Crystallization trials of refolded breast tumor kinase (BRK) and peroxisome proteins

CHAPTER 1 MACROMOLECULAR X-RAY CRYSTALLOGRAPHY 1.1 PROTEIN STRUCTURE DETERMINATION The cause of most human diseases, like cancer, malaria, Parkinson and Alzheimer's, are proteins.. 2.1

CHAPTER 1 MACROMOLECULAR X-RAY CRYSTALLOGRAPHY

1.1 PROTEIN STRUCTURE DETERMINATION

The cause of most human diseases, like cancer, malaria, Parkinson and Alzheimer's, are proteins As one of the basic components of a cell, proteins are responsible for regulatory mechanisms, defense systems and structural supports Consequently, in the factory of living cells, proteins are the workers, performing a variety of biological tasks Each protein has a particular three dimensional structure that dictates its function Thus, complete understanding of a protein structure will unveil its particular function and hand in hand this relationship between structure and function will give us a valid picture to design new drugs that can be potential therapeutics for treatment of diseases

Even with high-throughput approach, known as structural proteomics, elucidation of the three-dimensional structure of all natural proteins is likely to be a physically impossible task Recently, X-ray crystallography and Nuclear Magnetic Resonance (NMR) are widely used as the two primary and powerful techniques for protein structure determination at atomic details X-ray crystallography appears to overcome protein size restriction, a disadvantage of NMR Although at least 40,000 protein structures have been solved, this number is currently a small fraction of the thousands of proteins that remain to be understood in detail

1.2 PROTEIN CRYSTALLOGRAPHY

Crystallography originated as the science of the study of macroscopic crystal forms It was originally developed to study minerals and subsequently extended to crystals With the advent of the X-ray diffraction, the science has become primarily concerned with the study of atomic arrangements in crystalline materials, and the definition of a crystal was defined by Buerger (1956) as: “a region of matter within which atoms are arranged in a three-dimensional translationally periodic pattern” Furthermore, crystallization is one of the several means (including nonspecific aggregation/precipitation) by which a metastable supersaturated solution can reach a stable low energy state by reduction of solute concentration (Weber, 1991) The three stages of crystallization include: nucleation, growth, and cessation of growth

1.2.1 X-ray crystallography for proteins

Before the development of X-ray diffraction, the study of crystals was primarily based on external geometry In 1912, crystal structure determination was formulated by an experiment of Max von Laue, who produced an interference pattern

on a photographic plate when X-rays were passed through a crystalline sample The obtained diffraction pattern is due to the scattering of X-rays by the electrons in the sample With the recent development of computer and diffraction equipment, crystal structure determination has become a relatively easy process Interestingly, X-ray crystallography has become a primary method for structure determination of biological macromolecules, such as proteins

1.3 BASIC CONCEPTS IN CRYSTALLOGRAPHY

1.3.1 Unit cell and lattices

A crystal (Fig 1.1) consists of a large number of molecules, which are arranged in a particular manner

Figure 1.1 A single protein crystal (adapted from Mathias Klod)

A regular pattern of arrangement of an array of points periodically in three

dimensional spaces is known as a lattice In a crystal, a unique volume of space, which is repeated in three dimensions, is called a unit-cell If each box is represented

by a point, then the arrangement of all unit-cells will form a lattice Even though every crystal has a reduced unit–cell (minimum volume), in some crystals we select a bigger unit-cell (that would include two or more smaller unit-cells), which would satisfy the full symmetrical needs of the crystal The least volume unit-cell in all crystal systems is called the primitive unit-cell and the bigger unit-cell in some selected crystal systems is called a centered unit-cell The geometry of a unit-cell is defined by three non-coplanar axes (a, b, c) and their inter-axial angles (α, β, γ) A crystal system is named after the symmetrical requirements of that system and it adopts the corresponding unit-cell The seven systems are triclinic, monoclinic, orthorhombic, tetragonal, trigonal, hexagonal, and cubic While all other systems use

the corresponding unit-cell, the trigonal system uses either a hexagonal unit-cell or a rhombohedral unit-cell, depending on the symmetry of that particular crystal These seven crystal systems, depending on the use of the corresponding unit-cell, produce

14 Bravais lattices

Figure 1.2 Unit-cells and 14 Bravais lattices

1.3.2 Symmetry, point group and space group

Symmetry in a crystal can be defined as the arrangement of atoms occupying minimum volume, identical and repeats itself throughout the crystal There are three types of basic symmetry operations in crystallography: rotation, reflection and inversion

The rotational symmetry needs an axis to act upon and produces identical images of an object, around the axis The number of images generated in crystallography by the rotational symmetry can be 1, 2, 3, 4 or 6 The reflection

symmetry acts upon a plane and inversion occurs through a point The 32 point

groups in crystallography describe the unique combinations of these symmetry

elements (without any translational component applied to them) in a unit-cell When a translational component is applied to the symmetry operations rotation and reflection, two additional types of symmetry, the screw axis and the glide plane, are generated The complete description of a crystal, including the crystal system, lattice type and

symmetry elements, is known as the space group of that crystal There are 230 space

4 groups in crystallography and proteins crystallize only in 65 space groups (without inversion and reflection) that do not warrant the need for D amino acids

1.3.3 Crystals and X-rays

Visible light has the advantage of being focused by a lens and thus it can produce an enlarged image of an object X-rays, on the other hand cannot be focused However, the electrons in a crystal diffract X-rays and virtually we want to look at the electron distribution The objective in X-ray crystallography is to grow crystals to an optimum size and quality for diffraction study Crystals are generally grown to 0.1-

0.3mm by using different techniques Crystals for small molecules are easier to form than for proteins This is due to the complexity of protein molecules and their low availability Protein crystals are grown by several techniques, including the most common vapor diffusion (hanging drop and sitting drop method) and batch methods

1.3.4 X-ray diffraction

X-ray diffraction is best explained if the radiation is taken as a wave, Eq 1.1

where A is the amplitude, ω is circular velocity, t is time and E is the energy

of the wave X-rays interact with matter and get scattered in all directions These

scattered rays travel different lengths as they originate from different places in a crystal They differ from one another with respect to their phase and amplitude Two waves interact constructively when they are in phase (their amplitudes are magnified

as the sum of the two waves) while the resultant wave decreases in amplitude if the waves are out of phase (Fig 1.3)

