Molecular and biophysical analysis of the non-catalytic PH, TH, SH3 and SH2 domains of Bruton tyrosine kinase (Btk) protein
Michael P. Okoh (Scientific publications)
Department of Biosciences, Division of
Biochemistry, University of Helsinki;
To be presented
for public criticism, with the permission of the Faculty of Science of
the University of Helsinki, in the auditorium PIII in Portania, Yliopistokatu
2 Signal transduction
3 Btk non-catalytic domains
4 Prelude to experimental approaches: Spectroscopic methods in brief
5 Aims of the study
6 Experimental approaches
9 Future perspectives and conclusions
I. Okoh, M.P. and Vihinen, M. (1999) Pleckstrin Homology Domains of Tec Family Protein Kinases. Biochem Biophys Res Comm. 265, 151-157
II. Okoh, M.P. and Vihinen, M. (2002) Interaction Between Btk TH and SH3 Domain. Biopolymers 63, 325-334
III. Okoh, M.P., Kainulainen, L., Heiskanen, K., Nizam Isa, M., Varming, K., Ruuskainen, O. and Vihinen, M. (2002) Novel insertions of Bruton tyrosine kinase in patients with X-linked agammaglobulinemia. Human mutat. in press
IV. Hansson, H., Okoh, M.P., Smith, C.I.E., Vihinen, M., and Härd, T. (2001) Intermolecular interactions between the SH3 domain and the proline-rich TH region of Brutons tyrosine kinase FEBS Lett. 489, 67-70
V. Okoh, M.P., Lappalainen, I., Stenberg, K., Pasanen, T. and Vihinen, M. (2002) Ligand Binding and Stability of Four N-Terminal Domains of Btk. Manuscript
Bruton tyrosine kinase (Btk) belongs to the Tec family of non-receptor protein tyrosine kinases. Mutations in the gene coding for Btk lead to a hereditary immunodeficiency, X-linked agammaglobulinemia (XLA), in man. This family of tyrosine kinases shares a common domain composition, with an N-terminal Pleckstrin homology domain, the Tec homology (TH) domain, Src homology 3 (SH) and SH2 domain, and a C-terminal Src kinase domain. The TH domain adjacent to the SH3 domain in Btk consists of two motifs, the Btk motif and proline-rich regions (PRR) that conform to the canonical SH3 binding motifs.
This study aimed at elucidating the role of the N-terminal non-catalytic domains (PH, TH, SH3 and SH2) in the regulation of the kinase activity of the Btk protein, and exploring the nature of mutations in Btk encoding gene in Finnish patients with XLA. Molecular modelling of Btk and its family members with mutational analysis of Btk gave insights on the structural and molecular consequences of amino acids alteration in the PH domain of the Btk. Using base excision sequence scanning methodology followed by direct sequencing, several families with XLA were studied. The role of the SH3 domain was studied using circular dichroism (CD) and fluorescence spectroscopy by analysing binding of peptides corresponding to segments of the PRRs, to the SH3 domain. The N-terminal half of the PRR was shown to bind specifically, whereas the C-terminal peptide had no detectable affinity. The TH domain had about four times lower affinity for the SH3 domain (17.0 mM) than the N-terminal peptide (3.9 mM). For the PRR-SH3 domain, neither of the two peptides could compete for the binding to the construct. However, the TH domain binding to the PRR-SH3 domain had two times lower affinity than to the SH3 domain alone. By using intermolecular NMR cross-relaxation techniques combined with gel filtration chromatography profiles, a dimer formation involving the PRR and the SH3 domain was identified. Further, using CD, fluorescence spectroscopy and temperature gradient gel electrophoresis (TGGE), the unfolding process and stability of the N-terminal (PH, TH, SH3 and SH2) domains of Btk was studied. These results have helped us to gain insights into the function and regulation of the Btk protein. Moreover, the involvement of the non-catalytic domains in signalling and the concomitant disease phenotype upon amino acid alterations is further elucidated.
1.1 Primary immunodeficiency disorders (PIDs)
Primary immunodeficiency disorders (PIDs) affect the functioning of the immune system. They arise mainly due to mutations in genes, which are involved in the production of the components of the immune system. Due to these intrinsic defects, affected patients are highly susceptible to persistent and recurrent infections (Buckley et al., 1997; Smith and Notarangelo, 1997; Vihinen et al., 2001). PIDs were first identified some 50 years ago (Smith and Notarangelo, 1997). In molecular terms, the gene whose defects cause the X-linked form of chronic granulomatous disease (XCGD), was the first to be identified using positional cloning technique (Royer-Pokora et al., 1986). X-linked agammaglobulinemia (XLA) was the first reported human disorder in which a mutation in the gene encoding a cytoplasmic protein tyrosine kinase was found (Tsukada et al., 1993; Vetrie et al., 1993). Adenosine deaminase (ADA) deficiency was the first primary immuno-deficiency in which gene therapy was attempted (Blaese et al., 1995; Bordignon et al., 1995). Bovine enzyme replacement therapy has usually been accompanied with polyethelene glycol (PEG), in the treatment of patients with ADA deficiency, hence, the clinical impact of gene therapy has been difficult to assess. Cavazzana-Calvo et al. (2000) reported the first successful gene therapy trial for a patient with X-linked severe combined immunodeficiency. A significant advancement over the pioneering studies of Bordignon et al. (1995), using gene therapy in the treatment of ADA deficiency has also been recently reported (Aiuti et al., 2002a; Aiuti et al., 2002b).
Most PIDs are characterized by an increased susceptibility
of the individual to opportunistic infections (Lederman and Winkelstein,
1985; Arkwright et al., 2002). Patients with B-cell deficiency are liable
to bacterial infections, whilst patients with T-cell defects tend to be
susceptible to various parasitic, viral and fungal infections (Buckley
et al., 1997; Vihinen et al., 2001). Diseases that afflict phagocytes
cause mainly skin and oral bacterial infections and the formation of granulomas
due to the spread of the causative bacteria to such organs as the liver
(Vihinen et al., 2001). In these patients fungal infections tends to cause
severe complications. Patients with certain PIDs are known to be susceptible
to some specific pathogens. Patients with X-linked lymphoproliferative
syndrome (XLP), a disease caused mainly due to mutation in the gene (SH2D1A)
(Coffey et al., 1998; Nichols et al., 1998; Sayos et al., 1998), are prone
to Epstein-Barr virus (EBV) infections. Whereas those with mutations in
interleukin 12 (IL-12), with the disrupted interferon-γ signaling, are
susceptible to some forms of idiopathic mycobacterial and Salmonella infections
(de Jong et al., 1998).
Genes associated with PIDs are found on a number of
chromosomes (Table 1.1).
The majority of the genes code for multidomain proteins, although there are some e.g. ADA and SH2DIA with a single domain (for details see Vihinen et al., 2001 and references therein). Most of the protein defects implicated in PIDs have been found to be involved in diverse cellular processes ranging from signal transduction, transcription factors, phagocytosis, to cell surface receptors etc. However, for many of these proteins, with their diverse locations, their exact mechanisms of action have yet to be established.
1.1.1 X-linked agammaglobulinemia (XLA)
XLA is a disease resulting from defects in Btk protein. The disease is characterized by profound hypogammaglobulinemia, an early onset of bacterial infections, a decrease in the number of B-lymphocytes, and an almost complete lack of plasma cells (Sideras and Smith, 1995; Smith and Notarangelo, 1997). The immunoglobulin levels in affected individuals are very low. The disease afflicts about 1/200,000 males (Sideras and Smith, 1995; Ochs and Smith, 1996). The associated infections, particularly bacterial meningitis and pneumonia, are often life threatening and therefore, early diagnosis is critical for the management of XLA (McKinney et al., 1987; Ochs and Smith, 1996).
Btk was identified as the molecular defect in XLA by two different approaches, using positional cloning (Vetrie et al., 1993) and in a search for novel protein kinases expressed in B lymphocytes (Tsukada et al., 1993). The gene was mapped to the Xq21.3-22 region in the mid-portion of the long arm of the X-chromosome (Kwan et al., 1986; Ott et al., 1986). The human gene encompasses 37.5 kb and is organized into 19 exons (Hagemann et al., 1994; Rohrer et al., 1994), including a 5 untranslated region (exon 1). Btk is a 659 amino acid long protein in both man and mouse (Vetrie et al., 1993; Tsukada et al., 1993). It encodes a cytoplasmic protein tyrosine kinase (PTK).
