DEVELOPING AND OPTIMIZING A METHOD TO ANALYZE SUBSTRATE SPECIFICITY OF TYROSINE KINASES

Protein Tyrosine Kinases (PTKs) are crucial enzymes which aid in cellular signal transduction pathways as well as cell cycle regulation. This is accomplished by phosphorylating downstream targets specifically on the hydroxyl group of tyrosine residues. Equally important, extensive research has established a connection between overexpression of PTKs and the development of certain cancers. One example of this is seen in Chronic Myeloid Leukemia (CML) where a chromosomal translocation occurs in patients which results in a hybrid, overexpressed BCR/Abl fusion PTK ultimately leading to tumor formation. Thus, understanding PTKs along with what substrates they act on is of utmost importance. Along with understanding the structure and function of PTKs, identifying which substrates they phosphorylate is crucial as well. There are several methods both in vitro and in vivo which allow the study of PTK activity on their specific substrates. Each method has some setbacks. Some methods can be time-consuming to set up and use while other methods such as studying PTKs in mammalian cells can lack consistency due to other factors in the cell that can act on these enzymes and alter experimental data. A rapid and efficient system to allow characterization of PTK/substrate relationships would be highly valuable. In the following research, a bacterial system has been developed, optimized and tested which allows a researcher to screen both wild type and mutant protein tyrosine kinases for their phosphorylation activity on chosen substrates. In this screening model, PTKs Abl, Csk and Src and their respective substrates (crk l, kdSrc) were used to demonstrate the effectiveness of this method. Once demonstrated, chosen mutants of the above PTKs and substrates were then tested to further validate this screening method. In both wild type and mutant testing using this applied in vivo screening system, the expected phosphorylation activity was demonstrated. This system can be used to identify substrates for a known kinase, or kinase mutants with certain substrate specificity. It is a useful platform for understanding the mechanisms of PTK substrate specificity. Once this method was developed and proven, the protocol was optimized to allow rapid colony screening of PTKs for substrate specificity. This method can be used to screen mutant PTKs generated for the substrates they phosphorylate in a rapid, effective manner in an effort to further understanding PTKs.

Lastly, I would like to dedicate this master's thesis to Dr. Gongqin Sun, who took a chance on me and trusted me to work in his laboratory. He has shaped me with his wisdom and continued effort to mold me into a great researcher. I hope I have made his choice of having me work in his lab both a rewarding and positive experience for him as it was for me.

INTRODUCTION Protein Kinases and Signal transduction
There are a multitude of enzymes that occur in nature with a wide range of functions. Some enzymes such as proteases help breakdown targeted proteins while others like restriction enzymes recognize and cleave specific recognized regions of DNA. Protein kinases function by transferring a phosphate group onto a target substrate (phosphorylation) which allows for regulation of certain biochemical processes such as cell cycle and signal transduction pathways. [1][2][3] Several groups of protein kinases exist including but not limited to serine/threonine kinases, histidine kinases and protein tyrosine kinases. The main difference between these three groups of protein kinases is their method of action. Serine/threonine kinases function by phosphorylating the hydroxyl group of either serine or threonine side chains on their substrate. In contrast, histidine kinases first add a phosphate group from ATP onto their own histidine residue and then usually transfer it to an aspartate group on their substrate. Finally, protein tyrosine kinases transfer a phosphate group from ATP to the hydroxyl of tyrosine residues on their substrates. [2] Regardless of mode of action, stringent regulation of cell processes is achieved by the action of protein kinases. 2