Figure 1.3 The two types of interference

1.3.5 Bragg’s law

In crystals, atoms diffract X-rays Each reflection is the combined effect of waves diffracted by all atoms in the crystal, governed by a set of parallel and equally spaced planes that slice all unit-cells in that particular orientation According to Bragg’s law when X-rays with a wavelength λ are incident on a set of planes with

Miller indices hkl (where h, k, l are the integral divisions of the unit-cell axes a, b, c,

respectively) and interplanar spacing of dhkl at an angle θ, they will produce a diffracted beam only if θ meets the following condition, Eq.1.2

where n is an integer (Rhodes, 2000)

1.3.6 Reciprocal lattice and Ewald sphere

A set of parallel planes with Miller indices hkl in real space is related to a

point (hkl) in the reciprocal space The direction of the reciprocal vector corresponds

to the plane normal and the magnitude of the reciprocal vector is equal to the reciprocal of the interplanar spacing of the real space planes

1.3.6.1 Ewald sphere

Bragg’s law can be rearranged in the reciprocal space using Eq 1.4

sinθ = λ/2dhkl = (1/dhkl) / (2/λ) (Eq 1.4) The aim of the Ewald sphere is to determine which set of real space planes (represented by the grid points on the reciprocal space) will result in a diffracted signal for a given wavelength, λ, of incident radiation (Fig 1.4)

Figure 1.4 Ewald Sphere and radius equation

1.3.7 Fourirer transform, structure factor and phase problem

The diffraction pattern of atoms in a crystal is related to the atomic arrangement through their Fourier transforms Thus the electron density at any point

in the unit-cell can be calculated by Eq 1.5

ρ (x, y, z) =1/V ΣΣΣ F hkl e-2πi (hx+ky+lz) (Eq 1.5)

Through this equation, we transform the diffraction effect in inverse space to real space electron density at every point x, y, z In the above equation, if the structure factor Fhkl is known, we can calculate the atomic positions and thus the real structure However, in crystallography to calculate the structure factor Fhkl, we need to know the atomic positions The reverse Fourier transform of the structure factor equation, will give back the atomic position which is our ultimate aim, i.e to ascertain the location

of every atom in the structure from their diffraction pattern In order to calculate the position of an atom in a structure we need to know two parameters about a diffracted wave: amplitude and the phase While the amplitude is calculated from the intensity

of a reflection, the phase of the wave, which depends on the positions of all atoms with respect to the origin of the unit-cell, is not measurable This non-availability of phases is called the ‘phase problem’ in X- raycrystallography

1.4 DATA COLLECTION

For crystal structure determination, the intensities of most, if not all, diffracted beams must be measured All corresponding reciprocal points must be brought to diffracting position by rotating a crystal First, the geometry of diffraction which includes the shape, size and symmetry information, is confirmed This is followed by the measurement of intensities which is ultimately related to the distribution of diffracting electrons in a unit-cell The X-ray diffractometer (Fig 1.5) consists two parts, the mechanical part to rotate the crystal and the detector to measure the intensities of diffracted beams There are three independent axes (ω, χ and φ) through which a crystal can be rotated to bring a desired set of planes into a diffracting orientation Different physical devices, like photographic film, image plate and charged coupled device (CCD) are used to record X-ray reflections

Figure 1.5 X-ray diffractometer

1.5 STRUCTURE DETERMINATION

1.5.1 Phasing techniques

Four techniques are commonly used to derive phase information for structure determination These methods provide a rough estimate of phases initially which is further improved using repetitive model building and refinement

1.5.1.1 Direct method

This method can be used to solve structures containing 100 or less amino acids It is based on the assumption that the structure is made of similarly shaped atoms and that there is a statistical relationship between sets of structural factors The other requirement in case of direct method is the requirement for a very high resolution of data, at the order of 1.2Å or better

1.5.1.2 Molecular replacement

This method is generally preferred to solve the phase problem when a good model for a reasonably large fraction of the structure exists This means that the sequence similarity between the subject protein and the model must be at least 40% with the model being fairly complete in size This method is very useful when the structures of structurally homologous proteins are to be solved

1.5.1.3 Multiwavelengh isomorphous replacement

Developed in the early 1940s, this method makes use of heavy atoms like gold, mercury or platinum An initial diffraction pattern of a native crystal is collected, followed by soaking the crystal in two or more heavy atom solutions separately and collection of additional data sets These heavy atoms contain more electrons than normal protein atoms and hence they produce a significantly varying intensity for every corresponding reflection Therefore besides serving as spot markers, the change in spot intensities of these atoms help calculate initial phases which are further refined over successive refinement cycle The reason for use of more than one heavy atom is in the fact that different metals bind to different regions

in the protein, thereby aiding very much to resolve phase ambiguity

1.5.1.4 Anomalous dispersion

This method degenerates into single wavelength anomalous dispersion (SAD)

or multi wavelength anomalous dispersion (MAD), with the latter being the common method used to study protein structures When X-rays are incident on molecules heavier than carbon, nitrogen or oxygen part of the energy is absorbed and re-emitted

at the same wavelength but at a different phase This scattering is called ‘anomalous’ scattering Certain atoms produce substantial anomalous scattering when compared toothers in the useful wavelength range The most common atoms utilized in X-ray crystallography are sulphur for SAD or selenium which replaces the sulphur in the methionine of a protein in MAD The advantage of this method lies in the requirement for only one single good quality and well diffracting seleno-methionine crystal

1.5.2 Model building and refinement

After scaling and indexing a data set using a program like HKL2000 andsolving the phase problem by one of the above methods, an initial rough model of the structure is built There are several model building programs like O or Coot Once the initial model is built, the structure is further refined such that the atomic data is bestfitted Large numbers of systematic and random errors have an effect on the accuracy of the initial model Refinement is the process of adjusting the model to find

a closer agreement between the calculated and observed structure factors by squares methods or molecular dynamics This refinement is carried out several times until an accurate model of the structure is obtained

1.5.3 Validation and presentation

The structure is refined several times until a sufficiently low and acceptable R factor without affecting other parameters is achieved The final structure requires

validation before it can be presented There are two important parameters that must be verified

1.5.3.1 Ramachandran Plot

This powerful validation parameter is not used during the refinement process, but is used to check for the stereochemistry of a structure For good validation, residues in the disallowed region should be further refined to get at least ninety percentage of all the residues in the allowed region