The study of XLA has profoundly contributed in the understanding of the immunological basics of the humoral immunity. Btk is expressed throughout the development in B cells except the plasma cells (Sideras and Smith, 1995). In XLA, the production of Btk is blocked at the transitional stage from pro-B to pre-B cells resulting in B cell apoptosis (Anderson et al., 1996) (see Fig 1.1). Moreover, as was recently shown in xid mice, the first manifestation of Btk deficiency occurs in the transition from pro-B to pre-B stage (Cancro et al., 2001). Thus, the initial effects of xid in mice and XLA in man are at the same stage of B cell development (Cancro et al., 2001). Various types of XLA causing mutations have been identified in all Btk domains (Vihinen et al., 1999).
The analysis of Btk expression in primary cells of XLA patients has shown that
majority of the individuals do not express the protein, regardless of
the mutation type (Gasper et al., 2000). So far, it has not been possible
to predict how specific mutations at critical sites in Btk affect the
function and the concomitant XLA phenotype (Gasper et al., 2000; Gasper
et al., 1998). Further, correlation between the protein expression level
and differences in the clinical and immunological phenotypes have not
been possible. Many individuals previously diagnosed as having common
variable immunodeficiency or immunoglobulin subclass deficiency, have
been shown to have Btk deficiency (Futatani et al., 1998; Vórechvsky,
1994). The majority of the described mutations lead to a truncated protein.
Whilst the large majority of mutations are found in the coding region,
over 12% of mutations have been found to affect splice site recognition
sequences (Vihinen et al., 1995a; Conley et al., 1998). Several XLA causing
mutations in Btk have been described and are collected in the BTKbase
that is available at http://www.uta.fi/imt/bioinfo/Btkbase (Vihinen et
al., 1995a; Vihinen et al., 1999). In addition to describing the mutations,
the database also contains clinical information regarding to patients.
The term signal transduction implies the conversion of an input signal received at the extracellular face of the plasma membrane into an intracellular signal that, ultimately, leads to alteration of gene expression. The correct transmission of the signal is vital for both unicellular and multicellular organisms; the stimulation of cells from outside, due to signaling molecules, can trigger a cascade of events leading to diverse cellular responses such as, differentiation, growth and movement (Hunter, 1995). Various types of proteins, including transmembrane receptors, G-protein subunits, adaptor proteins, kinases and phosphatases, are involved in signaling.
The plasma membrane plays an important role as a meeting point for signaling proteins. The association of signaling proteins with the membrane can be direct or indirect (Mumby, 1997). The binding of a cytoplasmic protein to the membrane could be achieved, for example, by modification of the proteins by lipid (Mumby, 1997). Further, with specific protein-protein interactions, signals can also be transduced through the membrane via an indirect mechanism. These processes are regulated through covalent protein modification such as phosphorylation, one of the hallmarks of intracellular signaling (Carpenter and Cantley, 1996).
Btk protein is a non-receptor PTK known to be involved in B-cell survival, cell cycle progression and proliferation in response to B cell receptor (BCR) stimulation (Petro et al., 2000). The phosphorylation of a tyrosine residue in the activation loop of Btk is essential for its participation in BCR signaling (Kurosaki, 1997).
2.1 Non-receptor protein tyrosine kinases
Approximately 20% of the ~32,000 human genes are known to encode proteins that are involved in signaling (Blume-Jensen and Hunter, 2001). Among them are, for instance the transmembrane receptors, adaptor proteins, kinases and phosphatases. Included in the latter groups are the PTKs, a group of enzymes that have evolved to play key roles in transmitting signals in multicellular organisms. PTKs are found only in metazoans (Blume-Jensen and Hunter, 2001). In the human genome, over 90 genes encode for PTKs and these can be divided into two subgroups, receptor and non-receptor PTKs. Currently, there are 30 identified mammalian non-receptor tyrosine kinases which can be divided into 11 families based on amino acid sequence identities (Burkhardt and Bolen, 1996). In addition to the kinase domain, PTKs have other conserved domains, which are also found in several distinct signaling pathways. These conserved domains are thought to regulate signaling through their ability to mediate protein-protein interactions. These domains have consequently become the building blocks for the formation of complex networks of interacting proteins (Saouaf, 1995).
Signaling domains are generally dynamic and plastic, and can be modified. They mostly occur as part of domain assemblies in modular proteins. Although the catalytic domain of most PTKs has low substrate specificity, the non-kinase domains attached also interact with regulators and substrates in the context of specific signaling processes and the cellular environment. Moreover, intra and intermolecular interactions between domains can critically affect the kinase activity of the enzyme.
The role of modular proteins in signaling has been a focal point in signal transduction research over the last decade. Discoveries of proteins with variable domains such as PH, SH3, SH2, SH1 or the kinase domain, PDZ, C1 and C2 (Hurley et al., 2002) and elucidation of their properties and functions, have contributed greatly to the current understanding of signaling pathways. The importance of the domain concept is reflected in the fact that each domain mentioned above occurs in a large number of different signaling proteins. Hence, understanding the function of a particular domain in one protein can be used to infer its function in many other proteins that contain the same domain. Thus, domain information provides a simple and most powerful conceptual bridge in attempts to understand the vast and complex sequence data from the human genome (Hurley et al., 2002).
2.2 Tec family of cytoplasmic tyrosine kinases
The Tec family of PTKs is characterized by similar amino acid sequences (Fig. 2.1) and cells of the immune system express them predominantly. They have a characteristc domain architecture comprising the N-terminal PH domain followed by a Tec homology (TH) domain, which contains a Zn2+ binding Btk motif and a proline-rich region, a SH3, SH2, and the C-terminal kinase domain. Atypical member of the Tec family PTKs is Txk, which lacks the PH and part of the TH domain. Moreover, the Drosophila type 1 splice variant of Btk29A, also lack the PH domain. The structural similarity (Fig. 2.2) shared among the Tec family members suggest that these proteins may have overlapping functions.
Tec family kinase homolog are also expressed in other species, such as Drosophila melanogaster, skate and zebrafish (Roulier et al., 1998; Baba et al., 1999; Haire et al., 1997; Haire et al., 1998). Btk29A, the Drosophila member of Tec kinases, exists in two different splice variants i.e. types 1 and 2 (Baba et al., 1999). Type 2 of Btk29A is composed of 786 amino acids, hence it has the highest molecular weight among Tec kinases (Baba et al., 1999). Detailed information relating to the Tec family protein tyrosine kinases is presented in the Table 2.2
Table 2.2 Tec family PTKs
Figure 2.1 Sequence alignments of Tec family members. Mutations causing XLA are indicated above the sequence, X indicating nonsense mutations, # delections, and @ insertions.
2.2.1 The regulation of Btk
The Tec family proteins are involved in a vast array of signal transduction
pathways many of which form communication networks. The role of these
family members in signaling has been best characterised for Btk in the
context of BCR signaling (for review see Smith et al., 2001). The family
members are composed of similar domains, which are the most commonly used
domains involved in signaling (see Fig. 2.2).
Figure 2.2 Domain organisations of Tec family protein tyrosine
kinases. Txk contains in the N-terminus unique cysteine rich region.
Binding of an antigen ligand brings about the activation of BCR which, in turn, activates Btk through a phosphorylation cascade (Park et al., 1996; Oda et al., 2000). The stimulation of BCR is intimately linked to the activation of three cytoplasmic tyrosine kinase families, i.e. the Tec family, Src family, and the Syk family (Vihinen and Smith, 1996) (Fig. 2.3). Upon BCR cross-linking, PtdIns(3,4,5)P3 is generated by phosphatidylinositol 3-kinase (PI3K) which then leads to the PH domain-mediated translocation of Btk to the membrane (Li et al., 1997). This binding to the lipid intermediate is followed by the phosphorylation of Y551, a highly conserved activation loop tyrosine, by a transient association with Lyn, a Src kinase (Fig. 2.3), causing a marked increase in Btk activity. Subsequent autophosphylation of Y223 in the SH3 domain by the kinase domain induces the full activation of Btk (Rawlings et al., 1996; Park et al., 1996).
Recently it has been shown that antigen binding and oligomerization of the BCR results in rapid translocation of BCR into lipid rafts (detergent insoluble membrane microdomains enriched in cholesterol and sphingolipids) (Cheng et al., 1999). Lyn kinase a member of the Src kinase family is concentrated within the raft region. Thus lipid rafts can serve as platforms for signaling (Cheng et al., 1999). The translocation of the BCR into lipid rafts has been shown to result in the recruitment to BCR of several associated signaling components, such as signal cascades, BLNK (B cell linker protein), PI3 kinase, VAv (a guanine nucleotide exchange factor) and Btk (Cheng et al., 2001) (Fig. 2.3).