Oncogenes and Protein Kinases
Cancer research and treatment has changed gradually throughout the last century. Initially, research focused on treating and preventing the proliferation of cancerous cells. Treatment consisted of radiation and chemotherapy techniques that have limited success and often cause patients to become immune to these therapies and experience drug resistance. Overall, these treatments served as the most prevalent mode of action to treat cancer patients . [4][5][6] In contrast to treating the proliferation of tumor cells with the techniques listed above, a new wave of cancer treatments focuses on controlling the activity and effects of the underlying cause of a range of cancers, oncogenes. Oncogenes are the result of protooncogenes becoming mutated or overexpressed. Proto-oncogenes are genes that have the capacity to induce cancer development through their protein and enzymatic products, when they are either overexpressed or mutated. [5] The discovery of the first retroviral oncogene, v-src, in 1911 was responsible for the importance placed on understanding oncogenes and led to a shift in cancer therapy as well. [7,8] The discovery of the first oncogene v-src and its impact on cancer research In 1911, Francis Peyton Rous, injected cell-free extract from a cancerous chicken tumor into healthy chickens and observed that this was promoting cancer development (sarcoma) in the chickens injected. Further research implicated the Rous Sarcoma Virus (RSV) was the driving force behind the cancer generation in the afflicted chickens. [7,8] The Rous Sarcoma Virus genome contains three genes, gag, pol 3 and v-src and along with this, RSV has the capability to integrate its genes into the host chromosome (chicken) and induce overexpression of these viral genes. [9] Although overexpression of pol and gag is responsible for the replication of the virus, overexpression of the viral v-src leads to uncontrolled mitosis of the host chicken cells and ultimately tumor formation. The discovery of v-src was a breakthrough in science and due to this amazing discovery in cancer research; Peyton Rous was awarded the Nobel Prize in 1966. [10] The work of Peyton Rous and research related to his discovery not only led to a better understanding of the causative agent of cancer but also promoted research on a new type of protein kinase group, protein tyrosine kinases. After the discovery of the v-src oncogene, research on its function elucidated a new mechanism of protein phosphorylation in which a phosphate group is attached directly to the hydroxyl group on a tyrosine residue of the targeted substrate. Due to this, these protein kinases were named protein tyrosine kinases (PTKs). The impact of the discovery of a new group of enzymes and their ability to become oncogenic fueled researchers to discover, isolate and study other PTKs. [2] 4

Protein Tyrosine Kinases: Structure and Function
With the discovery of v-src's function as a protein tyrosine kinase as well as its oncogenic potential, this led to a booming increase in research driven toward understanding the structure and functions of PTKs as well as the discovery of new PTKs. This effort was applied in hopes that by understanding these unique enzymes and their mode of action, cancer development linked to oncogenic PTKs could be further understood, and therapeutic strategies can be developed.
Since the discovery of v-src, many other PTKs have been discovered including Abl, Csk and Frk. These PTKs all have conserved regions as well as similar modes of action, but their specific roles in a cell tend to vary. [2] Figure 1 shows the evolutionary relationship between both receptor PTKs and nrPTKs present in nature.

Structure homology of PTKs: Receptor vs. Non-Receptor PTKs
When it comes to the protein tyrosine kinase family, there are generally two types, receptor and non-receptor PTKs. The first type, receptor tyrosine kinases (RTK) span the cellular membrane and bind extracellular ligands. There are 20 distinct families of RTKs identified which can bind extracellular large and small ligands. [11] Binding of extracellular ligands to RTKs generally causes dimerization of the receptor and trans-phosphorylation of the kinase domain thereby activating it. This activated kinase will then phosphorylate a downstream target and lead to amplification of the signal. RTKs, such as EGFR, consist of three main domains including a transmembrane   [3] In general, the conserved SH2 domain of nrPTKs is made up of 100 amino acids and tends to be located upstream of the catalytic domain of nrPTKs. [3,12] The conserved SH2 domain can bind to phosphorylated tyrosine containing peptides and is also involved in localization to RTKs and other enzymes activated by tyrosine phosphorylation.   11 Amino acids outside the catalytic cleft can also play a huge role in increasing the efficiency of catalysis within PTKs. An example of this is when inactivation of Src is carried out by Csk on a specific C-terminal tyrosine 527. A specific stretch of amino acids on Csk's peptide binding lobe termed RSRGRS is crucial for Csk inactivation of Src. Without this region, inactivation of Src by Csk does not occur. [14] Despite similar modes of catalytic action, PTKs still achieve a high level of substrate specificity.

Model Protein Tyrosine Kinases
With the wide variety of nrPTKs that exist, it is a challenge for researchers to understand how cell signaling through PTKs is achieved. Although the catalytic domains and catalytic mode of action is generally conserved, they still manage to achieve a high level of substrate specificity. An example of this is Src PTK which demonstrates substrate specificity for its auto-phosphorylation site (Tyr416), but cannot phosphorylate its inactivation site (Tyr527), which is targeted by another PTK, Csk. [3,12,15] Based on this, researched have grouped nrPTKs into specific families which share similarities both structurally as well as the regulatory features they possess. Several of these model kinase families include the Src family kinases (SFKs), Csk family kinases, Abl family kinases (AFKs), Frk family kinases and finally the Tec family kinases. By grouping nrPTKs into subfamilies, it can provide an overall understanding of how signal transduction is achieved by these different nrPTKs. The three model PTK families Src, Csk and Abl that are relevant to the applied research will be discussed below.