1.5.3.2 Folding profile methods

A potential protein fold is assigned to the subject protein crystal by searching databases for proteins with similar fold Often proteins with similar sequence identity tend to show a similar fold This method was established by Eisenberg and co-workers The refined coordinates (positions of the atoms) are orthogonalized (arranged with respect to three orthogonal axes), even if the unit-cell has non-orthogonal axes Also, the temperature factor is a good indicator about the thermal vibration of an atom The solved structure is deposited at the Protein Data Bank (PDB)

CHAPTER 2 BIOLOGY BACKGROUND

For my Master thesis, I carried out two projects In the first part, I have obtained the refolding of Brk, an important kinase involved in lymphoma and then initiated crystallization of the refolded protein In the second part, in collaboration with Prof Suresh Subramani’s Lab (University of California, San Diego, USA) I have successfully overexpressed GST-Pex8 and GST-Pex20 (two important peroxins in

Pichia pastoris) and initiated their crystallization

2.1 BREAST TUMOR KINASE (BRK)

2.1.1 Protein tyrosine kinase in signal transduction

Protein tyrosine kinases (PTKs) are a wide variety of multigene family evolved to perform functions that regulate a range of cellular processes, including cell growth, differentiation, apotosis, motility, adhesion, and cell-to-cell communication (Pawson, 1994) Although phosphorylation of tyrosine residues in target proteins is essential for maintaining cellular homeostasis, this post-translational modification still provides a number of cellular oncogenes, deregulates various signaling pathways and induces transformation PTKs are therefore important targets for both basic research and drug development efforts (Levizki et al., 1995)

The PTK superfamily can be divided into two groups according to the presence of transmembrane and extracellular domains, which enable PTKs possessing them to recognize extracellular ligands, in particular, various peptide growth factors Specific ligands and intracellular signaling pathways induced by them have been

identified for many, albeit not for all, membrane-spanning PTKs (Schlessinger, 2000) PTKs lacking the transmembrane and extracellular sequences are referred to as non-receptor or non-transmembrane PTKs Thirty-two genes encoding for non-receptor PTKs clustered into 10 families are present in the human genome (Robinson, et al.,

2000, Manning et al., 2002)

Activation of the PTK domain of either class of PTK enzymes results in the interaction of the protein with other signal transducing molecules and propagation of the signal along a specific signal transduction pathway (van der Geer et al., 1994) Dysfunction of cellular phosphorylation is associated with several of human diseases, including cancer to diabetes Each PTK possesses a functional kinase domain that is capable of catalyzing the transfer of the γ-phosphate group of ATP to the hydroxyl groups of specific tyrosine residues in a protein Although phosphotransfer reactions that are catalyzed by various PTKs are similar with regard to their basic mechanisms, the recognition of substrates by PTKs and, therefore, subsets of proteins phosphorylated by them show a considerable degree of specificity

Abnormal kinase activity has been shown in a variety of human diseases, in particular those involving inflammatory or proliferative responses, such as cancer Nowadays, more than 400 human diseases have been connected to protein kinases The ability to modulate kinase activity therefore represents an attractive therapeutic strategy for the treatment of human illnesses However, despite a wealth of potential targets, only a few kinases are targeted by drug on the market Analysis of PTK expression in malignant cells, in general or in lymphomas, in particular will lead to a better understanding of oncogenesis, which in turn will lead to novel therapies based

on selective inhibition of these PTKs which are identified as involved in malignant transformation

2.1.2 Brk family non-receptor tyrosine kinases

Breast tumor kinase (Brk) belongs to a novel family of intracellular soluble tyrosine kinases and is distinct from the Src family kinases (Serfas and Tyner, 2003) This family is derived from the Src tyrosine kinase family tree early in evolution Although these kinases are thought to be critical for the normal regulation of many biological mechanisms including cell growth, proliferation, differentiation and metabolism in cancer, their physiological functions remain largely unknown

The Brk family of non-receptor PTKs has four members: Brk, Frk, Srms, and Src42A (Serfas and Tyner, 2003) They are defined by a highly conserved exon-intron structure that is quite different from other major intracellular tyrosine kinase families including c-Src (Lee et al., 1998) They have been cloned independently from human, mouse, and rat cells by several laboratories Brk is also known as PTK6 and Sik (Mitchell, et al., 1994;Vasioukhin, et al., 1995), whereas Frk is also known as Rak, Bsk, Iyk, and Gtk (Cance et al., 1994; Oberg-Welsh and Welsh, 1995; Sunitha and Avigan, 1996; Thuveson et al., 1995) Srms have been cloned and studied only in mice, but its ortholog is present in the human genome, as well (Kohmura et al., 1994)

Src42A, also known as Dsrc41, has been cloned and studied only in Drosophila, and

it shares 61% amino acid identity with its putative mammalian ortholog Frk (Serfas and Tyner, 2003; Shishido et al., 1991) Brk and Frk are expressed specifically in epithelial cells, intestinal tract, and their expression is upregulated in some epithelial tumors In contrast, Srms expression is ubiquitous, although found most abundantly in lung, liver, spleen, kidney and testis (Kohmura, et al., 1994) Src42A is expressed in a wide range of tissues during embryonic development

The Brk-family PTKs are highly homologous to the Src-family PTKs, even more so than are the Csk-family PTKs (Robinson, et al., 2000) Their domain

structure including three highly conserved domains followed by an SH3 domain, an SH2 domain, and a tyrosine kinase domain is very similar to that of the Src-family PTKs The SH2 and SH3 domains are reported to have interactions with phosphorylated tyrosine residues and proline rich sequences of target proteins, respectively (Songyang, et al., 1993, Cohen, 1995) Like the Src family PTKs, these domains are involved in both intermolecular associations that regulate signaling cascades, and intramolecular associations that autoregulate protein kinase activity (Sicheri and Kuriyan, 1997;Thomas and Brugge, 1997;Xu, et al., 1997) However, most Brk-family PTKs lack the N-myristoylation site, the main difference from the Src kinases and are thus not specifically targeted to membrane The only exception from this rule is rodent Frk, which retains the glycine residue in position 2 and is consequently myristoylated and localized to membrane (Sunitha and Avigan, 1996) Subcellular localization of Brk and Frk has been reported (Cance, et al., 1994; Derry, 2000; Haegebarth, et al., 2004) Frk, Brk and Src42A, unlike Srms, possess tyrosine residues near their C-termini, which might negatively regulate these PTKs in a Src-like fashion Frk and Src42A have been shown to be phosphorylated by Csk and dCsk respectively (Cance, et al., 1994;Read, et al., 2004)