Prior to the ligand stimulation Tec and Btk have both been observed to be constitutively associated with gp130, the signal-transducing subunit for the IL-6 cytokine (Matsuda et al., 1995). More recently, in the downstream of antigen receptors in both B and T cells, Tec was shown to be activated (Yang et al., 2001). As to the downstream effect of Btk, it is interesting that Btk has been shown to be present in the nucleus of hematopoietic cells, although in a much lower amount compared to the cytoplasm (Mohamed et al., 2000). Under certain experimental conditions e.g. due to the SH3 domain deletion, Btk has been localized to the nucleus and perhaps being involved in regulating gene expression (Mohamed et al., 2000). In fact, both Btk and Tec are known to modulate transcription of cytokine genes in mast and T cells (Yamashita et al., 1998). Evidence linking B cell receptor stimulation with Btk nuclear response was recently found when Btk was shown to be involved in the activation of the transcription factor NF-κB (Petro et al., 2000). Apart from these implied roles in transcriptional activation and reprogramming gene expression in the nucleus, Btk family kinases are also thought to have an effect on cytoskeletal reorganization and cell motility (Qui and Kung, 2000). In DT-40 cells lacking Btk, the pattern of actin expression and its ability to aggregate was shown to differ from the wild-type cells (Nore et al., 2000), suggesting that Btk is involved in cytoskeletal reorganization.
Tec and Btk share common functions during B cell development (Ellmeier et al., 2000). Itk and Txk co-exist in T cells (Schaeffer et al., 1999). The activity of Tec/Btk is required for the proper generation of immature B cells. However, Btk function seems to be more important than that of Tec as in Btk deficient mice a reduction of pre-B cells was observed, but not in those lacking Tec (Ellmeier et al., 2000). Btk has also been found to fully compensate for the loss of Tec, but Tec is not able to fully compensate for the loss of Btk in mice cells (Ellmeier et al., 2000). Itk, on the other hand, has been shown to associate directly with CD28, a T-cell costimulatory molecule (August et al., 1997). Further, Tec like Itk is activated downstream of CD28, upon the phosphorylation of Dok (a molecule that interacts with the GTPase activating protein of Ras and Ras-GAP) (Yang et al., 1999). Moreover, upon reconstitution of Btk deficient DT40 cells Itk and Tec have been shown to have an analogous function to Btk in at least PLC-γ2 signaling (Tomlinson et al., 1999).
Btk has earlier been implicated in a regulatory role in tyrosine phosphorylation
of PLC-γ2 and subsequent calcium mobilization in BCR mediated
signaling (Fluckiger et al., 1998) (see Fig. 2.3). In this cascade, the
SH2 domain-containing inositol-polyphosphate 5-phosphate (SHIP)
(Fig. 2.3) has been implicated as an on/off switcher of Btk catalytic
activity (Nore et al., 2000). In chicken DT40 cells the patterns of tyrosine
phosphorylation in wild type and Btk deficient cells were found to be
similar (Takata and Kurosaki, 1996). Syk (spleen tyrosine kinase) family
kinase as shown in Fig. 2.3, is involved in Akt activation, and it has
been shown to act as an upstream regulator of BCR induced PI3-kinase,
with Btk regulating the BCR-mediated Akt activation downstream of PI3-kinase.
Akt is known to be a target of PI-3K (Chan et al., 1999). Further, the
activation of other Tec family members e.g. Itk upon Src/CD28-induced
activation in response to interleukin-6 stimulation has been found to
also require PI3-kinase activity (Lu et al., 1998). The evidence that
null Btk and xid mice are similar to p85a null mice further supports the
concept that Btk and PI3-kinase are within the same pathway(s). Both mice
were found with reduced numbers of mature peripheral B cells and diminished
proliferative response to anti-IgM, anti-CD40, and liposaccharide (Fruman
et al., 1999).
The Src family members Blk, Fyn, Lck, and Lyn are present in B cells, where they are involved in signal transduction induced by binding of an antigen ligand to the BCR (Reth and Wienands, 1997). They interact via their SH2 domain with Syk, and also bind to other signaling molecules. Activation of the Src family kinases and Syk are the key events in the early steps of BCR signaling (Fig. 2.3). It is known that Syk phosphorylates tyrosine residues in the ITAM (immunoreceptor tyrosine based activation motif) motifs in BCR (Flaswinkel and Reth, 1994), and hence sets into motion the activation of PTK signaling cascades (Flaswinkel and Reth, 1994). The two SH2 domains of Syk are likely to be involved in the binding to the double tyrosine-phosphorylated ITAMs of the BCR (Fig. 2.3.). The deletion of the gene coding for Syk in a chicken cell line clearly implicates Syk in the signaling pathways between BCR and phospholipase Cg (Craxton et al., 1999). Src family kinases are also involved in the phosphorylation of Btk (Mahajan et al., 1995).
3 Btk non-catalytic domains
As discussed above, the Tec family kinases are predominantly expressed in cells of the immune system. The expression patterns demonstrate their ability to play diverse regulatory roles in various physiological processes. Table 3.1 summarizes some of the molecules known to interact with the non-catalytic domains. Apart from the subtle sequence differences, it is most likely that the versatile nature of the constituent domains and their various binding partners are what permits the Tec family kinases to participate in such diverse actions. Below, the Btk non-catalytic domains are discussed in more detail.
Table 3 A summary of some of the molecules known to interact with
the N-terminal domains of Tec family cytoplasmic protein tyrosine kinases.
Some binding partners have only been shown to interact with specific Tec
kinases, and this is indicated in parentheses.
The PH domains consisting of about 120 residues are highly divergent, and have been found in a number of signaling and cytoskeletal proteins including protein tyrosine and serine/threonine kinases and their substrates, phospholipase C, GTPase activating proteins, guanine nucleotide releasing factors, and adaptor proteins (Shaw, 1996). Although the precise function of the PH domain is not fully understood, several lines of evidence suggest a critical role in Btk function. In many cases, it has been found to act as a membrane localization domain (Fukuda et al., 1996; Hyvönen and Saraste, 1997).
The structure for several PH domains including Btk has been determined (Hyvönen and Saraste, 1997). Despite very low sequence identity, the structures have the same fold consisting of a β-barrel formed of two β-sheets and a C-terminal α-helix that caps one end of the β-barrel. PH domains are highly polar (Blomberg and Nilges, 1997). The structure of the Btk PH domain reveals R28 to be one of the most highly conserved residues in this motif. Several charged residues including R28 and E41 form a cleft near the N-terminus of the domain. These residues are directly involved in the ligand binding (Baraldi et al., 1999). The inositol compound binds between loops β1-β2 and β3-β4 where both the R28 and E41 are located (Baraldi et al., 1999). The binding of E41K mutant was studied in a cocrystal with Ins(1,3,4,5)P4 (Baraldi et al., 1999). In another study, R28C substitution was found to have reduced binding to Ins(1,2,3,4)P4, presumably because the substitution removes the basic arginine from the ligand-binding cleft and this affects the strength of the electrostatic polarization (Fukuda et al., 1996).
Residues implicated in the binding of the PH domain to phosphoinositide compounds have been localized in Btk, pleckstrin, and PLC δ1 (Baraldi et al., 1999; Harlan et al., 1995; Ferguson et al., 1995). The positively charged surface of the PH domain binds to the negatively charged phosphate groups of the phosphoinositol molecules (Baraldi et al., 1999). Several XLA-causing mutations including the Xid mutant R28C are located within the PH domain (Vihinen et al., 1998; Vihinen et al., 1999, de Weers et al., 1994). The knockout variant of R28C in mice exhibited a much milder phenotype (Thomas et al., 1993; Kerner et al., 1995). In humans, patients with missense mutation of the corresponding residue (R28) as in xid have a severe B cell deficit and classical XLA (de Weers et al., 1994).
By using a retroviral passage mutagenesis scheme, a gain of function mutant (E41K) of Btk, located in the PH domain, was isolated (Li et al., 1995). The mutant allows the constitutive expression of Btk due to its increased affinity for phosphoinositol compounds. The expression of this mutant was shown to drive the growth of NIH3T3 cells (Li et al., 1995). It was found to enhance transphosphorylation of Y551 by endogenous Src family tyrosine kinases, and autophosphorylation at position Y223 within the SH3 domain. The combined phosphorylation(s) and autophosphorylation(s) lead to constitutive membrane association via binding by phosphoinositol products of PI3K, e.g PI(3,4,5)P3 and 1,3,4,5-tetrakisphosphate resulting in the activation of Btk (Rawlings et al., 1996; Baraldi et al., 1999). The interaction between the PH domain and PIP3 is presently the best-characterized requirement for membrane targeting of the protein. Btk PH domain has also been found to interact with proteins, including F-actin and focal adhesion kinase (FAK), suggesting multiple mechanisms for regulating Tec kinases (Qiu and Kung, 2000; Lewis et al., 2001). Further, the carboxy terminus of the PH domain has been shown to modulate binding to the bg subunit of heterotrimeric G proteins and, in conjunction with the Btk motif, to facilitates the interaction of Btk with the Gα12 subunit of heterotrimeric G proteins (Tsukada et al., 1994; Jiang et al., 1998).
