Background: Syk is a protein tyrosine kinase that plays an important role in intracellular signal transduction in hematopoietic cells (1-3). Syk interacts with immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of immune receptors (4). It couples the activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis (4). There is also evidence of a role for Syk in nonimmune cells and investigators have indicated that Syk is a potential tumor suppressor in human breast carcinomas (5). Tyr323 is a negative regulatory phosphorylation site within the SH2-kinase linker region in Syk. Phosphorylation at Tyr323 provides a direct binding site for the TKB domain of Cbl (6,7). Tyr352 of Syk is involved in the association of PLCγ1 (8). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain; phosphorylation at Tyr525/526 of human Syk (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (9).
Background: Microglia cells are resident macrophages of the brain that survey the brain environment and dynamically respond to maintain brain homeostasis. Microglial responses include phagocytosis of cellular debris, restricting sites injury or pathology, and/or releasing inflammatory signals to initiate an immune response. Such responses are important during normal development and during diseased states (1).Recently, the role of microglia in neurodegenerative disease pathology, particularly Alzheimer’s disease (AD), has been of intense investigation. Much of this work is driven by human genetic data that links microglia-enriched genes with AD progression (2). The triggering receptor expressed on myeloid cells 2 (TREM2) protein is an innate immune receptor that is expressed on the cell surface of microglia (3). TREM2 plays a role in innate immunity, and a rare functional variant (R47H) of the TREM2 gene is associated with the late-onset risk of AD (3,4). How TREM2 contributes to disease function is currently an active area of research (4,5), but might drive a number of microglial cellular functions ranging from microgliosis, phagocytosis, and cytokine release via a variety of signaling cascades triggered by TREM2.The TREM2 receptor is a single-pass type I membrane glycoprotein that consists of an extracellular immunoglobulin-like domain, a transmembrane domain, and a cytoplasmic tail. Ligands for TREM2 include phospholipids, apolipoproteins, and lipoproteins. Upon activation, TREM2 interacts with the tyrosine kinase-binding protein DNAX-activating protein 12 (DAP12, TYROBP) to form a receptor-signaling complex (6). Ligand binding by DAP12-associated receptors, including TREM2, results in phosphorylation of tyrosine residues within the DAP12 immunoreceptor tyrosine-based activation motif (ITAM) by Src family kinases; ITAM phosphorylation leads to activation of spleen tyrosine kinase (Syk) and downstream signaling cascades (7). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain and phosphorylation at these residues (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (8). Syk phosphorylation is also a readout for β-amyloid triggered TREM2 activity (9). Phosphoinositide-specific phospholipase C γ 1/2 (PLCγ1/2) is reported to be down stream of Syk (10). Tyr352 of Syk is involved in the association of PLCγ1 (11); Syk-mediated phosphorylation PLCγ1 at Tyr783 activates PLCγ1 enzymatic activity (12). Interestingly, mutations in the microglia-enriched PLCγ2 gene are associated with AD (13,14,15).
Background: Cluster of Differentiation 3 (CD3) is a multiunit protein complex expressed on the surface of T-cells that directly associates with the T-cell receptor (TCR). CD3 is composed of four polypeptides: ζ, γ, ε and δ. Engagement of TCR complex with antigens presented in Major Histocompatibility Complexes (MHC) induces tyrosine phosphorylation in the immunoreceptor tyrosine-based activation motif (ITAM) of CD3 proteins. CD3 phosphorylation is required for downstream signaling through ZAP-70 and p85 subunit of PI-3 kinase, leading to T cell activation, proliferation, and effector functions (1). Cluster of Differentiation 8 (CD8) is a transmembrane glycoprotein expressed primarily on cytotoxic T cells, but has also been described on a subset of dendritic cells in mice (2,3). On T cells, CD8 is a co-receptor for the TCR, and these two distinct structures are required to recognize antigen bound to MHC Class I (2). Cluster of Differentiation 4 (CD4) is expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells, and plays an important role in the development and activation of T cells. On T cells, CD4 is the co-receptor for the TCR, and these two distinct structures recognize antigen bound to MHC Class II. CD8 and CD4 co-receptors ensure specificity of the TCR–antigen interaction, prolong the contact between the T cell and the antigen presenting cell, and recruit the tyrosine kinase Lck, which is essential for T cell activation (2). Granzyme B is a serine protease expressed by CD8+ cytotoxic T lymphocytes and natural killer (NK) cells and is a key component of the immune response to pathogens and transformed cancer cells (4). Forkhead box P3 (FoxP3) is crucial for the development of T cells with immunosuppressive regulatory properties and is a well-established marker for T regulatory cells (Tregs) (5). CD19 is a co-receptor expressed on B cells that amplifies the signaling cascade initiated by the B cell receptor (BCR) to induce activation. It is a biomarker of B lymphocyte development, lymphoma diagnosis, and can be utilized as a target for leukemia immunotherapies (6,7). F4/80 (EMR1) is a heavily glycosylated G-protein-coupled receptor and is a well-established marker for mouse macrophages (8). CD11c (integrin αX, ITGAX) is a transmembrane glycoprotein highly expressed by dendritic cells, and has also been observed on activated NK cells, subsets of B and T cells, monocytes, granulocytes, and some B cell malignancies including hairy cell leukemia (9,10).
Background: A-Raf, B-Raf, and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway (1). Activation of c-Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338, Tyr341, Thr491, Ser494, Ser497, and Ser499 (2). p21-activated protein kinase (PAK) has been shown to phosphorylate c-Raf at Ser338, and the Src family phosphorylates Tyr341 to induce c-Raf activity (3,4). Ser338 of c-Raf corresponds to similar sites in A-Raf (Ser299) and B-Raf (Ser445), although this site is constitutively phosphorylated in B-Raf (5). Inhibitory 14-3-3 binding sites on c-Raf (Ser259 and Ser621) can be phosphorylated by Akt and AMPK, respectively (6,7). While A-Raf, B-Raf, and c-Raf are similar in sequence and function, differential regulation has been observed (8). Of particular interest, B-Raf contains three consensus Akt phosphorylation sites (Ser364, Ser428, and Thr439) and lacks a site equivalent to Tyr341 of c-Raf (8,9). Research studies have shown that the B-Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma (10). Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301, and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation. The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events (11).