2.1.3 Brk

The intracellular tyrosine kinase Brk was cloned from a human metastatic breast tumor (Mitchell, et al., 1994; Qiu H and Miller WT., 2004) Brk (also known as PTK6) shares 80% amino acid sequence identity to Sik, a non-receptor tyrosine kinase in the mouse intestinal epithelial cell (Vasioukhin, et al., 1995) Both Brk and Sik are members of the Frk family of tyrosine kinase and distantly related to Src-kinases (Serfas MS and Tyner AL., 2003; Kasprzycka et al., 2006) While Brk

expression is restricted to normal mammary epithelial cells of skin, melanocytes, gastrointestinal tract and prostate, its highest level is expressed in breast carcinomas, melanomas, colon carcinomas, T-cell lymphoma and in various types of squamous cell carcinomas (Vasioukhin et al., 1995; Derry et al., 2003; Petro et al., 2004; Kasprzycka et al., 2006) Interestingly, Brk expression is developmentally regulated

It is detected late in gestation in mouse, at mouse embryonic day 15.5 (E 15.5) in the differentiating granular layer of the skin and at E 18.5 in the differentiating intestine (Vasioukhin, et al., 1995) Brk expression is initiated as cells migrate away from the proliferative zone and begin the process of terminal differentiation Overexpression of Brk in mouse keratinocytes resulted in increased expression of the differentiation marker filaggrin during calcium-induced differentiation (Vasioukhin and Tyner, 1997) Brk is expressed in many breast carcinoma cell lines and primary breast tumors, but has not been detected in normal human breast tissue (Barker, et al., 1997;Mitchell, et al., 1994), or at any stage of mammary gland differentiation in the mouse (Llor, et al., 1999) Modest increases in Brk levels have been detected in colon tumors and Brk expression increases during the differentiation of Caco-2 colon adenocarcinoma cells (Llor et al., 1999) In prostate cancers, although the expression

of Brk is not significantly elevated, its localization is altered from the nucleus of normal cells to cytoplasmic with gradual progression of tumors (Derry et al., 2003)

While Brk is distinctly related to Src (structurally with the SH3-SH2-YP motif, and the regulatory C-terminus) its amino acid sequence does not contain an

NH2-terminal myristoylation signal that localizes Src to cell membrane, and therefore

is not specifically targeted to membrane (Fig 1) (Vasioukhin, et al., 1995) In fact, its intracellular localization is flexible and can be present in the nucleus as well as the cytoplasm or at membrane (Haegebarth, et al., 2004) The Src homology 3 (SH3)

domain plays an important role in intramolecular interactions that regulate kinase activity, interactions with substrates, cellular localization, and association with other protein targets (Pawson, 1995; Qiu and Miller, 2004) The SH3 domain binds the proline-rich sequences, with the consensus PXXP motif, in substrate proteins or interact with polyproline linker region between the SH2 and kinase domain (Sichery and Kuriyan, 1997) The SH2 domain, on the other hand, is essential in controlling interactions It recognizes and binds to phosphorylated tyrosine residues, with the specificity being determined by the 3-5 amino acids following these tyrosine residues (Songyang et al., 1993)

Like the Src family members, the SH3 and SH2 domains of Brk are also involved in intramolecular interactions with the kinase domain to form an autoinhibited conformation (Qiu and Miller, 2002) Brk activity is down-regulated by phosphorylation of its C-terminal tyrosine residue, Tyr-447 in mouse, similar to that

of the Src-family PTKs (Fig 2.1) However, it remains to be determined how this tyrosine becomes phosphorylated in Brk, since it is phosphorylated neither by Brk itself nor by Csk (Qiu and Miller, 2002) Csk is playing this role for the Src-family PTKs (Liu, et al., 1993) Phosphorylation of Tyr-447 in Brk causes an intramolecular interaction of this tyrosine with the SH2 domain, which leads to the binding of the SH3 domain to the linker region connecting the SH2 domain and the tyrosine kinase domain However, the question is how tightly this phosphorylation site is associated with the SH2 domain (Qiu and Miller, 2004) Recently, a study has reported that this SH2 domain of Brk is not more functionally similar than most SH2 domains and does not show a high affinity for the proposed autoinhibitory tyrosine of Brk (Y447) (Hong

et al., 2004; Julie et al., 2007) In fact, the intramolecular interactions in Brk prevent the binding of ATP to critical catalytic residues, rendering its inactive conformation

Mutation of the carboxy-terminal tyrosine of Brk to phenylalanine (Y447F), which is analogous to Y527 in Src results in increased enzyme activity when overexpressed in epithelial cells, supporting a role for this residue in autoinhibition (Derry et al., 2000; Qiu and Miller, 2002) However, mutation of this regulatory tyrosine resulted in a decrease in the ability of Brk to induce anchorage–independent growth of fibroblasts (Kamalati et al., 1996)

Thus, physiological regulation of Brk plays a significant role in kinase activity, localization and cell growth The SH3-SH2-YK motif presents still remains several unanswered questions relative to the expression, conformation and intramolecular interactions of Brk

Figure 2.1 Structure of Src and Brk tyrosine kinases Brk shares

44% amino acid sequence identity to Src Like Src, Brk contains three

domains including SH3, SH2 and catalytic domain The lysine at 295

in Src and at 219 in Brk correlates with the ATP binding site In

contrast to Src, mutation of Tyr-447 to Phe in Brk results in decreasing

the ability of Brk to induce anchorage-independent growth

Localization of the Brk tyrosine kinase, similar to Src, has been correlated with its activities and plays an essential role in oncogenesis Brk is localized in the nucleus of normal luminal prostate epithelia as well as of well-differentiated prostate carcinomas, but mainly in the cytoplasmic in poorly differentiated tumors and more aggressive tumor cell lines (Derry, et al., 2003) Therefore, relocalization of the Brk kinase during the progression of prostate tumor supports its function in maintaining the normal phenotype of prostate epithelial cells which might involve in an unknown

signaling pathway In addition, a correlation between tumorigenicity and the subcellular localization of Brk has also been found in oral squamous cell carcinomas (OSCC) (Petro, et al., 2004) Brk is detected in both the cytosol and nucleus of normal oral epithelium (NOE) and mainly presents in moderately differentiated OSCC cells Nonetheless, in poorly differentiated OSCC cells, Brk was localized in perinuclear regions, supporting the notion that subcellular localization of this tyrosine kinase plays a role in determining its growth regulating functions and tumorigenesis (Petro et al., 2004)