3.2 TH domain
The TH domain in Btk consists of two motifs, the Btk motif and the proline rich region (PRR) (Vihinen et al., 1994a; Vihinen et al., 1997). The complete TH domain can be found only in the Tec protein family. The Btk motif follows the PH domain in several forms of Ras GTPase-activating protein 1 and also in a putative interferon γ-binding protein (Vihinen et al., 1995b). The function of the TH domain is not fully understood, although SH3 domains bind to PRR in vitro and the Btk motif seems to be essential for interactions with G proteins (Clapham and Neer, 1997).
The Btk motif consists of a highly conserved stretch of amino acids found in Tec kinases, Ras GAP, and IGBP (Vihinen et al., 1997). It structure has been determined in a cocrystal with the PH domain (Hyvönen and Saraste, 1997; Baraldi et al., 1999). The motif has a novel fold with a coordinated Zn2+ ion binding which packs against the ß-sheet of the PH domain. This motif seems to be crucial for the function of Tec family PH domains (Hyvönen and Saraste, 1997). A finding that in Txk, which is devoid of the PH domain, the Btk motif is also missing also supports functional linkage.
Btk contains a duplicated sequence of proline rich stretches at the C-terminal portion of the TH domain. In other Tec family kinases (except for Bmx), a homologous stretch is also found. Bmx contains a canonical SH3 domain-binding PXXP motif, however, the surrounding residues do not conform to the SH3-binding fingerprint. The proline rich region in the TH domain of Itk was shown to make an intramolecular contact with the SH3 domain (Andreotti et al., 1997). The two PRRs in the TH domain of Btk interact with the SH3 domains of Fyn, Lyn and Hck (Yang et al., 1995). Site-directed mutagenesis of the proline residues in the PRRs of Btk abolishes the interaction with SH3 domains (Yang et al., 1995).
3.3 SH3 domain
The SH3 domain consists of approximately 60 amino acids. They are present in a wide variety of proteins ranging from cytoskeletal proteins, such as myosin 1 and the a-chain of spectrin, to signal transduction proteins, e.g non-receptor tyrosine kinases and phospholipase C. Recent data from the genome sequencing projects further suggest that 63 proteins in Drosophila, 55 in Caenorhabditis elegans, and 25 in Saccharomyces cerevisiae contain at least one form of this domain (Rubin et al., 2000). SH3 domains bind to proline rich peptides and proteins. Both biochemical and genetic evidence have demonstrated that SH3 domain-mediated interactions are necessary for proper signaling by e.g. receptor tyrosine kinases (Pawson, 1995). The solution structure of the Btk SH3 domain has been determined using nuclear magnetic resonance (NMR) spectroscopy (Hansson et al., 1998). The structure of the Btk SH3 domain consists of two tightly packed anti-parallel b-sheets that form a sandwich-like fold.
The structural basis for the interactions between the proline-rich peptide ligands and SH3 domains are well understood (Lim et al., 1994). Based on the mode of binding, two classes of ligands (class 1 and 2) have been defined, distinguished by the orientation of the bound ligand (Mayer and Eck, 1995). Ligands of both classes contain a PXXP core sequence that anchors the ligand to the receptor, in the polyproline type II (PPII) helical conformation. The core sequence of the ligand, provides the promiscuity of binding to different SH3 domains, the flanking residues are unique to individual SH3 domains and bind to specific pockets (Lim et al., 1994). These findings combined with successive phage display experiments have provided a framework for an algorithm to predict specific binding sites for any SH3 domain from its primary sequence (Brannetti et al., 2000). In the Btk SH3 domain the potential ligand-binding site is formed of residues Y223-Y225, D232, W251, P265, and Y268 (Hansson et al., 1998).
Recent studies show that SH3 domains, under certain circumstances bind ligands, which lack the PxxP core binding motifs. For example, the peroxisome Pix-SH3 domain in Pak, the sequence that recognizes the SH3 was found to be PPPVIAPRPETKS (Manser et al., 1998). On the other hand, PxxDY provides the site for the binding of the SH3 domain of the epidermal growth factor receptor substrate Eps8 (Mongiovi et al., 1999). The SH3-domain of the immune receptor adaptor molecule SKAP55, recognizes a novel tyrosine based RKxxYxxY motif (Kang et al., 2000). Further, in YAP VPMRLR is the minimal sequence requirement for YAP recognition by the SH3 domain of p53 protein (Gorina and Pavletich, 1996). Conversely, as many SH3 ligands containing the PxxP core motifs have been detected by direct filter binding and yeast two hybrid systems, it is safe to assume that for most SH3 domains the PxxP core motif is the target recognition sequence. The recent comparison of the yeast SH3 domains with several other SH3 domains showed the SH3 domains, which recognize canonical PxxP motifs, to be the most numerous (Cesareni et al., 2002).
In Itk, a intramolecular regulatory association has been identified between the PRR and the adjacent SH3 domain (Andreotti et al., 1997). More recently, by using 2D-NOESY in combination with HSQC spectra measurements, an intermolecular interaction between the SH3 and SH2 domains of Itk was found (Brazin et al., 2000). The interaction provides an insight on the possible mechanism for displacement of the intramolecular interaction between the PRR and SH3 domain (Brazin et al., 2000; Ogawa et al, 2002). Possibly, the intermolecular interaction releases the SH3 domain-binding site that could be blocked by the intramolecular interaction, a step that may be necessary for the full activation of the Tec family members. A point mutation in the Itk SH3 domain, W208K, completely abrogates the intermolecular interaction and, also eliminates self-association of Itk (Brazin et al., 2000). Similarly, by introducing two additional lysines i.e. N383K, A384K (present in the Btk SH2 domain), using C-terminal rational modification of Btk SH3-SH2 protein, combined with C337 substitution (C337S and C377M) in the surface of the SH2 domain, the solubility and homogeneity of the Btk SH3-SH2 construct was improved (Nera et al., 2000). The addition of lysines at the N- or C-terminus has previously been shown to increase protein solubility (Ingley and Hemmings, 1999). The engineering of the Btk SH3-SH2 domains benefited from the observations gathered from the Btk SH2 model structure (Vihinen et al., 1994b). The dimer formation observed between Btk SH3-SH2 proteins could be intramolecular due to disulfide bridge formation, rather than intermolecular, since mutations C377S and C377M within the SH2 domain abrogate dimerization (Nera et al., 2000). The SH3-SH2 dimerization requires in addition to proper sequence, complementarity in structure and also complementarity outside the actual recognition site (Vihinen and Smith, 1998). Obviously, it will be essential to carry out further analysis on Btk SH3-SH2 to study the nature of this interaction.
A critical event in the catalytic activation of Btk is transphosphorylation of Y551 by Src in the catalytic domain. This is followed by autophosphorylation of Y223 in the SH3 domain. The observed self-association described above that involves the proline rich region open up the possibility that the autophosphorylation of Y223 could occur in either trans or in cis. The substitution Y223F in the Btk SH3 domain results in enhanced fibroblast transformation (Park et al., 1996), suggesting that the SH3 domain has a negative regulatory role (Park et al., 1996). The scenario of trans or cis activation does have implications for the understanding of the activation of Btk and other Tec family proteins. Thus, targeted mutants changing some of the residues within this domain and the adjacent TH domain would be vital to reveal the important residue(s) that are critical for either trans or cis activation. However, as recently shown (Hansson et al., 2001), changing the critical prolines in both the N-terminal PRR (Btk residue P189 and P192) and the C-terminal PRR (P203 and P206), that are thought to be critical in making van der Waals contact in the binding pockets, do not completely abrogate intermolecular interactions. This implies that other residues distal from these might contribute to the SH3 domain interactions.
