La secundaria

In normal B-cells, when the immunoglobulin molecule is ligated, signalling is triggered by phosphorylation of the cytoplasmic immunoreceptor tyrosine- based activation motifs (ITAMs) of CD79a and CD79b (Packham and Stevenson, 2010) (Niiro and Clark, 2002) (Dal Porto et al., 2004). This

phosphorylation is predominantly catalysed by the Src family kinase Lyn and spleen tyrosine kinase (SYK) (Kulathu et al., 2008) (Geahlen, 2009). This phosphorylation and activation is a critical event in BCR signalling (Jiang et al., 1998) (Takata et al., 1994) leading to the formation of a plasma

membrane-associated signalling complex (signalosome) which includes many kinases and adaptor proteins, including the kinases SYK, Bruton tyrosine kinase (BTK), and Lyn, the guanine exchange factor Vav proteins, and the adaptor proteins Grb2 and B-cell linker (BLNK) which mediate activation of

downstream signalling pathways (Woyach et al., 2012). This activation is amplified by several protein kinases. SYK phosphorylates both CD79a/CD79b and Lyn provides amplification of the signal through recruitment of other protein tyrosine kinases together with formation of a complex with

costimulatory molecules including CD19 that reduce the threshold of B-cell activation (Yamamoto et al., 1993) (Rolli et al., 2002) (Fearon et al., 2000). This results in BCR aggregation and formation of a microcluster or lipid raft on the plasma membrane (Cheng et al., 1999). Signal propagation from the BCR occurs via multiple pathways, predominantly through phospholipase C-γ2 (PLC-γ2) and phosphatidalyinositol-3-kinase(PI3K). After initial

phosphorylation of ITAMs by SYK and Lyn, BLNK is phosphorylated by SYK when it is recruited to the non-ITAM tyrosines of CD79a, where it binds via its Src homology2 (SH2) domain (Engels et al., 2001) (Kabak et al., 2002). BTK then binds to this complex and together BTK and SYK activate PLC-γ2 by dual phosphorylation. Activation of PLC-γ2 produces the second messengers diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3) from the plasma membrane lipid phosphatidylinositol 4,5-bisphophate (PIP2) (Dal Porto et al., 2004). DAG activates protein kinase C, and IP3 releases calcium from the endoplasmic reticulum and the extracellular compartment (Roos et al., 2005) Calcium release directly activates a number of transcription factors, including NF-κB, Jun, and nuclear factor of activated T-cells (NFAT). NFAT proteins are indirectly activated by calcium through the calcium dependent activation of the phosphatase calcineurin. Dephosphorylated NFAT proteins are translocated to the nucleus and subsequently regulate cytokine production and other

effectors of the immune response (Rao et al., 1997). NF-κB plays a broad role in B lymphocyte proliferation and class switching and also mature B-cell survival (Ruland and Mak, 2003) (Stadanlick et al., 2008). The canonical NF- κB pathway is also an important survival effector in BCR signalling. NF-κB exists in inactive form in the cytoplasm as dimers consisting of p50, p52, p65/RelA, RelB, or c-Rel with the most usual dimers being the p50/p65 heterodimer and the p50/p50 homodimer (Ghosh et al., 1998). In the inactive form it is also bound to I-κB. On stimulation via BTK,PI3K or Akt the I-κB kinase complex causes phosphorylation and subsequent proteasomal degradation of I-κB. This results in nuclear translocation of NF-κB and gene transcription. NF-κB activates a wide variety of genes responsible for

inflammation, proliferation and B-cell survival (Ghosh et al., 1998) (Stadanlick et al., 2008).

Increased intracellular calcium (iCa2+) along with PKC anddirect activation by Vav and Grb2 activates mitogen-activated protein kinase (MAPK)-family kinases, including extracellular regulated kinase (ERK), c-JUN NH2-terminal kinase (JNK) and p38 MAPK. The MAPK pathway regulates a number of transcription factors, including Elk1 and c-Myc through Erk, c-Jun and ATF2 through JNK, and ATF2 and Max through p38 MAPK (Stadanlick et al., 2008) (Vigorito et al., 2005) (Johnson and Lapadat, 2002).