A recent study has also demonstrated the influence of different subcellular localization of Brk in mammalian cells on oncogenic properties HEK 293 cells, which express Myr-HA-PTK6 and the Brk protein is targeted to the plasma membrane

by the Src myristoylation signal (Myr), show increased cell proliferation, apoptosis, migration and anchorage-independent growth (Kim and Lee, 2009) However, targeting a variant of Brk (NLS-HA-PTK6) to the nucleus of HEK 293 cells suppresses such oncogenic function These results suggest that the identification of Brk at plasma membrane could be used as a medical prognostic indicator in the progression of various tumors Nuclear Brk on the other hand may provide a molecular profiling marker for long-term survival in patients with PTK6-positive cancers

anti-2.1.4 Role of Brk in lymphoma

Although Brk is expressed in several types of normal and cancer cells, its actual role in cell physiology and malignant transformation still remain poorly understood (Kasprzycka et al., 2006) Recently, Brk expression has been documented

in normal T-cell-rich PBMCs on their activation with mitogenic or T-cell

receptor-targeting stimuli as well as transformed B lymphocytes (Kasprzycka et al., 2006) Three different types of T-cell lymphoma, CTCL, ALK+, and lymphoblastic has been detected with strong expression of Brk (Kasprzycka et al., 2006) This finding suggests that Brk plays a key role in the physiology of such cells, especially in response to antigens However, identification of the exact role of Brk in T and, possibly, B lymphocyte as well as conclusion for the comprehensive expression pattern of the kinase in normal and malignant lymphocytes or other immune cells has

to be further studied

Interestingly, subcellular localization of Brk as well as its retranslocation in some malignant epithelial cells in vivo contributes to important functions involving in either cell survival or apoptosis and in cell growth, migration and invasion (Kamalati

et al, 1996; Derry et al., 2003; Petro et al., 2004; Haegebarth et al, 2005) This is demonstrated in the regulation of signaling by epidermal growth factor (EGF) and serum, via EGFR, erbB3 and possibly other cell-surface receptors to changes in phosphoinsitide 3-kinase, Akt, paxilin and Rac 1 activities and interactions In the T-cell lymphoma and other lymphoid cells, nuclear localization of Brk is mainly observed and supports to be different from that of cells in which Brk is located in the cytoplasm It suggests that the nuclear localization of Brk in such cells may be related

to the interactions of Brk with a different set of intracellular partners as well as involved in binding with nuclear proteins from the Star family, possibly impacting on cell proliferation (Kasprzycka et al., 2006)

In addition, Brk expression also plays a key role in the oncogenicity and pathogenesis of lymphomas Similar to empty vector-transfected BaF3cells, the kinase-silent K219M mutation fails to effectively compensate for the loss of cytokine (IL-3) or serum-mediated stimuli Furthermore, this kinase-negative mutant was

unable to preserve proliferative and survival characteristics of the BaF3 cells on cytokine and serum withdrawal (Kasprzycka et al., 2006) This result suggested that Brk may have different roles between malignant transformation of lymphoid and mammary cells

Thus, the exact mechanism of Brk and its expression in lymphoma remain to

be elucidated The structural study will establish Brk as a potential therapeutic target and help the patients with long-term breast carcinomas for survival (Kasprzycka et al., 2006)

2.2 THE IMPORTANT ROLES OF PEX8 AND PEX20 IN PICHIA

PASTORIS

2.2.1 Peroxisome biogenesis and degradation

2.2.1.1 Peroxisomal constituents and its functions

Peroxisomes are multifunctional organelles present in nearly all eukaryotic cells Their diameter ranges from 0.1 to 1.0 µM and they are denser (1.21 – 1.25 gm/cm3) than mitochondria (1.18 gm/cm3) (Subramani, 1993) They are delimited by

a single membrane which is impermeable to protons and small metabolites, creating

an enzymatically and chemically unique microenvironment within the cell (Dansen , 2000) Peroxisomes contain at least one hydrogen-peroxide (H2O2)-producing oxidase and catalase to decompose the hydrogen peroxide (Lazarow and Fujiki, 1985) Peroxisome functions are often specialized by organism, cell type and environmental milieu The most widely distributed and well-conserved functions are peroxisomes is the H2O2-based respiration Other functions include ether lipid (plasmalogen) synthesis and cholesterol synthesis in animals, the glyoxylate cycle in germinating seeds (“glyoxysomes”), photorespiration in leaves, glycolysis in trypanosomes (“glycosomes”), and methanol and/or amine oxidation and assimilation in some yeasts (Fukui and Tanaka, 1979; Tolbert, 1981; Veenhuis et al 1983; Opperdoes 1987; van den Bosch et al., 1992)

In human, there is a wide range of metabolic pathways that is involved with peroxisomes (Fig 2.2), like ß-oxidation of fatty acids, elimination of hydrogen peroxide, synthesis of plasmalogens and cholesterol The importance of peroxisomes

is underscored by several diseases of peroxisomal dysfunction This is most clearly demonstrated in the human genetic disorder Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum's disease which are characterized by

mental retardation, severe neurologic, hepatic and renal abnormalities, and premature mortality (Lazarow and Moser, 1989)

Figure 2.2 Electron micrograph of rat liver Ultrastructure of rat

liver peroxisomes (P), mitochondria (M) and smooth ER (SER) The

peroxisomal core composed of crystalline urate oxidase is indicated by

arrowheads (Stanley and Paul, 2007)

2.2.1.2 Basic views of Lipid and Protein Import into Peroxisomes

Similar to other organelles such as mitochondria and chloroplasts, peroxisome biogenesis also requires at least three conceptually distinct processes: the formation of the lipid bilayer, the insertion of membrane proteins into this bilayer, and the import

of soluble proteins across the membrane into the matrix The peroxisomal membrane contains a phospholipid composition that is distinct from that of other organelles and peroxisome has to import these phospholipids (Lazarow and Fujiki, 1985) Due to the lack of nucleic acids, peroxisomes must import all of their protein content (Fig 2.3) Proteins destined for the peroxisomal membrane are synthesized on free ribosomes in the cytoplasm (Fujiki, 1984; Imanaka, 1996) and posttranslationally imported into the peroxisome in an ATP-independent manner (Diestelkotter, 1993; Imanaka, 1996) Peroxisomal matrix proteins are also synthesized on free ribosomes and posttranslationally imported, although transport into the matrix is an ATP-requiring

