 and 2 act cooperatively as negative regulators in T cells These mice showed elevated TD response but were normal towards TI antigen CD4+ T cells from Dok-1 or 2 displayed increased proliferation as well as IL-2 production Furthermore, CD4+ T cells from DKO mice responded even more vigorously to TCR cross-linking The loss of Dok-1 and 2 resulted in enhanced phosphorylation of ZAP-70, LAT and ERK in CD4+ T cells The authors overexpressed Dok-1 and Dok-2 bearing a mutation in the PTB domains in 25-14

T cells implied that they may compete with ZAP-70 for binding to the ITAMs of TCRζ and CD3ε upon TCR signaling Interestingly, DKO mice exhibited high titers of antibodies to dsDNA, together with deposits of immune complexes in kidney glomeruli and development of lupus-like renal disorder

1.4.5.2 Dok-3

Two research groups independently reported the identification of Dok-3 (aka Dok-L), a member of the Dok family of adaptors expressed in B cells, myeloid cell and macrophages (Cong et al., 1999; Lemay et al., 2000) Cong et al used Abl in a yeast-two-hybrid screen to clone a new gene which they coined Dok-like (Dok-L) while Lemay et

al utilized the same approach but used Csk as a bait in the presence of Src kinase to clone a novel adaptor which they termed Dok-3 Like its relatives Dok-1 and Dok-2, Dok-3 possesses an amino-terminal PH domain, a PTB region, and a long carboxyl-terminal segment with potential sites of tyrosine phosphorylation Dok-1, 2 and 3 share extensive homology in the PH and PTB domains but there are little similarity in their C terminus tail region In particular, the repeated YXXP motifs that constitute interaction between RasGAP or Nck with Dok-1 and 2 are absent in Dok-3

Like Dok-1 and 2, Dok-3 expression is mostly restricted to immune organs It is abundantly expressed in spleen and bone marrow but not in thymus (Lemay et al., 1999) (Figure 1.1) Further analysis of the expression pattern of these three family members using sorted primary cells reveal that, like Dok-1, Dok-3 is distinctively expressed throughout B cell development in the bone marrow and by peripheral B cells, while both Dok-1 and 2 transcripts are upregulated during thymocytes development (Yasuda et al., 2007) (Figure 1.2) These results strongly suggest the possibility of overlapping roles for Dok-1 and 2 in regulation of thymocytes development, while both Dok-1 and 3 may be required for B cells development Indeed, Dok-1-/-/-2-/- DKO exhibited increased number

of thymocytes and CD4+ or CD8+ T cells in the spleens (Yasuda et al., 2007) However

the phenotype for a Dok-1-/-/-3-/- DKO is not known as Dok-3-/- mice has not been generated at the start of this work

Figure 1.1 mRNA expression of Dok-1, 2 and 3 in various (A) mouse tissues and (B) cell lines by Northern hybridization (obtained from Lemay et al., 2000)

Figure 1.2 Expression of Dok-1, 2 and 3 genes in hematopoietic cells shown using RT-PCR (obtained from Yasuda et al., 2007)

Dok-1 and Dok-3 binds directly to Abl in a kinase-dependent manner through its PTB domain (Cong et al., 1999) However comparison shows that unlike Dok-1, overexpression of Dok-3 strongly inhibited v-Abl-stimulated MAP kinase activation,

while Dok-1 had no effect Furthermore, overexpression of 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., 2000) Overexpression of Dok-3 in the A20 B-cell line caused an inhibition of BCR-induced release of IL-2 (Robson et al., 2004) Mutating four critical carboxyl-terminal tyrosines into 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., 2004) 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 lacking SHIP-1, implying that Dok-3-mediated recruitment of SHIP-1 or an analogous mechanism is a physiologically relevant mode of JNK inhibition Interestingly, Dok-1 was shown to bind to SHIP-1 (Sattler et al., 2001b; Dunant et al., 2000; Abramson and Pecht, 2002)

Another interesting Dok-3 binding protein was identified recently Dok-3 was overexpressed together with Src in 293T cells and immunoprecipitated The eluted proteins was sequenced and identified to be the already known binding partner Csk and a novel one, Grb2 (Honma et al., 2006) Mutational studies showed that phosphorylation of Dok-3 at Tyr-398 and Tyr-432 by Src enables binding to SH2 domain of Grb2 Since Grb2 is known to form a stable complex with SOS, the authors proposed that Dok-3 could sequester Grb2-SOS complex from Shc and therefore inhibits the Ras-ERK pathway

Astoundingly, while analyzing the signaling role of Grb2 in B cells using Grb2DT40 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., 2007) They isolated this unknown protein, sequenced and determined it to be an avian ortholog

-/-of Dok-3 Previously Grb2 was shown to be an inhibitor -/-of Ca2+ mobilization (Stork et al., 2004) 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-331, a residue critical for binding

to SH2 domain of Grb2, is essential and sufficient to render enhanced Ca2+ mobilization Incidentally, this amino acid together with Dok-3 PTB domain, is important for association with SHIP-1 SHIP-1 is a well-known inhibitor of BCR-induced Ca2+elevation However the authors found that the loss of Dok-3 did not affect SHIP-1 activation but enhanced PLCγ2 activation and therefore concluded that SHIP-1 and Dok-

