Btk, indentified for the first time in 1993 (Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia, 1993), is a non-receptor protein Tyrosine-Kinase, belonging to the Tec family (The Tec family of cytoplasmic tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk and homologs in other species, 2001). The sequence of the gene is located in the 21.3-22 region of the X chromosome long arm (Xq21.3-22) and it is composed by 19 exons. The protein is constituted by 659 amino acids and it has a molecular weight of 77 KDa (Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia, 1993).
CHEMICAL STRUCTURE AND IMAGES
1 MAAVILESIF LKRSQQKKKT SPLNFKKRLF LLTVHKLSYY EYDFERGRRG SKKGSIDVEK
61 ITCVETVVPE KNPPPERQIP RRGEESSEME QISIIERFPY PFQVVYDEGP LYVFSPTEEL
121 RKRWIHQLKN VIRYNSDLVQ KYHPCFWIDG QYLCCSQTAK NAMGCQILEN RNGSLKPGSS
181 HRKTKKPLPP TPEEDQILKK PLPPEPAAAP VSTSELKKVV ALYDYMPMNA NDLQLRKGDE
241 YFILEESNLP WWRARDKNGQ EGYIPSNYVT EAEDSIEMYE WYSKHMTRSQ AEQLLKQEGK
301 EGGFIVRDSS KAGKYTVSVF AKSTGDPQGV IRHYVVCSTP QSQYYLAEKH LFSTIPELIN
361 YHQHNSAGLI SRLKYPVSQQ NKNAPSTAGL GYGSWEIDPK DLTFLKELGT GQFGVVKYGK
421 WRGQYDVAIK MIKEGSMSED EFIEEAKVMM NLSHEKLVQL YGVCTKQRPI FIITEYMANG
481 CLLNYLREMR HRFQTQQLLE MCKDVCEAME YLESKQFLHR DLAARNCLVN DQGVVKVSDF
541 GLSRYVLDDE YTSSVGSKFP VRWSPPEVLM YSKFSSKSDI WAFGVLMWEI YSLGKMPYER
601 FTNSETAEHI AQGLRLYRPH LASEKVYTIM YSCWHEKADE RPTFKILLSN ILDVMDEES
Protein Aminoacids Percentage:
Btk has some domains essential for the interaction with other proteins, these domains are the Pleckstrin Homology ( PH), the Tec Homology ( TH), the Src-Homology 2 and 3 ( SH2 and SH3) and the catalytic domain. The PH domain binds specifically the phosphatidylinositol 4,5-bisphosphate (PIP 2), allowing the Btk recruitment on the cell membrane, where occurs the Btk- phospholipase C- γ (PLC-γ) mediated activation through phosphorylation in tyrosine (Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells, 1999). The TH domain is composed by the Btk Homology ( BH) region and by a proline rich region. The former is characterized by the presence of a zinc-binding site, needful for the GTPase proteins activation; the latter is responsible for the interaction with the SH3 domain and regulates the kinase activity. The SH2 domain recognizes specifically the phosphorylated tyrosine, allowing Btk to bind the activated tyrosine-kinase receptors; the SH3 domains recognizes the proline rich sites. After all the catalytic domain, localized at the C-terminal of the protein, is characterized by tyrosine-kinase activity.
Figure 1: Bruton’s Tyrosine Kinase (Btk) structure (Image adapted, Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain, 2009)
Ca 2+ SIGNALING IN B LYMPHOCYTES AND THE ROLE OF BTK:
After an antigenic stimulus, the B lymphocyte actives a chain of intracellular events that determine the cellular response to that stimulus. The B Cell Receptor (BCR), located on the lymphocyte membrane, recognizes this antigenic stimulus and transduces it through some second messengers. Important is the role of the calcium
( Ca 2+) in the initial phase of the B lymphocyte activation. The sudden increment of the intracellular calcium concentration (Ca 2+i), observed after BCR stimulation, is a irreversible change to the lymphocyte activated state and to those intracellular events that are fundamental for the humoral immunity response regulation. For instance, Ca 2+ regulates the activation and/or the translocation of transcription factors (Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression, 1998), the lymphocyte shape and polarity (Mapping the sensitivity of T cells with an optical trap: polarity and minimal number of receptors for Ca(2+) signaling, 1999), the gene expression (Gene regulation mediated by calcium signals in T lymphocytes, 2001).