C B

A

Figure 2.3 Peroxisome morphology on different growth media

(Pichia pasteris) A Glucose media; B Methanol media; C Oleate

media As growing from glucose media to methanol or oleate,

peroxisomes in cells are consequently induced to adapt with surviving

environmental change: peroxisomes proliferate and import the

necessary metabolic enzymes

Peroxisomal matrix proteins are also synthesized on free ribosomes and posttranslationally imported, although transport into the matrix is an ATP-requiring process (Lazarow and Fujiki, 1985; Subramani, 1993) One curious feature of peroxisomal matrix protein import is that it appears to accommodate folded proteins (Glover JR et al., 1994;McNew JA, Goodman JM., 1994) and even internally cross-linked proteins and gold particles (Walton et al., 1995), indicating that the translocation apparatus can induce the formation of a large pore in the peroxisomal membrane

2.2.1.3 Components of the peroxisomal matrix and membrane protein import

machinery

Like the sorting of proteins to other subcellular compartments, protein targeting to peroxisomes is signal dependent The peroxisome targeting signal 1

(PTS1) and PTS2 signals direct proteins to the peroxisomal matrix, whereas membrane PTS (mPTSs) specify a peroxisomal membrane location (Lazarow, 2003) (Fig 2.4) These PTSs are recognized by soluble, cytosolic receptors – Pex5p for PTS1 (McCollum et al., 1993; Terlecky et al., 1995), Pex7p and its co-receptor, Pex20p, for PTS2 (Leon et al., 2006; Rehling et al., 1996; Stein et al., 2002; Titorenko

et al., 1998; Zhang et al., 1996) and Pex19p (and/or other undefined components) for mPTSs (Jones et al., 2004; Snyder et al., 2000) Following cargo recognition, receptor/cargo complexes are delivered to the peroxisomal membrane for further action

The peroxisome membrane has many peroxins that facilitate the import of matrix and membrane proteins Two subcomplexes, known as the docking (Pex8p, Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) subcomplexes, are bridged by Pex8p or another protein, Pex3p, to form a larger complex known as the importomer (Agne et al., 2003; Hazra et al., 2002)

Figure 2.4 Targeting signals used by peroxisomal proteins PTSs

are located in the boxes along with the consensus sequences, where

applicable and conserved variants are shown below these sequences In

each case, an example of a protein containing the PTS is given

The importomer plays a role in matrix, but not membrane, protein import The PTS1 and PTS2 receptors and their accessory proteins (e.g Pex20p) ferry cargo from the cytosol and first interact with the docking subcomplex (Heiland and Erdmann, 2005) The receptor/cargo complexes then either enter the matrix, or are deeply embedded in the peroxisome membrane (Dammai and Subramani, 2001; Leon et al., 2006; Miyata and Fujiki, 2005; Nair et al., 2004) This is followed by cargo release into the peroxisome matrix, export/release of the receptors on the peroxisome membrane (Leon et al., 2006; Miyata and Fujiki, 2005; Platta et al., 2005), followed

by dislocation/recycling of the receptors from a peroxisome-associated state to the cytosol (Leon et al., 2006; Miyata and Fujiki, 2005; Platta et al., 2005)

Figure 2.5 Membrane protein complexes of the peroxisomal protein import machinery Peroxin-13 (Pex13), Pex14 and Pex17 are

constituents of the docking complex for cycling peroxisomal import

receptors Another protein assembly in the peroxisomal membrane

comprises the RING-finger-motif-containing peroxins Pex2, Pex10

and Pex12 This motif is a characteristic element of E3 ubiquitin

ligases, and this subcomplex is linked to the docking complex by Pex8,

which is peripherally attached to the lumenal side of the peroxisomal

membrane (Erdmann and Schliebs, 2005)

Mutations in any component of the importomer affect the import of peroxisomal matrix proteins, suggesting that the whole importomer is somehow involved in protein translocation across this membrane (Agne et al., 2003) However, certain transient residents of the peroxisomal matrix, such as Pex5p and Pex20p, become peroxisome-associated and protease protected, even in the absence of the RING subcomplex of the importomer, but their entry into the peroxisome is Pex14p-dependent (Leon et al., 2006; Zhang et al., 2006) These data suggest that the docking subcomplex may be the true translocon, at least for these proteins, if not for other matrix cargo as well The RING subcomplex proteins are required for the export/release of receptors on the peroxisome membrane (Leon et al., 2006; Zhang et al., 2006) The dislocation/recycling of the receptors from the peroxisomes to the cytosol requires the action of a receptor recycling machinery, comprised of an E2-like ubiquitin-conjugating enzyme, Pex4p, two AAA-ATPases, Pex1p and Pex6p, that interact with each other in an ATP-dependent manner, and a peroxisomal membrane

protein (Pex15p in S cerevisiae or PEX26 in mammals), which provides a docking

site for Pex6p (Leon et al., 2006; Miyata and Fujiki, 2005; Platta et al., 2005) When this receptor recycling machinery is affected, a peroxisomal RADAR (acronym for Receptor Accumulation and Degradation in the Absence of Recycling) pathway becomes evident (Collins et al., 2000; Kiel et al., 2005a; Koller et al., 1999; Leon et al., 2006; Platta et al., 2004) This involves polyubiquitylation (Fig 2.6) of peroxisome-membrane-associated Pex5p and Pex20p, most likely by redundant UBCs

(Ubc1/Ubc4p/Ubc5p in S cerevisiae), followed by their degradation by proteasomes

(Kiel et al., 2005a; Kiel et al., 2005b; Kragt et al., 2005; Leon et al., 2006; Platta et al., 2004)

Figure 2.6 Models for the role of ubiquitylation in receptor

recycling (or dislocation) from the peroxisome membrane to the

cytosol, and in degradation by the RADAR pathway (Leon et al.,

2006)