Src family kinases (SFKs)
Being the first PTK discovered and having oncogenic potential, much is known about Src and related SFKs such as FYN and Yes. The SFK contains 9 members and tend to regulate important cell processes such as cell growth, migration, differentiation, and survival throughout various tissues in the body. [2] They directly act on growth factors and play a huge role in growth factor signaling pathways. Although 13 each SFK plays important roles in normal cell development and function, elevated levels of SFKs have been associated with oncogenesis in humans, specifically in colon cancers and other cancers such as lymphoma and melanoma. [2] SFKs have a structure containing an SH3 domain, an SH2 domain, a kinase domain, an N-terminal variable site which can be modified by myristoylation or palmitoylation and finally a flexible tyrosine containing C-terminal tail. The SH2 domain plays a role in binding phosphotyrosine-containing peptide sequences while the SH3 domain has been shown to bind type II polyproline helix ligands. Meanwhile, the N-terminal variable region plays a role in membrane recruitment. Finally, the highly conserved Cterminal tail serves as a regulator of SFKs through phosphorylation or dephosphorylation of the tyrosine located on the tail. [2,12,15] Although SFKs have been shown to be oncogenic, there has to be strict regulation of SFKs to prevent this from always occurring inside the cell. This is achieved by a stringent regulation on two important residues contained in SFKs. The first residue, tyrosine 416, is the site of autophosphorylation of SFKs, which when phosphorylated activates the enzyme. This activating tyrosine is present in the autophosphorylation loop, a region highly conserved in amino acid sequence(RLIEDNEYTARQGAK) among SFKs and closely related members. [3] In contrast, phosphorylation by Csk on tyrosine residue 527 on the C-terminal flexible tail of Src inactivates the enzyme and renders it inactive. When phosphorylated, the phosphorylated tyrosine residue 527 interacts with Src's SH2 domain and allows the SH3 domain and linker region to come together keeping the enzyme in an inactivated state. [2,3,12,14] Because Src is a substrate for both Csk and Src, but on different tyrosine residues, it is an ideal 14 substrate for testing the substrate specificity for Csk and Src. The structural organization and regulation of Src is shown below in Figure 3.

Csk family kinases(CFKs)
The Structurally, Csk family kinases share a 40% sequence identity when compared to Src.
They share a conserved SH2, SH3 and catalytic domain with Src but significant 15 differences do exist. Missing from their structure is an N-terminal lipid anchoring domain, an activation loop tyrosine and finally a C-terminal tyrosine-containing tail.  [3] 18 This suggested to researchers that Csk was a distant relative of Src with a different mode of regulation. [3,12] It has been established that CFKs have limited and highly specific substrate targets, usually of the SFK. Due to such a high level of substrate specificity, this lends support as to why Csk was one of the first full-length protein tyrosine kinase to be overproduced efficiently in E.coli with the addition of chaperonins GroES and GroEL.
By having a stringent level of substrates it targets, Csk proved to be less toxic when introduced to foreign hosts such as E.coli. Due to the ability to overproduce the Csk enzyme with this novel strategy, it has been widely studied and serves as a model PTK. In contrast, efforts to purify Chk enzyme have been unsuccessful in the past making it hard to study as well. [1,3,12] Csk's structure and regulation of Src PTKs are shown below in Figure 4.