3.4 SH2 DOMAIN
SH2 domains contain about 100 amino acids. They play a central role in a number of cellular processes, including growth, immune response, metabolism, mitogenesis, motility and gene transcription (Kuriyan and Cowburn, 1997). SH2 domain-containing proteins take part in the coordination of signal transduction pathways by binding to phosphotyrosine molecules. Binding specificities of several SH2 domains have been determined by using phoshotyrosine (pY) peptide libraries and phage display (Songyang et al., 1994; Cochrane et al., 2000). Most of the XLA-causing mutations in Btk SH2 domain disrupt the pY peptide binding sites (Vihinen et al., 1994; Vihinen and Smith, 1996; Vihinen et al., 1995).
The three-dimensional structures of several SH2 domains have been determined (e.g. Waksman et al., 1992; Waksman et al., 1993; Xu et al., 1995; Eck et al., 1993). The conserved fold contains a large central antiparallel a-sheet and an associated smaller a-sheet, flanked by a-helices on each side. The pY-containing ligand binds to the SH2 domain in an extended conformation. The crystal structures of SH2 domains in complex with high-affinity peptides have shown that several residues are involved in the phosphotyrosyl peptide binding. The number of residues binding, in addition to pY, varies from three to six or seven. Regions outside the actual pY binding region can be essential for affinity and specificity (Vihinen and Smith, 1998). The most important conserved interaction between the SH2 domain and its ligand is the direct interaction between the pY phosphate group and the guanidinium group of an arginine (R307 in Btk). Other important residues for pY binding in Btk are R288 and S309. Residues forming a second ligand-binding pocket for the residue pY+3 are found in aA, bB, bD, and the loop connecting strands B and C. These residues provide the specificity for pY+1 and pY+3 affinity (Kuriyan and Cowburn, 1997).
The Btk SH2 domain contains a single cysteine residue, C337. According to the model of SH2 domain, C337 is situated at the surface of the domain (Vihinen et al., 1994). In Grb2, oxidation and subsequent dimer formation impair protein crystallization (Cochrane et al., 2000). Using mutagenesis to replace the Btk C337 with Ser or Met, dimer formation was prevented (Mattsson et al., 2000). The C337S or C337M substitutions do not impair phosphotyrosine binding nor do they affect the secondary structure of the Btk SH2 domain (Mattsson et al., 2000).
A recently identified B-cell linker protein (BLNK or SLP-65) as one of the major Btk SH2 binding proteins explains the role of Btk SH2 domain in calcium signaling (Hashimoto et al., 1999; Su et al., 1999). Upon BLNK phosphorylation by Syk (Spleen tyrosine kinase), BLNK is thought to provide docking sites for Btk SH2 domain as well as for PLC-g2. Thus, this provides a scenario where, Btk and PLC-g2 are brought into close proximity (Hashimoto et al., 1999; Kurosaki and Tsukuda, 2000). When Btk is activated via the mechanisms earlier discussed (see also Fig. 2.3) it phosphorylates PLC-g2 leading to its full activation (Kurosaki and Tsukuda, 2000). In this process, phosphorylated BLNK was found to be vital further implying that BLNK is the direct connector between Btk and PLC-g2. However, the effect of Btk on PLC-g2 was abrogated when a mutant variant of Btk SH2 domain was used in a heterologous transfection system (Hashimoto et al., 1999).
4 Prelude to Experimental approaches: Spectroscopic methods in brief
CD Spectroscopy: Circular dichroism (CD) spectroscopy is a powerful tool for the studies of biochemical systems in solution. CD is sensitive for the shape of a protein molecule. The principles of CD spectroscopy are based on the chirality of a protein backbone and the side chains (For a complete details see Fasman, (1996)). This method can provide information about the secondary and tertiary structure of a protein molecule.
Fluorescence spectroscopy: Fluorescence spectroscopy can reveal information about the intrinsic environment of a biological molecule. The method is a sensitive mode to view the structural and dynamic characteristics of macromolecules (for details see Gore (2000)). Fluorescence measurements are based on the ability of a molecule to absorb electromagnetic radiation. Upon the absorption via a quantum mechanical process, the molecule is transformed from a ground state to an excited state. The absorption of light results in an electronic transition in the molecule. Through a process that is determined from the Beer-Lambert law, the electron is transformed from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO).
The fluorescence properties of proteins are highly individual; hence, it has
long been viewed as a sort of fingerprint of the individual protein. The aromatic
amino acids, phenylalanine, tyrosine, and tryptophan absorb light in the ultraviolet
spectral range. Among these, Y with molar absorptivity of 270= 1405 m-1 cm-1
and W 280 = 5579 m-1 cm-1 has been used extensively.
5 Aims of the study
Bruton tyrosine kinase plays a crucial role in B cell development. Both genetic and biochemical methods have been used to illustrate the physiological importance of Btk in B cell signaling. This has been clearly demonstrated with the finding that mutations in the genes encoding Btk lead to immunodeficiency in both man (XLA) and mice (xid).
The present study focuses on the role of the non-catalytic PH, TH, SH3 and SH2 domains in the cooperative regulation of the activity and interactions of the full-length protein.
The detailed aims of the study were:
1) to extend the knowledge relating to the structural dynamics of Btk.
6 Experimental approaches
6.1 Mutation scanning using base excision sequence scanning (BESS) method (I and III)
The upstream primer for each exon (1-19) was radioactively labelled as described [I and III]. Samples were incubated at 37°C for 30 minutes, and then inactivated by heating at 70°C for 5 minutes. Each of the nineteen BTK exons was amplified using the improved primers as described by Vorechovský et al. (1995). The PCR reactions were carried out as described in ref. I & III.
The amplified products were excised using BESS excision enzyme reaction (Epicentre Technologies, USA), and subjected to 8% polyacrylamide sequencing gel electrophoresis. Exons revealing differences after scanning with G- and T-tracker were reamplified without radiolabel. The PCR products were resolved in a 1% agarose gel stained with ethidium bromide. The band of interest was excised and purified using QIAquick PCR purification kit (Qiagen, Germany). DNA sequencing was carried out for both strands using an automated ABI PRISMTM 310 Genetic Analyser (PE Applied Biosystems), with the same primers as used in the PCR reactions. Detected mutations were confirmed by comparison of the resulting sequence with the reported coding sequence (Vetrie et al., 1993).
6.2 Molecular cloning and expression (II, IV and V)
cDNAs encoding the desired domains were amplified using polymerase chain reaction (PCR). Cleavage sites for selected endonucleases were included in the designed primers. The amplified PCR fragments were digested and cloned into a vector with a poly-histidine (His)-tag for the TH domain and glutathione-S-transferase (GST)-fusions for the SH3, PRR-SH3 and PH-SH2 domains, respectively. The PH-SH2 constructs were cloned into a pBAT vector (Peränen et al., 1996). The authenticity of all constructs was verified by DNA sequencing. Cloning was carried out in Escherichia coli DH5a strain.
The different domains were expressed in E. coli strain BL21 (DE3) or BL21/pUBS20 strain for the PH-SH2 construct. The domains were expressed by fermenting the cells in 2xTY medium containing 2% glucose as a catabolic repressor at 37°C, or 30°C for the TH domain. Expression was induced with isopropyl-D-thiogalactopyranoside (IPTG) when absorbance at 600 nm reached 1.8 nm. Induction was carried out for 4 hours with the exception of the PH-SH2 construct, which had 20 hours induction. The harvested cells were lysed using Bead beater cell breaker.
6.3 Protein purification using chromatography (II, IV and V)
The cDNAs encoding the recombinant proteins were cloned to GST/His-tag fusion vectors except for the PH-SH2 construct. The over-expressed proteins in the supernantant were first purified using an affinity column containing GSH-Sepharose or Ni2+-NTA-agarose respectively. The bound fusion products were digested overnight with thrombin (for GST fusion) at room temperature, and the proteins eluted with PBS (phosphate buffer saline) buffer. After the affinity purification, the proteins were further purified by gel-filteration on a Superdex 75 column. Then, SDS polyacrylamide gel electrophoresis and N-terminal amino acid sequencing were used to confirm the identity of the purified proteins. For further studies, the proteins were concentrated using membrane filters with a 10 kDa cut-off. The protein concentrations were determined as described in (II).
6.4 Molecular modeling/sequence analysis (I and III)
Molecular modeling was carried out essentially as described in reference I. Briefly, the computer assisted modeling of the Tec family PH domain was done by utilizing the 3D structure of the Btk PH domain (Hyvönen and Saraste, 1997) as a template. The models were generated using InsightII. Upon searching loops from the database that contained a representative selection of the PDB files, deletions and insertions were modeled. Sequence analysis was carried out using programs such as gap as contained in the GCG sequence analysis package (Devereux et al., 1984). ClustalW (Higgins et al., 1994) was used for multiple sequence alignment analysis.