2.2.2 Peroxisomes and human diseases

2.2.2.1 Peroxisome biogenesis disorders

The importance of peroxisomes for human health and normal development is underlined by the existence of several inherited diseases in humans, so called peroxisomal disorders A defect in a peroxisomal gene can lead to a single enzyme deficiency which might affect one specific peroxisomal function or metabolic

pathway However, when the affected protein is a peroxin, which is involved in the

biogenesis and maintenance of peroxisomes, several or all peroxisomal functions can

be affected, and peroxisomes can be completely absent This is the case in peroxisome biogenesis disorders (PBDs) (Braverman et al., 1995; Hannah et al., 2006) PBDs lead

to progressive metabolic diseases as well as developmental abnormalities that produce

distinct dysmorphic features Peroxisome biogenesis defects are genetically heterogeneous diseases with an autosomal recessive mode of inheritance They include the Zellweger syndrome (ZS, also called cerebrohepatorenal syndrome), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) The clinical pictures of these disorders show similarities, but an important difference is a difference in severity, the clinical course being most severe in ZS and mildest inIRD Exceptional patients present with a still milder phenotype (Marjo and Valk, 2005)

2.2.2.2 Peroxisomal single protein defects

The most common of the single enzyme defects, in which peroxisomes are present but a single enzyme function is deficient, is X-linked adrenoleukodystrophy (XALD) XALD (estimated incidence between 1:40,000 and 1:100,000) is based on mutations in the ALD gene encoding an ATP-binding cassette (ABC) transporter protein of the peroxisomal membrane, which is involved in the import/activation of saturated, unbranched very long chain fatty acids (VLCFA) (Mosser et al 1993; Netik

et al., 1999) Defects or a loss of ALD protein lead to an accumulation of VLCFA, and clinically to progressive demyelination/neurodegeneration in the central nervous system, adrenal insufficiency and death within a few years (Aubourg et al, 1993)

2.2.2.3 Diagnosis and therapy

Most understanding in the molecular defects and pathophysiology of the peroxisomal disorders has been made by studying peroxisome biogenesis in yeast mutants and analyzed in vivo by the generation in knock-out mice Great promise for the early diagnosis of PBDs lies in the molecular analysis of PEX genes, and molecular testing is evolving Laboratory diagnosis usually involves blood and urine

analysis (e g., plasma VLCFA analysis, analysis of plasmalogens in erythrocytes, oxidation of phytanic acid) followed by detailed biochemical and morphological studies in patient’s fibroblasts An alternative approach is the so called pharmaco-logical gene therapy, which uses certain drugs (e g., 4-phenylbutyrate) to increase the expression of peroxisomal genes which can either complement the function of the disease gene or increase the number and matrix protein content of peroxisomes (Haan

α-et al., 2006)

2.2.3 Pichia pastoris Pex8 and Pex20: role in peroxisomal matrix protein import

machinery

2.2.3.1 Pichia pastoris Pex8 (Pex8)

Pex8 has been described above as a central organizer of the importomer, a multisubunit complex of peroxisomal membrane and associated proteins, which is essential for matrix protein import into peroxisomes Interestingly, Pex8 is necessary for peroxisomal matrix import of proteins carrying either a PTS1 or a PTS2 sequence (Lan et al., 2006) To release the PTS2 cargo within the peroxisomes, Pex8p interacts with the cargo-loaded pex20 and pex7 complex The interaction nature of these three proteins is still unknown Which protein in this complex can first bind to Pex8 and cotranslocate into the peroxisome or both proteins in cargo complex interact with Pex8p are to be verified To perform these critical functions, Pex8 must be translocated into the peroxisome matrix The question is how Pex8 is itself released from Pex7 and Pex20 cargo inside peroxisome

2.2.3.2 Pichia pastoris Pex20p (Pex20)

Pex20 has also been related to the PTS2 pathway and cotranslocates with Pex7 and Pex20 into the peroxisome However, this interaction mechanism and the cycle between the cytosol and the peroxisome as part of an “extended cycle” of this

complex are still unknown Recently, a conserved cysteine residue of this Pichia pastoris Pex20 has been identified to play an essential role for its recycling from

peroxisome to the cytosol (Leon et al., 2006) Nevertheless, this residue is not completely necessary for the function of the protein because similar to what happens

in the recycling mutants, Pex20 (C8S) is constitutively degraded by the RADAR pathways It may be relative to the ubiquitylation mechanism but whether or not this cysteine is ubiquitylated remains unknown Identification of the Pex20 structure as well as the components and interactions mediating the recycling and RADAR

pathways will help in the global understanding of peroxisome biogenesis in Pichia pastoris as well as shed light to treat human peroxisomal biogenesis defects

2.3 OBJECTIVE

I have undertaken two independent projects for my research The 3D structure

of the full length wild type Brk protein will provide a good background to understand the tumorigenesis progression cell proliferation and survival In addition, the combination of this backbone structure and its mutants may be helpful to analyze their significant roles in the pathogenesis research of lymphoma and breast cancer researches, in general Some preliminary proteomic studies have been performed with

a variety of comprehensive expression patterns of Brk and its partner as well as its

probable interactions either in vitro or in vivo However, the exact mechanisms

responsible for the Brk-mediated control over cellular functions remains to be elucidated

The solution structure of the SH2 domain has been solved However, the structure full length Brk only will offer promising answer to how this tyrosine kinase expresses and influences in malignant cell transformation of lymphocytes Along with some of the interaction studies and substrate binding mechanism, Brk may shed light

on some specific signal transductions, which are related to most long-term breast cancer patients Furthermore, structure determination of this enzyme, coordinated with functional analysis, will help in the development of morphological and molecular markers for cancer therapy

Pex8 plays an important role in peroxisomal matrix protein import mechanism Without the understanding of the structures of the involved proteins, the whole map of all pathways related to peroxins will not be complete Interestingly, the

interaction between Pex8, Pex20 and Pex7 in the PTS2 path-way in Pichia pastoris

still remains a mystery Hence, each full length structure (Pex8, Pex20 and Pex7) will

be useful in addressing questions like which parts of them play a significant role in

interaction and what are their actual biological functions in Pichia pastoris These

structures and full understanding of their complexes will be excellent models for elucidating the potential links between the cellular import-deficiency of yeast mutants and corresponding pathways in human cell lines In turn, potential cure for human peroxisomal biogenesis disorders can be systematically attempted

CHAPTER 3

MATERIALS AND METHODS

3.1 EXPRESSION AND PURIFICATION OF RECOMBINANT WT-BRK 3.1.1 Construction of expression for WT-Brk

The humanized cDNA encoding breast tumor kinase gene was synthesized using reverse transcriptase polymerase chain reaction (RT-PCR) This reaction was used with oligo(dT) primers and total mRNA extracted from human breast tumor cells (T47D) as template A 5μg- 20μl RT reaction with 2μl DTT (0.1 M, Invitrogen), 4μl 5X RT buffer (Invitrogen), 2μl dNTP (10 mM, Roche), RNAase OUTTM (Invitrogen) 1μl, 1μl Oligo dT (50 μg/μl, Invitrogen), 5μl RNA (1 μg / μl), 1μl Superscript3 RT (Invitrogen), 4μl nuclease-free reaction water was used