Abl family kinases(AFKs)
The Abl family of non-receptor tyrosine kinases (AFKs) includes both c-Abl (cellular Abl) encoded by ABL1 and its relative Arg (Abelson-Related Gene) encoded by ABL2. ABL genes are found in all metazoans, suggesting that both their structure and function were fixed early in tyrosine kinase evolution. AFKs function in such processes as cell growth, cell survival and DNA repair. [16,17] Structurally, both members of AFK share conserved N-termini containing an Nterminal cap, myristoylation site, SH3 and SH2 domain, and similar kinase domains. Sciences. [3] 21 In contrast, the C-terminus is quite different in that c-Abl contains nuclear localization and export signals as well as a DNA binding domain that Arg does not possess. Both members do however contain an F-actin binding domain located at their C-termini. [17,18] AFKs have been shown to be phosphorylated by members of the SFKs and have been shown to possess autoinhibitory mechanisms as well. These include a myristoyl group that can bind to the surface pocket in AFKs kinase domain and contribute to an autoinhibitory fold. Along with this, AFKs contain an amino-terminal cap which can stabilize the inactive conformation of these enzymes using specific surface interactions. Further experiments have demonstrated that disruption of these autoinhibitory mechanisms result in an increase in kinase activity of AFKs. [16,18] Another similarity to SFKs is that AFKs are proto-oncogenic and can become auto-phosphorylated causing it's catalytic activity to increase heavily. Due to these three consequences, several cellular effects occur including uncontrolled cell proliferation, reduced DNA repair, and ultimately Chronic Myeloid Leukemia. [16,19] Although many signaling pathways are activated by BCR/Abl, only a few proteins appear to be necessary for BCR-Abl-dependent transformation. These include the proteins Gab2, Myc, CrkL and STAT5. Out of these 4, CrkL is among the most preferred substrates of both wild type Abl and BCR/Abl and its binding to BCR/Abl PTK is necessary for oncogenic transformation to occur in CML patients. [16,20] Being a substrate for both Abl and BCR/Abl, crk-l has become heavily studied. It functions in numerous biological processes including cell adhesion and migration, cell proliferation, apoptosis, and regulation of gene expression.

Studying PTKs and drawbacks of developed methods
Although much is known about PTKs, there are several drawbacks to the methods designed to study these intricate enzymes. In vivo methods such as studying PTKs in mammalian cells can produce complex and hard to interpret results. The main reason for this is that in a mammalian cell, there are many other factors that can play a role in mediating or altering the activity of the PTK being studied. One example of this occurs when researchers attempt to study inactivation of the SFK and FGFR families by reactive oxygen species (ROS) in vivo. Although studies showed that these PTKs might have been inactivated by the presence of ROS, they were unable to directly correlate it to the addition of ROS. This is due to the possibility of other 25 regulatory factors such as members of CFK inactivating these PTKs rather than the ROS being the direct cause of inactivation. [21] A similar problem occurs generally when studying most PTKs due to these other regulatory factors being present inside the cell and influencing the study of these enzymes by their regulatory actions.
Along with in vivo methods to study PTKs, in vitro methods such as radioactive kinase assays have their drawbacks as well. Radioactive kinase assays consist of using radioactive ATP along with both purified enzyme (PTK) and substrate in an intricate time-based reaction. [14] The drawbacks to this analysis method is that first, the researcher must be able to purify both PTK and substrate to a very high purification level; otherwise the experiment conducted will not be valid. Purification methods do work for many PTKs but some PTKs fail to express and purify correctly such as seen in attempts to purify Chk enzyme. [1] Also, some PTKs and substrates do purify but can contain contaminants when purified. Lastly, radioactive kinase assays require the use of radioactivity and its use has strict regulations and can be difficult to work with.

Focus of this thesis
The experimental data, results and discussion pertinent to this work focuses on two related projects, both of which have a similar goal of developing and optimizing systems to understand and rapidly screen PTKs for substrate specificity. The first project involves developing and optimizing an in vivo bacterial system which allows a researcher to rapidly screen PTKs for substrate specificity. The second project focuses on a colony screening method which allows for the screening of mutant PTKs in a rapid, effective manner. Both projects are aimed at allowing researchers to efficiently study PTKs and their substrates in an effort to further understand their function and roles, specifically in oncogenesis.

Developing a rapid, efficient bacterial system to analyze substrate specificity in PTKs
In an effort to overcome drawbacks experienced by researchers in analyzing PTKs as well as the substrates they target, a novel, bacterial system has been developed and optimized which allows for rapid analysis of substrate specificity in PTKs. The system developed consists of co-expressing both PTK and substrate to be analyzed in a bacterial host and following an optimized, rapid protocol which has been shown to produce expected substrate specificity when tested. By using this developed system, researchers now have another method to study PTKs with few of the drawbacks that other methods tend to present such as outside factors acting on PTKs and purification problems as seen in radioactive kinase assays.