Initial phosphorylation and complex formation also activates the PI3K pathway. PI3K has two subunits, the p85 subunit, which is a regulatory

component and p110 subunit, which is a catalytic subunit. At rest they remain in close association but on activation of the BCR complex, the p85 subunit is recruited to the plasma membrane where it complexes with the Src kinases Lyn and Fyn (Woyach et al., 2012) (Pleiman et al., 1994). p85 also binds to CD19, and this complex activates the p110 subunit, which then

phosphorylates PIP2 to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 recruits a number of BCR signalling molecules with a pleckstrin homology domain to the plasma membrane, like the serine/threonine kinase AKT, BTK and other kinases. Active AKT is important for BCR-induced survival and proliferation pathways. It inactivates the pro-apoptotic BCL2 family protein, BAD, and forkhead family transcription factor FOX03a. It enhances activation of NF-κBthrough phosphorylation and inhibition of glycogen synthase kinase 3 which is also a negative regulator of MYC and D-type cyclins (Downward, 2004).

BTK is a member of the tyrosine protein kinase (Tec) family of kinases and also has a critical role in the amplification of the BCR signal. This is

exemplified by profound BCR signalling defects in X-linked

agammaglobulinemia (XLA) (also known as Bruton’s Agammaglobulinemia or Congenital Agammaglobulinemia) and its mouse counterpart X-linked

immunodeficiency (XID). In these conditions, there is a failure in B-cell

development at the pre-B to immature B-cell stage and subsequent defective B-cell signalling and reduced immunoglobulin production, all leading to profound humoral immune deficiency (Tsukada et al., 1993) (Vetrie et al., 1993). The major molecular defect is a mutation in the pleckstrin homology domain of BTK which prevents effective membrane recruitment by PIP3. This will cause defect in calcium flux associated with BCR signalling and thereby the downstream signalling (Roos et al., 2005).

BTK is mainly involved in the initial phosphorylation events and deficiency produces defects in early BCR phosphorylation, whereas increasing

intracellular calcium can restore downstream effects of BCR signalling (Khan et al., 1995). SYK and Lyn phosphorylate BTK at the Y551 site of the kinase domain. This step is usually followed by amplification through auto

phosphorylation of the Y223 site in the SH3 domain (Park et al., 1996). In addition, BTK recruits phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) which are responsible for synthesis of PIP2, and after phosphorylation by PI3K to PIP3 results in continued recruitment of BTK. BTK also activate IκB kinase, which phosphorylates the NF-κB inhibitor I-κBα, allowing NF-κB to translocate to the nucleus (Saito et al., 2003).

The activation of positive BCR signalling pathways is tightly regulated by inhibitory signals to prevent the unrestrained activation that can result in development of autoimmune conditions and malignancies (Woyach et al., 2012). This is mainly mediated by inhibitory regulators, such as CD22 or FcγRIIb (CD32) and various phosphatases, including SH2 domain-containing tyrosine phosphatase-1 (SHP-1) and SH2 domain-containing phosphatidyl 5- phosphatase (SHIP) -1 and -2 and kinases with differential activation and inhibitory properties, like Lyn. CD32 when co-clustered with BCR induces a negative signal by recruiting SHIP to the plasma membrane which eliminates the membrane binding of PLC-γ2, BTK, and Akt by hydrolysing PIP3 (Ono et al., 1996). SHP-1 can associate with ITIM-containing molecules, and activated SHP-1 dephosphorylate various substrates (Scharenberg et al., 1998) (Carver et al., 2000) (Bolland et al., 1998). Coligation of the BCR and CD32 results in the reversal of SHP-1 autoinhibition, SHP-1 is also associated with the BCR at rest, which gets disrupted by BCR stimulation, suggesting that SHP-1 is involved in preventing signal transduction in resting B-cells (Pani et al., 1995). These phosphatases are activated downstream of Lyn, and therefore Lyn plays both positive and negative roles in signal transduction via the BCR. It has been shown that Lyn knockout mice demonstrate BCR hyper-

responsiveness and develop lethal autoimmune glomerulonephritis (Hibbs et al., 1995) (Nishizumi et al., 1995).This is due to the fact that Lyn is required for phosphorylation of both SHIP and FcγRIIb making it a crucial kinase in the regulation of the BCR (Hibbs et al., 2002) (Nishizumi et al., 1998).