The RT reaction was followed by a normal PCR reaction to amplify the gene

of interest A 50μl PCR reaction mix with 3μl MgCl2 (25mM, Promega), 5μl Mg free 10X PCR buffer (Promega), 1.5μl dNTP (10 mM, New England Biolab), 2μl each forward and reverse primers (10 μM), 1μl cDNA (2ng), 34.5μl dH2O, 1μl Taq Polymerase (Promega) was used The PCR reaction was carried out for 35 cycles The forward and reverse primers BRK14FP (TTCTT CATATG ATG GTG TCC CGG GAC CAG) and BRK14RP (TTCTT GGATCC TCA GGT CGG GTT CTC GTA GCT), respectively, provided WT-Brk gene with the NdeI and BamHI restriction enzyme sites, respectively (underlined) Briefly, the initial denaturation of the PCR cycle was set at 94°C for 4 minutes and followed by 35 cycles of: denaturation at 94

°C, annealing at 55°C and extension at 72 °C, for 30 sec at each step The final extension was carried out at 72 °C for 10min PCR products were analyzed by 1%

agarose gel electrophoresis and the PCR product at the expected size was cloned to the pGEM T-EZ vector for sequencing and subsequent cloning

3.1.2 Bacterial expression

The WT Brk gene was sub-cloned into a modified pET 14b (Novagen) vector (with His-tag) for expression The construct was transformed into BL21 (DE3) pLysS competent cells The BL21 (DE3) pLysS cells that contain the pET 14b: Brk construct, which confer ampicillin and chloramphenicol resistance Hence the transformants were plated onto an LB agar plate containing 100 μg mL-1 ampicillin and 34 μg mL-1 chloramphenicol for selection

Initially, a series of experiments were carried to check and optimize protein expression Expression was tested using 50 ml cultures before it was scaled up to higher volumes The first series of trials involved testing the construct using time based expression experiments Four 50 ml cultures were inoculated with 2 ml of culture grown overnight Bacteria were initially grown to an optical density (OD600)

of 0.6 (log phase) at 37 °C, and induced with different concentrations of IPTG (0.25, 0.5, 0.75 and 1 mM) Full length WT-Brk showed protein expression but most of the protein formed inclusion body and very less soluble protein

Normally, very low expression temperatures (less than or around 30 °C) and lower IPTG concentration (less than or around 0.1mM) can increase the solubility of expressed protein Six 1L culture was grown to log phase at 37°C and the temperature was lowered to 16°C before induction with 0.1 mM IPTG The cultures were grown for 16 hours and then the cells were harvested by spinning them at 5,000g for 20 minutes

3.1.3 Solubilization, purification and refolding from inclusion body

3.1.3.1 Solubilization

The inclusion body fraction of WT-Brk is defined as the component that gets pelleted during centrifugation of the cell lysate The pellet from the previous section was lysed two times by French press in 50mL denaturing buffer A containing 8 M urea, 20 mM Tris-HCl (pH 8.5), 0.5 M NaCl, 100 μM PMSF The suspension was centrifuged at 40,000g for 30 minutes at 4°C The supernatant was then applied to the purification step

3.1.3.2 Purification

The supernatant was mixed with 5 ml of the nickel-NTA agarose (Quigen) resin and rotated on a rocker for overnight binding at 4 °C The resin containing bound WT-Brk protein was subjected to washing with 30 mL of 20 mM Tris-HCl, 0.5

M NaCl (pH 8.5), 100 μM PMSF, 5 mM 2-mercaptoethanol, 1% Tween 20 and 5 mM imidazole The protein was eluted with 10 mL of buffer A containing 250 mM imidazole as elution buffer

3.1.3.3 Rapid dilution

The eluted supernatant was then refolded by 30 fold rapid dilution in a redox refolding buffer [20 mM Tris-HCl (pH 8.5), 0.5 M NaCl, 0.5 M arginine, 0.5 mM oxidized glutathione and 5 mM reduced glutathione] The refolding solution was incubated for 24 hours at 4°C with continuous stirring and then dialyzed for 36 hours against 20 mM Tris-HCl (pH 8.5), 0.5 M NaCl without urea The dialyzed solution was clarified and concentrated to 5ml before being further purified by Fast protein liquid chromatography (FPLC)

3.1.3.4 Size-exclusion chromatography

The His-tag refolded WT-Brk protein was purified by size exclusion chromatography on a pre-equilibrated Hi-Load 16/60 Superdex-75 column (GE Healthcare) with buffer [20 mM Tris-HCl (pH 8.5), 0.5 M NaCl in an AKTA Explorer 10 system (GE Healthcare) at a flow rate of 0.8 ml/min Protein peaks were detected by UV absorbance at 280nm The fractions under the peak were analyzed by SDS-PAGE for purity The most pure fractions were pooled together and concentrated until 0.5, 1, 1.8, 2, 3 mg/ml, respectively and frozen using liquid nitrogen for long term storage

3.1.3.5 Western blot analysis for denatured and refolded WT-Brk

The denatured and refolded WT-Brk proteins were verified by Western blot analysis using a Brk specific primary antibody The eluted denatured protein after purified by affinity chromatography and the refolded protein after rapid dilution and purification by size exclusion chromatography, were run on 12% SDS-PAGE with PageRulerTM Prestained Protein Ladder as a standard marker The SDS-PAGE gel was passively transferred to a PVDF membrane using a Mini Trans-Blot cell (Bio-Rad) following the manufacturer’s instruction The non-specific sites were blocked by incubating the membrane overnight at 4 °C in PBST buffer (1X PBS containing 0.05% Tween 20) plus 5% skimmed milk After the incubation, the membrane was washed three times with PBST buffer without skimmed milk for total 30 minutes Then, a rabbit monoclonal anti-Brk antibody Sigma-Aldrich was used as a primary antibody for binding overnight at 4 °C in PBST containing 5% skimmed milk The next morning, the primary antibody was removed and the membrane was washed three times with PBST buffer without skimmed milk The membrane was incubated