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Screening of generated PTK mutants for substrate specificity using a rapid colony screening method In an effort to understand PTKs function and role, PTK mutants have been generated and studied using similar techniques as when studying non-mutated PTKs.
Regardless of whether mutant or non-mutant, there are several problems that arise. There are several in vivo and in vitro methods to analyze substrate specificity of PTKs but they tend to be time-consuming and can produce unreliable results. In vivo methods in mammalians are important but generally involve too many outside factors and influences when trying to analyze substrate specificity of a certain PTK. In contrast, in vitro methods such as radioactive kinase assays do produce accurate results but require the purification of substrate and enzyme as well as using radioactivity, a general hazard.
The following research presents a rapid and efficient bacterial system for analyzing

Introduction:
Protein tyrosine kinases are important enzymes in mammalian signal transduction pathways as well as many other important cellular processes such as cell cycle regulation. They phosphorylate specific substrates on their tyrosine residues using a phosphate group derived from ATP. [1][2][3] By doing this, these enzymes regulate specific cellular activities and cell function. Although generally sharing several conserved structures, PTKs still manage to exhibit very specific substrate specificity even when comparing homologous PTKs such as Src and Csk. [1][2][3] Recent breakthrough research has linked several PTKs to the development of certain cancers. An example of this is in Chronic Myeloid Leukemia (CML) where a chromosomal translocation produces a mutated, overexpressed BCR/Abl PTK which leads to unregulated cell proliferation and subsequent tumor formation in patients with CML. [4] In contrast, PTKs such as Csk have been shown to be tumor suppressors, inactivating PTKs of the Src family. [3] Regardless of whether oncogenic or tumor suppressing, this recent research has placed utmost importance on understanding substrate specificity of these enzymes. The hopes are that by discovering and understanding the targets of PTKs, this information can be used to develop drugs that target PTKs or their substrates and regulate the effects of these mutated or overexpressed enzymes in afflicted cancer patients.
There are several developed methods to test substrate specificity of PTKs but regardless of the methods used, all remain time-consuming and can produce erroneous results. Using in vivo mammalian cells to analyze substrate specificity of PTKs can be performed, but the results can be misleading due to the amount of other proteins and enzymes in a cell that can alter substrate specificity of PTKs. [5] Relevant to this, in vitro methods such as radioactive kinase assays require the purification of both PTKs and substrates and involve using radioactive compounds. [5,6] Regardless whether in vivo or in vitro, both ways tend to present a myriad of problems to a researcher.
In the following research, we sought to develop and optimize a bacterial system which can be used to analyze substrate specificity of PTKs in a rapid, accurate manner. Using this developed system, three specific model nrPTKs (Abl, Csk and Src) were expressed in combination with their preferred substrates, either crk-l or kdSrc, in an effort to achieve the predicted substrate specificity demonstrated by these protein tyrosine kinases. Once predicted substrate specificity by these PTKs was verified, both mutant PTKs and substrates were also tested to further validate the system developed.

Reagents and Chemicals
Consumables and culture media, or media components were purchased from Fischer Scientific. The PY20 anti-phosphotyrosine antibody was purchased from Santa Cruz Laboratories. The P-Src (Y527), P-Src (Y416) and Anti-Rabbit IgG was purchased from Cell Signaling Technology. The p-Tyr antibody(PY20) antibody was purchased from Santa Cruz laboratories. The Phospho-Crkl (207) antibody was purchased from Abcam and the anti-mouse IgG secondary antibody from Sigma-Aldrich. All other chemical reagents used were purchased from Sigma-Aldrich.

Cloning of model PTKs and Substrates into selected plasmids
After optimization by Yixin Cui, it was shown that expression of selected PTK coding regions of wild-type Abl, Csk(full-length), Src(aa 83-533) and mutants into low copy number plasmid pCDF-1b was shown to work best in achieving proper substrate specificity in this system. The substrates tested kdSrc and its mutants were previously cloned into low copy number plasmid pRSET-A. [5,6] Substrate crk-l was previously cloned into pGEX-4t-1. [4] To clone the selected coding regions of the PTKs Abl, Csk, Src and mutants into pCDF-1b, a general sub-cloning procedure was followed. Generally, the plasmids that contained the selected PTK coding regions and the pCDF-1b plasmids to be cloned into were digested in 50 µl Eppendorf tubes with specific restriction enzymes at 37° Celsius for 1 hour. The digestion products were loaded onto a 1% agarose DNA gel and the proper-sized bands gel-extracted using Qiagen's QIAquick gel extraction kit and quantified with Thermo-Scientific's Nanodrop 8000. Once quantified, specific quantities of both digested insert and vector were used to set up calculated insert to vector ratios and these were ligated overnight @ 16°C. Next, these ligation products were transformed into electro-competent E.coli Bl21-Gold(DE3) cells using electroporation(2mm cuvettes) and plated on LB plates containing appropriate antibiotics and incubated overnight in a 37°C non-shaking incubator. The following day, colony-PCR was performed on the colonies produced using a combination of primers specific for pCDF-1b plasmid (pCDF-1b +) and the insert cloned (ex. Src Y454A -). Restriction enzyme digest analysis was also performed using the same restriction enzymes initially used to clone in the insert to further verify correct sub-cloning of desired insert into pCDF-1b. Finally, the newly made insert-containing plasmid products were sent for sequencing to the University of Rhode Island Genomics and Sequencing Center and subsequently verified by sequence analysis.