3 do not function together in a common Ca2+ signaling pathway, and PLCγ2 as an effector protein of Dok-3/Grb2 signaling module

Most of the work prior to the completion of this PhD thesis project had been focused on studying the role of Dok-3 in BCR signaling using either A20 mouse B lymphoma cell line or the DT40 chicken B cell line Although these studies indicate a role for Dok-3 in negative signaling of the BCR, a definitive role of Dok-3 in primary B cells has yet to be established, and awaits future experimentation

Diagram 4 Amino acid sequences of chicken, mouse and human Dok-3 orthologs aligned using ClusterW algorithms (obtained from Stork et al., 2007)

Conserved amino acids are depicted in red High or weak biochemical and structural similarity of amino acids positions are indicted in green and blue, respectively Non-conserved amino acid positions are in black The degree of similarity is further indicated below the alignment by asterisk for fully conserved and, colon or dot for strong and weak structural similarity, respectively The PH and PTB domains are accentuated by a single

or double underscore, respectively

* 2 D

Binds effector molecules

* 3

* 4

* 2 D

Binds effector molecules

* 3

* 4

Diagram 5 Domains of Dok-3 and its binding partner

Dok-3 has a PH domain, a PTB domain and 4 consensus tyrosine phosphorylation sites These regions mediate Dok-3 binding to Csk, SHIP-1, Abl and Grb2 The domains responsible for Dok-3 interactions with other proteins are denoted by domain A, B C and

D (red) The SH2 domain of Csk binds at Dok-3 at domain C; the SH2 and proline-rich region of SHIP-1 binds to Dok-3 at domain B and D; the SH2 domain of Grb2 binds to

Dok-3 at domain D; while the region responsible for binding of Abl to Dok-3 at B is still unknown

1.4.5.3 Dok-4, Dok-5 and Dok-6

Dok-4, 5 and 6 constitute a subgroup of Dok family members that are coexpressed with c-Ret, a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), in various neuronal tissues Murine Dok-4 and 5 was first identified using c-Ret as a bait in yeast-two-hybrid screen (Grimm et al., 2001), and

by searching the EST data base for new molecules with homology to the PTB domain of Dok-1 (Bedirian et al., 2004a) whereas human Dok-4 and Dok-5 genes were cloned while searching the human genome data base for new genes encoding PH-PTB domains with potential roles in receptor tyrosine kinase signaling (Cai et al., 2003) Cai et al termed these two newly identified proteins IRS5 and IRS6 for Dok-4 and Dok-5, respectively, as they also acted downstream of insulin receptors Using RT-PCR, Favre et al showed that human Dok-4 and 5 are expressed in T cells but their function remain unclear to this date (Favre et al., 2003) Dok-6 was isolated through EST data base searching using mouse Dok-4 and 5 nucleotide sequences (Crowder et al., 2004) It is approximately 75 to 78% identical to Dok-4 and 5, respectively

Northern blot analysis reveals both human and murine Dok-4 mRNA to be strongly expressed in non-hematopoietic organs, particularly heart, intestine, brain, lungs, kidneys and skeletal muscle These are interesting expression patterns in term of insulin action as muscle and liver are two of the most important systemic targets of insulin mRNA expression patterns of Dok-5 in mice are dramatically different from what was found in human While murine Dok-5 mRNA was only found to be expressed in regions

of the brain including cerebral cortex, cerebellum and brainstem but not in the skeletal muscle (Grimm et al., 2001; Bedirian et al., 2004b), human Dok-5 mRNA was mainly expressed in skeletal muscle and less so in the brain tissue (Cai et al., 2003) Dok-6 mRNA co-expressed with Ret in several locations, including sympathetic, sensory, and parasympathetic ganglia, as well as in the ureteric buds of the developing kidneys Dok-4,

5 and 6 not only share high homology in their PH and PTB domain but also in a short ten amino acid motif in the C terminus tail This highly conserved motif, as PRSAYWHHIT

in Dok-4 (where Y is Tyr-269), PRSAYWQHIT in Dok-5 (where Y is Tyr-267) and PRSAYWHHIT in Dok-6 (where Y is Tyr-268), may indicate a conserved function or mode of regulating for this subclass of Dok proteins

During development of the central and peripheral nervous systems, neurite extension mediated via GDNF and its receptor c-RET is critical for neuronal differentiation Activated c-Ret recruits signaling proteins like Grb7, Grb 10, Src, PLCγ, Shc and Grb2, which bind to phosphorylated tyrosine residues in the C-terminus cytoplasmic region It is known that activated c-Ret promotes neurite outgrowth and Tyr-

1062 in c-Ret is essential All three Dok proteins bind to the phosphorylated c-Ret at

Tyr-1062 residue and shown to mediate neuronal differentiation and neurite outgrowth (Crowder et al., 2004; Grimm et al., 2001; Uchida et al., 2006) While all three Dok proteins mediate c-Ret signaling only Dok-4 and 5 play a positive role in insulin receptor signaling (Cai et al., 2003) Dok-4 and 5 are tyrosine-phosphorylated in response to insulin and IGF-1 in transfected cells, although the kinetics and responses differ Dok-4 is rapidly and heavily phosphorylated in response to insulin and, once phosphorylated, binds a set of SH2 domain proteins, including RasGAP, Crk, Src and Fyn, and activates