6.5 Biophysical studies of molecular interactions
6.5.1 CD spectroscopy (II and V)
The CD measurements were made on a Jasco J-720 instrument. The measurements
were performed as described in ref II & V respectively. CD spectra are often
reported in terms of molar ellipticity. In the work presented in this thesis,
the ellipticity data were converted to molar ellipticity (q) (in units of deg
cm2 dmol-1) using the equation:
6.5.2 Determination of dissociation constant (Kd) (II and V)
A simple binding association scheme was assumed for the reaction. The binding of the SH3 domain to either the peptide or the TH domain was assumed to be reversible and to form a complex such that SH3 + P D SH3P. The fluorescence changes (DF) were directly related to the fraction of SH3P or protein complex formed.
The differences in fluorescence due to ligand binding were analysed using the plot of A/V against A with the equation:
A = Km + 1 . A
where A is the concentration, V is the fluorescence change due to ligand binding and KM is the Michaelis constant. This plot has been shown to be more accurate than the double reciprocal plot.
6.5.3 NMR Spectroscopy (IV)
NMR measurements were carried out using Varian Inova 500, Bruker DRX 500 and DRX 600 spectrometers equipped with 5 mm triple-resonance 1H/15N/13C probes respectively. The measurements were performed as described in (IV).
6.5.4 Temperature gradient gel electrophoresis (V)
The temperature gradient gel electrophoresis (TGGE) was performed on a Biometra TGGE system. 8% polyacylamide gels with 0-4 M concentration of urea were cast on a polybond film as recommended by the manufacturer (Biometra, Göttingen, Germany). The gel was placed on the horizontal gradient block and connected to the electrode tanks using pre-soaked electrode wicks. The temperature gradient was set from 28 to 65°C. The sample was loaded onto the polyacrylamide gel attached to the polybond film, and the sample subjected to a pre-run electrophoresis for 10 minutes at room temperature enabling the sample to migrate onto the gel. The system was disconnected, the empty slot of the gel rinsed with the running buffer, and the polyacrylamide gel covered with a pre-cut film. The electrode wicks were placed on top of the cover film and the sandwich covered with an acryl-glide treated cover glass plate. After completion of the electrophoresis, the gel was stained using silver staining method.
7.1 Mutations XLA causing in the non -catalytic domains of Btk (I and III)
Btk contains the PH, TH, SH3, SH2, and the kinase domains. Mutations in the
genes encoding the Btk domains lead to XLA in man. Several XLA-causing mutations
in genes encoding the above mentioned domains in Btk have been described (Vihinen
et al., 1999; Vihinen et al., 2001). Using BESS method combined with direct
sequencing, we studied the prevalence of mutations in patients with XLA disease.
We identified XLA causing mutations in seven families out of eight. In seven
unrelated families we observed distinct mutations of which six were novel. Carrier
detection was performed in four families. Four of the seven mutations affected
the kinase domain gene region, three were found in PH and one in SH2 domain
(Table 7.1). There was a high proportion of insertions in the investigated patients.
There were a frameshift causing insertions in six patients, four were in introns,
one in the upstream noncoding region and one in exon 18 leading to a frameshift
mutation and the consequent truncation of the protein. In addition to the disease-related
mutations a previously described silent polymorphism, C/T (Bradley et al., 1994;
Rodriguez et al., 2001), at the 2031 cDNA position was identified. The clinical
profiles of all the patients showed classical XLA. Some of the patients showed
a positive family history of the disease, but in one case the mutation was found
to be new.
Table 7.1 BTK mutations in XLA patients and carriers
Pleckstrin homology domains are known to be involved in different interactions including binding to inositol compounds, protein kinase C isoforms, and heterotrimeric G proteins. Most Tec family PTKs contains a PH domain. The sequence alignment of the Tec family PH domains and the modeling of the three dimensional structure of the domains enabled us to compare their similarity and gene mutation pattern. The majority of the disease-causing changes in Btk PH domain are located in the inositol-binding region. Many of the alterations within this region either reduce or reverse the electrostatics of the domain. In the four Tec family members that we compared, similar fold and electrostatic properties were observed but the binding regions were dissimilar.
Many PH domain alterations have been shown to alter the binding of the PH domain to IP4 because of the removal of a positive charge from the binding pocket. IP4 interacts with Btk via residues in b1-3 and in the loop between b1 and b2 of PH (Baraldi et al., 1999). The minimal PKC binding region (Yao et al., 1997) contains residues R28 and Y39 of this region. Thus, this region contains essential amino acids that impair Btk function when altered.
The IP4 molecule was docked to the domain models according to the binding site in Btk. Using the structural models we interpreted the effects of the mutations. It was found that the substitutions K12R and S14F most likely prevented binding to IP4 due to the introduction of a bulky side chain into the binding pocket. The alterations T33P, V113D, S115F, T117P all were found to caused a sterical clash with the ligand. L19 is in the immediate proximity to the ligand-binding pocket and Y39 is a ligand-binding residue. Thus, the alterations L19E, Y40C or Y40R disturb the electrostatic properties of the domain, and hence reduce binding.
7.3 TH/SH3 domain partners in the downstream regulation of Btk interaction (II and IV)
Enzymatic activities of protein kinases are regulated by various intra- and
intermolecular interactions. By applying biophysical techniques we investigated
the role(s) of SH3 domain binding sequences present in the TH region of Btk.
7.3.1 interactions of Btk TH and SH3 domains (II)
The PRR sequence/motif of the TH domain in Tec family proteins can interact with several SH3 domains (Yang et al., 1995). Tec PTKs have the SH3 domain adjacent to the TH domain. We analysed the binding of the SH3 domain of Btk to the proline rich segments by using synthetic peptides. Peptides corresponding to the two Btk PRR repeats, peptide 1 TKKPLPPTPE (Btk residues 184-193) and peptide 2 LKKPLPPEPA (residues 198-207), were synthesized. CD and fluorescence scanning spectroscopy showed, that the Btk SH3 domain interacts specifically with the N-terminal proline rich region. Similar interaction has been previously reported for the Itk (Andreotti et al., 1997). The CD spectra of both peptides were measured and both had a negative extrema at about 202 nm. Competition assays with the peptides was used to test the interaction in the PRR-SH3 construct. The CD spectra of the Btk SH3 domain (amino acids 212-275) and the N-terminally extended version (amino acids 178-275) between 190 and 250 nm were measured. The sums of the CD spectra of Btk SH3 domain, peptide 1, as compared to the mixture of the two, indicated that the combined spectrum does not obey simple additive rules. The TH domain in competition with the Btk SH3 domain was used to further characterize the interaction.
The binding affinity of the interaction between the TH and SH3 domains of Btk was determined by using fluorescence scanning spectroscopy. The affinities measured for peptide 1 binding to the SH3 domain, TH domain binding to the SH3 domain, the TH domain binding to the extended PRR-SH3, and peptide 1 binding to the PH-SH2, were found to be 3.9 mM, 17.0 mM, 36.2 mM, and 350 mM respectively. Peptide 2 did not show binding even when used in high concentrations. These results indicate that interaction between the TH and SH3 domain contribute to the function and regulation of the Tec family PTKs.
7.3.2 Intermolecular interactions involving the TH domain (IV)
The dimer equilibrum of the PRR and the SH3 domain was studied using gel permeation
chromatography, intermolecular NMR cross-relaxation measurements, and [15N]-NMR
relaxation measurement. We showed monomer-dimer equilibrium of the Btk PRR-SH3
with a Kd of 60 mM. The gel chromatography profiles showed the Btk PRR-SH3 domain
retention to be concentration dependent. The titration of a synthetic peptide
corresponding to peptide 1 onto a [15N] labelled sample and monitored using
an NMR spectrum supported a monomer-dimer equilibrum in the PRR-SH3 that is
CD spectroscopy and temperature gradient gel electrophoresis (TGGE) were used to analyse the stability and unfolding of the PH, TH, SH3 and SH2 domains. The CD spectrum of the protein showed intense negative minimas at 208 and 222 nm, with a positive peak close to 195 nm.
The temperature-induced denaturation using TGGE for the multidomain protein gave a single Tm of 45°C. The result indicates that a single unfolding initiation process leads to the coordinated denaturation of the four domains. The ellipticity at 222 nm as a function of temperature was followed to understand the corporate dependance of the different domains. The thermal transition curves indicate that at high temperatures, the molar ellipticity is not zero: (q)222 = -10,10 mdeg at 75°C. This implies the presence of some residual secondary structure even at high temperatures as previously reported (Venyaminov and Yang, 1996).