When the BCR is activated by the binding with an antigen, the Lyn tyrosine-kinase, belonging to the Src family, phosphorylates the ITAMs ( Immunoreceptor Tyrosine Activation Motifs) situated on the Igα e Igβ chains (Antigen and Fc receptor signaling. The awesome power of the immunoreceptor tyrosine-based activation motif (ITAM), 1995) of the BCR receptor complex, promoting the Syk kinase recruitment (Signal transduction via the B-cell antigen receptor: the role of protein tyrosine kinases and protein tyrosine phosphatases, 2000). Then Syk binds the phosphorylated ITAMs through its SH2 ( Src-Homolgy 2) domains activating itself by this binding. Subsequently Syk phosphorylates the Y204 tyrosine of the Igα chain and an adapter protein called SLP-65 ( Src-homology 2 domain-containing Leukocyte Protein-65), known also as BLNK ( B cell Linker protein) (BLNK: a central linker protein in B cell activation, 1998), activating its interaction sites and binding itself to BLNK. This protein lacks of catalytic activity; its task is to facilitate the protein-protein and protein-cytoplasmic membrane interaction among those elements involved in the initial phase of this signaling pathway (Adapter proteins in lymphocyte antigen-receptor signaling, 2000). BLNK can anchors itself to the cytoplasmic membrane thank to a leucine motif located on its N-terminal. When the B Cell Receptor (BCR) is activated, occurs a chain of events that actives the phosphatidylinositol-3 kinase (PI 3 K) which generates PIP 3 (Signalling of Bruton's tyrosine kinase, Btk, 1999). It binds Btk, through the PH domain (A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains, 1997), causing its transfer from the cytosol to the cytoplasmic membrane; this process is facilitated by BLNK through its SH2 domain. Furthermore BLNK recruits the PLCγ2 in proximity of the Syk-BLNK-Btk complex. PLCγ2, that is activated through the concomitant phosphorylation by Btk and Syk, hydrolyzes the phosphatidylinositol (4,5)-bisphosphate (PIP 2) in diacilglicerol (DAG) and inositol phosphate 3 (IP 3). IP 3 binds the IP 3 receptors ( IP 3 R) located on the endoplasmic reticulum (RE), leading the Ca 2+ channels opening (Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes, 1999); so Ca 2+ flows in the cytosol. When the Ca 2+ concentration decreases over a threshold value, the N-terminal portion of a protein called STIM1 undergoes a conformational change that induces the dimerization. These dimers go in a ER region that allows the binding among the STIM1 C-terminal and Orai1 N- and C-terminal. This interaction induces the aggregation of the Orai1 monomers to tetramers forming a channel through which flows a Ca 2+ current from the extracellular space into the cell (Calcium signaling in immune cells, 2009). Moreover, the calcium mobilization, activates transcription factors like NF-κB (Nuclear factor- kappa B) that is involved in the cell cycle regulation and in the cell survival (Transitional type 1 and 2 B lymphocyte subsets are differentially responsive to antigen receptor signaling, 2002).
Figure 2: B Cell Receptor (BCR) Signaling (Image adapted, B-lymphocyte calcium influx, 2009.).
One of the mechanism of regulation of the Btk tyrosine-kinase activity depends by the Inhibitor of the Bruton’s Tyrosine Kinase ( IBtk) activity. The genetic locus codifying for IBtk is localized in the 14.1 region of the chromosome 6 long arm (6q14.1) (Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk, 2008). In this region is usual the verification of chromosomal aberrations that cause many times lymphoproliferative diseases (Chromosome 6 suffers frequent and multiple aberrations in thymoma, 2002). The IBtk gene has 77,56 kbp and it is constituted by 29 exons. From this sequence derives three different mRNA that codify for three proteins: IBtkα that has a molecular weight of 150,53 kDa, which is localized in the cytoplasm; IBtkβ that has a molecular weight of 133,87 kDa, which is localized in the nucleus; IBtkγ that has a molecular weight of 26,31 kDa, which is localized in the cytoplasm. From the study of the gene sequence that codifies for the three isoforms is deduced an evolutive conservation; expression analysis showed the absence of the β isoform in the murine proteome (Widespread mRNA polyadenylation events in introns indicate dynamic interplay between polyadenylation and splicing, 2007). Moreover further analysis revealed a conservative domain (aa 1286-1320 of IBtk, aa 173-207 of IBtkγ) that leads the interaction of IBtkγ with the Btk PH domain. This interaction, demonstrated through in vitro (Tyr223 self-phosphorylation) and in vivo (Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein, 2001) experiments, causes a negative dose-depending regulation of the Btk kinase activity. IBtkγ is highly expressed in the human lymphoid cells and in the murine spleen. Knockout mice IBtk -/- show an abnormal B cells development and activation. Recently has been shown a mechanism of regulation of IBtkγ on Btk. Particularly, the BCR stimulation causes the phosphorylation of some IBtkγ serine residues by the Protein Kinase C (PKC). The IBtkγ phosphorylation-PKC mediated causes the destabilization of the Btk/IBtkγ complex and the subsequent dissociation of Btk from its inhibitor. The mass spectrometry analysis has identified in the Serine 90 (S90) a critical residue that leads this mechanism.
Figure 3: Inhibitor of Bruton’s Tyrosine Kinase (IBtk) structure (Image adapted, Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk, 2008).
AGAMMAGLOBULINEMIA X-LINKED (XLA):
Studies demonstrated that mutations in the Btk gene cause an hereditary immunodeficiency called X-linked agammaglobulinemia ( XLA), discovered by Ogden C. Bruton in 1952 (Agammaglobulinemia, 1952). This pathology is characterized by the lack of the B lymphocyte development, observing a low level of plasmic gamma-globulins and a normal level, or lightly increased, of T lymphocyte. The patients show frequent bacterial or viral infections. Btk is expressed principally in the B lymphocytes and in all hematopoietic cells, comprised myeloid and erythroid progenitors, whereas in mature cells is observed a low expression (Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. Implication of phosphatidynositol 3-kinases, 2002). It is fundamental for the B lymphocyte development, differentiation and signaling (Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain, 2009).