Gold(DE3)
Through cloning as described above, the PTK-containing plasmids were already present in the strain of choice, E.coli BL21-Gold(DE3) and these stocks were then separately made electro-competent using a general high-efficiency electrocompetent cell protocol. The substrate-containing plasmids, either pRSET-A or pGEX-4t-1, were isolated from previously made stock cultures using Qiagen's 37 miniprep kit, and transformed into the appropriate strains using electroporation in 2mm cuvettes. The transformed products were recovered in 500µl of LB media for 1 hour in a 37°C shaking incubator(225 rpm) and this recovery product was then plated on LB plates with appropriate antibiotics added. The following day, the transformant colonies were tested for presence of both PTK and substrate-containing plasmids using colony PCR with primers specific for each plasmid and insert. Table 1 indicates exactly which PTK/Substrate combinations were made to be tested using the developed screening system. Table 2 indicates the PTK/Substrate combinations tested using the colony screening method. Once tested, the expression and activity of the PTK/substrate strains were verified by performing a western blot using P-tyr antibody(PY20,) which detects phospho-tyrosine containing proteins.  Preparation of samples to be tested volts in an SDS-PAGE buffer system. Once complete, the gel produced was used for western blotting purposes as described below. Duplicate gels were also produced and subsequently Coomassie stained for 30 minutes to vefiry the quantitation of samples.
Following de-staining and equilibration in water, gels were analyzed with the Bio-Rad GelDoc system.

Western Blotting of Samples and Exposure
For western analysis, samples were first subjected to SDS-PAGE as described above. The resolved protein gels produced were incubated in transfer buffer along with the appropriate filter paper and membranes used. The protein gels were then After incubation in the clarity reagent, the membranes are placed in between laminating sheets and exposed for multiple time points using high resolution chemiblot protocol in Bio-Rad's Universal Hood II GelDoc imager system.

Optimized colony screening sample preparation
To prepare samples, the strains to be tested are used to inoculate 5 ml of LB 10 minutes to lyse cells and release proteins. After heating of membrane, membrane is quickly rinsed in TBST and followed up by an optimized western blot protocol specific for this developed method.

Optimized Colony Screening Western Blot protocol
Upon quickly rinsing membrane with 15 ml TBST buffer, membrane is blocked in 5% non-fat milk with gentle shaking for 30 minutes. After blocking, membrane is washed 3 times (2mins/wash) with 15ml TBST buffer by gentle agitation. Next, the membrane is incubated for 1 hour at room temperature in the appropriate primary antibody concentration diluted in TBST buffer with shaking.
After incubation with the primary antibody, membrane is washed 3 times (3mins/wash) with 15 ml TBST buffer per wash. Following, membrane is incubated in 43 the appropriate concentration of secondary antibody diluted in 5% non-fat milk for 1 hour at room temperature. Next, membrane is washed 3 times (3mins/wash) in TBST buffer than membrane is dried and exposed to BioRad's clarity reagent for 2 minutes.
After incubation in the clarity reagent, the membranes are placed in between laminating sheets and exposed for multiple time points using high resolution chemiblot protocol in Bio-Rad's GelDoc imager system.

Demonstrating substrate specificity of model kinases Abl, Csk and Src
To and Csk which showed minimal activity as shown by the western blot in Figure 2 below.
Finally, Csk must demonstrate high substrate specificity toward kdSrc on tyrosine residue 527 when compared to Abl and Src. As expected, substrate specificity of Csk toward kdSrc on tyrosine residue 527 was in fact observed in comparison to both Abl and Src as shown in Figure 3. The approximate kdSrc band is indicated by the arrow. Antibodies used to perform blot were 1° Phospho-Src (Y416) at a 1:5,000 and 2° anti-rabbit IgG at a 1:10,000. All samples were produced and tested according to the developed screening method protocol as described in materials and methods section. Antibodies used to perform blot were 1° Phospho-Src (Y527) at a 1:5,000 concentration and 2° anti-rabbit IgG at a 1:10,000 concentration. All samples were produced and tested according to the developed screening method protocol as described in materials and methods section.
52 plasmid in this system is sufficient for demonstration of correct substrate specificity of these model PTKs toward their respective substrates. Once substrate specificity of Abl, Csk and Src toward their known substrates was demonstrated using this system, the next step was to validate the system further using both mutant PTKs and mutated substrates.