Based on the CD measurements we calculated a number of parameters. The thermodynamic parameters defined include the Tg; which is the upper temperature at which DG0 (T) is zero; DHg, which is the enthalpy of denaturation at Tg; and DCp, the change of heat capacity upon protein unfolding. We calculated the transition point of the enthalpy of denaturation (Tg) of the PH-SH2 protein and the vant Hoff enthalpy upon denaturation to be 322K and Hm 323 kcal/mol, respectively. Further, with the use of the vant Hoff analysis, the DCp was calculated to be 16.5 ± 1.9 kJmol-1K-1. These parameters were analysed with the assumption that the denaturation process follows a two state N D D transition. Previously, it has been shown that small proteins, like the SH3 domain usually unfold in a two state transition (Knapp et al, 1996; Viguera et al 1994; Fersht, 1999). Thus far, only the stability and peptide binding specificity of the Btk SH2 and SH3 domain has been reported (Tzeng et al., 2000; Knapp et al., 1998).
8.1 XLA causing mutations
Single stranded conformational polymorphism (SSCP) followed by direct sequencing has this far been widely used for molecular diagnosis of XLA and several other immunodeficiencies. In our study, base excision sequence scanning (BESS) method (Hawkins and Hoffman, 1997) coupled with direct sequencing was used for the mutation analysis in BTK gene. By using this approach, we studied eight unrelated families. The XLA causing mutations identified in our study were located in the PH, SH2 and the kinase domain (see Table 7.1). From the families we studied, different mutations were found: two insertions, two inframe insertions, an unknown mutation in the intron 14 leading to amino acid change in the tyrosine kinase domain, and a two base substitution in the intron 4. All the patients showed XLA phenotype. All the disease-causing mutations are novel. The silent polymorphism C/T at 2031 in one patient had previously been described (Bradley et al., 1994; Rodriguez et al., 2001). Previous work has described, XLA-causing mutations in all the Btk domains encoding gene and are collected in the BTKbase (Vihinen et al., 1999; Vihinen et al., 2001).
Most of the mutations available in the database are located in the PH, SH2 and the kinase domain and only few has thus far been seen in the TH and the SH3 domain. Indeed, it is not clear if a missense mutation in the PRR-region of the TH domain could cause the disease. A substitution has been found in this region, E205D, but this patient also had another mutation leading to truncation in the PH domain (Vórechovský et al., 1995). Another known mutation replaces T184 (in the beginning of the first PRR) with a proline. However, in this case there is again a frameshift mutation truncating a major part of the protein (Vórechovsky et al., 1997). Although, a large number of XLA-causing gene mutations have been identified, so far no causative missense mutations in the gene encoding the SH3 domain region have been found.
Inter and intramolecular interactions play an important role in the regulation of many PTKs. Studies have shown that the PRR of Btk interacts with the SH3 domains of Src family kinases i.e. Fyn, Lyn, and Hck (Cheng et al., 1994). In Itk the PRR was shown to interact in an intramolecular fashion with the SH3 domain (Andreotti et al., 1997). We were interested to study the nature of this interaction(s) in Btk. With different spectroscopic techniques we were able to bring into focus the interaction pattern(s) of the TH and the SH3 domains. Since both the tested proteins and peptides were optically active, it was possible to determine binding affinities. To accurately determine equilibrium constants for binding of ligands to SH3 domain fluorescence measurements were used (Otto-Bruc et al., 1993., Cussac et al., 1994, Vidal et al., 1999). With this method, the affinity for the interaction between the peptides, TH and the SH3 domains were determined. We also used NMR spectroscopy, which is particularly useful in the study of protein structure and dynamics in solutions. When a protein structure is known, NMR can be used to study interactions between the protein and ligands. Usually, this is achieved by observing the perturbation of the protein chemical shifts upon isotope labeling. In our case, this method was used to study the intermolecular interactions between the TH and the SH3 domains. Both [15N]-HSQC and NOESY-HSQC experiments were carried out, combined with gel chromatography profiles. The results showed a monomer-dimer equilibrum involving the PRR suggesting that interaction between the TH and SH3 domain contribute to the function and regulation of the Tec family kinases.
The affinity of the PRR and the SH3 domain in Tec has been studied (Pursglove et al., 2002). The N-terminal PRR (KTLPPAP), residues 155-161 in Tec, and the C-terminal PRR (KRRPPPPIPP) residues 165-174, were independently titrated into 15N-labeled Tec SH3 domain. The changes in the chemical shifts of resonances in the HSQC spectrum were measured as a function of ligand concentration. The interaction surface on the SH3 domain was defined and the binding affinity estimated. For the intramolecular interaction of Tec SH3 domain with the N-terminal PRR the Kd was found to be between 1.7-2.0 mM (Pursglove et al., 2002).
For the Tec SH3 domain, an intermolecular interaction involving the PRR has also been reported by applying surface plasmon resonance (Pursglove et al., 2002). The wild-type PRR-SH3 protein was found to bind to the SH3 domain with a Kd of 68 mM (Pursglove et al., 2002). This is consistent with our measurements for Btk (Kd 60 mM). In Tec, the disruption of the N-terminal PRR by mutagenesis had no significant effect on the affinity, whereas the alteration of the C-terminal PRR had a marked effect (Kd 2.2 mM) on the ability to bind the SH3 domain surfac indicating that this region is critical for the intermolecular interaction (Pursglove et al., 2002). Thus, in Tec the C-terminal PRR is responsible for the interaction while in Btk the N-terminal PRR is essential (Fig. 8.2).
Our results clearly indicate that the N-terminal PRR of Btk interacts with
the SH3 domain. The lack of spectral shift in the PRR - SH3-peptide complex
suggests that Tec family members might share a similar mode of internal regulation
by binding of the TH domain to the SH3 domain. Dimer formation of these domains
could prevent the binding of low affinity ligands. Recently, the C-terminal
PRR in Btk was shown to play a more robust role in dimer formation as compared
to the N-terminal PRR (Laederach et al., 2002) (Fig.8.2 C). Although, the N-terminal
PRR competed for dimer formation, it had a remarkably weaker affinity (Laederach
et al., 2002). Three conformational states for dimer formation have been proposed
to coexist at equilibrium (Hansson et al., 2001), i.e.
The SH3 domain in the PSD-95 (post-synaptic density protein 95 kDa) of the MAGUKS (membrane-associated guanylate kinases) family has been shown to have the ability to adopt different modes in its interaction and regulation (McGee et al., 2001). In PSD-95 the SH3 domain interaction with the GK fragment differs from the other SH3 domains so that the interaction occurs via 3D domain swapping involving the C-terminal portion of the SH3. 3D domain swapping involves the exchange of complementary structures by proteins to generate dimers or higher order oligomers (Newcomer, 2001). Previous studies of protein systems that undergo swapping have indicated that the hinge or linker region between two interacting domains plays an important role in determining the preference for intra/intermolecular interaction (Newcomer, 2001; 2002; Schlunnegger et al., 1997). It would be interesting to ascertain if this also happens in the Btk TH/SH3 interaction.
To further elucidate this interaction in Tec family members, the three-dimensional
structure of the full-length protein is essential. However, dimerization
behavior has been observed for the Btk PH domain (Baraldi et al., 1999).
These findings indicate that self-association plays a central role in
the regulation of the Btk/Tec family protein tyrosine kinases.
Figure 8.2 Schematic representations of the Btk PRR interactions. (A) The N-terminal PRR interacts with the SH3 domain-binding site in an intramolecular fashion. (B) Mutation of the C-terminal PRR does not show any significant effect on the interaction. (C) Proposed dimerization mode of the protein. (D) The interconvertion of the dimer in a symmetry mode.
8.3 The biological implications of Btk SH3 domain interactions
Our results show that the Btk SH3 domain plays a number of regulatory roles, these include inter and intramolecular regulation. Obviously the interaction of this domain with other domains within Btk is important for the overall function of Btk. Most of the PTKs exhibit similar patterns of regulation. Two different microarray chips were used to monitor gene expression patterns in a cell line with non-functional Btk. Btk itself was induced with Src kinases (Islam et al., 2002) implying similar regulation pattern for these proteins.
The interaction(s) of the Btk TH and SH3 domains, as shown by the results presented in this study, suggests a mechanism by which the accessibility of the TH and SH3 domains regulates the kinase activity of Btk. Recently, the deletion of the PRR sequence in Itk (Hao and August, 2002) was shown to result in a 50% reduction of basal activity of this protein, further implying that this region is essential for the regulation of Tec family kinases. The modulation of the SH3 domain by Y223 autophosphorylation has been predicted to block binding to the domain (Park et al., 1996; Rawlings, 1999; Yang et al., 2000). Exogenous partners that may disrupt the intra/intermolecular interactions between the TH and SH3 domains can potentially participate in the regulation of the Btk family members.
One of the roles suggested for the Btk TH and SH3 domain interactions is to
regulate and suppress the kinase activity (Ma and Huang, 1998; Yang et al.,
2000). The Src family kinases are known to interact with Btk TH domain via their
SH3 domain (Ma and Huang, 1998). Tec family kinases have been shown to recruit
other proteins, for example Src and Syk kinases, that uncouple initiated signaling
via their SH3 domain (Mano, 1999). Another regulator of Btk interactions is
Sab, an SH3-domain binding protein that preferentially associates with Btk by
binding the Btk SH3 domain via its PxxP motif (Yamadori et al., 1999). The interaction
between Sab and Btk SH3 domain results in the inhibition of Btk activity with
a dramatic reduction in both early and late BCR-mediated events including calcium
mobilization, IP3 production, and programmed cell death (Yamadori et al., 1999).
Such interactions with proteins that have the ability to regulate the phosphorylation
state of the full-length protein gives insight on the mechanism required to
sustain enzyme de-/activation via the interaction between the TH and the SH3
In c-Src kinase the regulation of the kinase domain by phosphorylation of the C-terminal tail was found to require both the SH3 and SH2 domains as elucidated by a coexpression experiment of Src mutants and Csk in yeast (Superti-Furga et al., 1993; Okada et al., 1994). The main intramolecular contacts that stabilize the inactive conformation of Src are those of the SH2-kinase linker with the SH3 domain, and the linker between SH2 and the N-terminal kinase lobe (Sicheri and Kuriyan, 1997). These interactions hold the enzyme in an inactive state due to limited conformational freedom (William, et al., 1998). An accessible ligand binding surface and RT (arginine and threonine) loop are essential for the function of SH3 domains (Superti-Furga et al., 1993; Okada et al., 1994; Erpel et al., 1995; Barila and Superti-Furga., 1998 ).
Recent data (Lowry et al., 2001) suggest that down-regulated Btk has a higher basal activity than down-regulated c-Src, possibly due to different structural conformations of the active sites and activation loops. The finding that the Btk catalytic domain cannot autophosphorylate at Y551 in the absence of the SH3/SH2 or the PH/TH modules, whilst the c-Src can autophosporylate at its equivalent site Y416 (Lowry et al., 2001), implies that the Btk catalytic domain relies on these domains to recognize the substrate or to modulate substrate accessibility. In this respect, further work is required to fully comprehend the role(s) of the TH/SH3 interaction(s) on the one hand, and the interaction between the SH3 and SH2 domains on the other hand.
8.4 The dynamics of the Btk N-terminal domains
We studied the unfolding and the stability of the Btk N-terminal domains. Thermal and chemical induced denaturation was followed at 222 nm using CD spectroscopy and TGGE. The reverse of the unfolding experiment was not done as it has previously been reported that the refolding process of multidomain proteins in vitro occur at a lower rate due to the slown correct pairing of the folded units (Jaenicke, 1987). When applying CD spectroscopy to protein denaturation and unfolding studies, it is assumed that the changes observed occur due to the sensitivity of the system to the changes in the environment. And also due to the changes of chromophoric amino acids resulting from temperature perturbation and the resultant local conformational changes in the full-length protein. These observations are supported by e.g. an earlier study on myosin (Holtzer and Holtzer, 1992), where the effect of temperature on the protein was attributed to the local changes in the helical content.
Local interactions at the secondary structure level and interactions between domains and subunits are thought to contribute to the net free energy of protein stabilization (Jaenicke, 2000). Understanding the individuality of the different Btk domains in concert with stability studies would profoundly increase the understanding of the biological functions of the full-length protein. In recent times, studies of protein folding, unfolding and in particular of misfolding have been implicated in a number of so called conformational diseases i.e. transmissible spongiform encephalopathies and Alzheimers disease (Ellis and Pinheiro, 2002) thus providing a wider relevance for such studies.
The three dimensional structure of Btk is vital to study the functional mechanisms and regulation in detail. The structure would help in illuminating the in vitro and in vivo observations made thus far. Moreover, the nature of the intra/intermolecular interactions between the TH/SH3 domains needs to be further addressed, as does the role of the Btk SH3 domain phosphorylation site. As revealed in this study among others, the interactions of these domains are essential for cell signalling, protein interactions etc. Using molecular and biophysical means we have successfully shown the biological significance of these domain interactions in Btk regulation. Specifically, we showed that the Btk SH3 domain can interact with the PRR in the N-terminal TH domain. To understand these interactions in the Tec family kinases further experiments are needed to elucidate the molecular determinants of the various conformational states of the Tec family kinases. Experiments, which explore the differences in the SH3 domains, the linker region, and the proline rich region would be vital in this respect.
The diversity of the molecules interacting with Btk and other family members suggest functions, which are significant and strictly controlled. Several signal mediators are now known as effectors of Tec family signalling. An understanding of the molecular interactions of the Tec family is essential for addressing issues related to the basis of signal transduction and diseases arising from mutations in the involved genes. In this regard, the combination of multiple methods will be vital in attempting to unravel the true complexity of these proteins. One of the fundamental questions this study answered is, how the N-terminal Btk (PH, TH, SH3 and SH2) domain interactions are coordinated and regulated to achieve both spatial and specific interaction.
This study was carried out at the Department of Biosciences, Division of Biochemistry, University of Helsinki. The academy of Finland, EU BIOTECH BIO4-CT98-0142, the Sigrid Juselius Foundation, and the Medical research fund of Tampere University hospital provided the financial support. Also the National graduate school in informational and structural biology is acknowledged for the financial assistance.
I am deeply indebted to my supervisor Professor Mauno Vihinen, who provided me with the opportunity to study in Helsinki. The driving engine behind this work from the inception has been your exemplary skills as a researcher and your eventful ideas. Your friendship during the bad and good times of this study and your confidence in my ability has been inspiring. You are to me a good friend indeed and for this I thank you without reservations. My sincere gratitude goes to Professor Carl G. Gahmberg, the head of the Division of Biochemistry for providing me with an unfettered access to the excellent facilities in the Division. I also wish to thank Professor Kari Keinänen for his supportive and friendly attitude toward my work and me.
I would like to thank Professors Veli-Pekka Lehto and Dennis Bamford for their quick but critical review of the manuscript. Their suggestions and comments were invaluable in improving this work. Professor Lehto is especially thanked to have introduced me to the world of protein signaling. Dr Sarah Coleman is also acknowledged for the language corrections and advice.
To our group members Ilkka, Juha, Kaj and Johanna, I thank you guys for I would not have been able to make it without your cooperations. To Ile and Juha I am particularly grateful for if not for you guys my work would have been more difficult knowing how naive I was in computer applications when I came from Oulu. To the other group members in Tampere especially, Jukka, Tomi, Hannu, Bairong, Jouni, Mansour and Martti I say thank you all for the wonderful times we always manage to have on the few occasions that we get together.
I cannot thank enough the people at the Division of Biochemistry, for their
friendly attitude and comradeship. Especially I owe thanks to Drs Mathias, Ragna,
Erkki, Pirjo and Matti. Also Drs Tian and Leena formally of our Division are
thanked for their friendship. Most shout out to Aki, Asko, Esa, Heli, Mikael
and Michaelis for your friendship. To L Kuoppasalmi, I say thank you for your
help. My sincere gratitude is also extended to the secretarial staff of the
My sincere gratitude also goes to Mensu Ize-Okhai for believing in my ability and for the elderly advice. I also wish to thank all my friends that have shared their time with me both before and during this study.
My story is about to end but not without mentioning the people who are the most important in my life; Mrs Q, Zakari, Mary and Dora; my most senior, immediate senior and junior sisters respectively. Thank you all for your understanding and support...I love you all. Mohan-Klem, Okoh my senior brother and my best friend thank you for everything so numerous to narrate here. Suffice though to say I am glad I have you as my best friend, my mentor and a brother....I love you. To my Mother thank you for your unwavering support throughout the period of this study. Thaddeus, can you see me? RIP boy. I wish my Father was alive he would have upon seeing this thesis say My Dear....See Meeeeee. Thank you Dad for everything, most of all thank you for your unconditional love and support to me throughout your lifetime even though I was not a very easy son. To you I dedicate this thesis.
This thesis is a testimonial to the biblical saying that
all things worketh good for those that beliveth. To God be the glory for
providing me with the inspiration, I thank him for making me to be just
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