Testing of mutant PTKs to further validate system
The next step to further validate the developed method to analyze PTKs for substrate specificity was to test two specific mutant PTKs generated; Csk R281A, + R283A and Src RSRGRS+ K458F. Both of these mutants have had amino acids mutated which have previously been shown to have altered phosphorylation activity on the substrate kdSrc when compared to their wild-type forms. (6) The first mutant PTK tested, Csk double mutant (DM) R281A + R283A, has had arginine residues replaced with alanine residues at amino acids 281 and 283. This has been shown in vitro, using radioactive kinase assays, to cause this mutant to dramatically lose its ability to phosphorylate kdSrc at its inactivation site, tyrosine residue 527, when compared to wild type Csk. [6] Therefore, it is expected, once tested using the proposed screening method, that this mutant should demonstrate a loss of phosphorylation activity on kdSrc at tyrosine residue 527, when compared to wild type.

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In fact, this is what was observed as shown in Figure 4. Once this Csk R281A + R283A mutant demonstrated the expected phosphorylation activity when tested, the next mutant analyzed in this system was Src RSRGRS + K458F.
The Src RSRGRS + K458F mutant has had amino acids 354-359 (KGETGK) replaced with the amino acid sequence RSRGRS as well as a K458F mutation. These seven residues are collectively referred to as the substrate-docking site of Csk and have been shown to be crucial for Csk phosphorylation of Src at tyrosine residue 527. [6] To figure out which amino acids of Src to replace with the residues crucial for Csk's recognition of Src at tyrosine residue 527 (RSRGRS), a sequence alignment was performed between Csk and Src's peptide binding lobe (figure 5). Once determined which amino acids to mutate, this specific Src mutant was made and tested, in vitro; showing much higher phosphorylation activity at tyrosine residue 527 of kdSrc when compared to wild type Src, which phosphorylates this residue minimally. (6) Therefore, this altered phosphorylation activity should be demonstrated when tested in this in vivo optimized system to further establish its accuracy. It was shown that this Src mutant does indeed phosphorylate substrate kdSrc on tyrosine residue 527 at a much greater level when compared to wild type form as seen in Figure 6.
To show that the Src RSRGRS +K458F, mutant still retained its auto-phosphorylation ability at tyrosine residue 416 of kdSrc despites the mutations, a western blot was performed using antibodies specific for this residue. As seen in Figure 7 below, this mutant still retained its phosphorylation activity at tyrosine residue 416 of kdSrc, although it is less active, when compared to Src (wt).  Lee et al. [6] 58  replaced with a phenylalanine greatly reduced Src's ability to phosphorylate this generated mutant as seen in Figure 8 below. concentration and 2° anti-rabbit IgG at a 1:10,000 concentration. All samples were produced and tested according to the developed screening method protocol as described in materials and methods section.
The second mutant tested within this developed system was kdSrc (Y527F), a mutant lacking the crucial tyrosine necessary for its inactivation at this site. As expected, when co-expressed with Csk, the kdSrc (Y527F) mutant substrate demonstrated a greatly reduced phosphorylation level at tyrosine 527 when compared to kdSrc in the same setup, as shown in figure 9. This is a further indication of the effectiveness of the optimized method for analyzing substrate specificity in PTKs.

Optimization of developed colony screening method
Initially, the first attempt when establishing the colony screening method was to use the strains to be tested directly from an isolation streak plate with isolated colonies. These individual colonies were aseptically spotted with toothpicks directly onto nitrocellulose membranes, and these membranes were placed in a petri dish containing 20ml 0.4mM IPTG for 1 hour to promote induction of cells contained on membranes. Following this, membranes were transferred to another petri dish containing approximately 15ml of 10% SDS and heated in a 95°C oven for 10 minutes, to promote lysing of cells and release of proteins to be blotted. After lysing, membranes were dried and blotted using the optimized western blot protocol described in the materials and methods section. concentration and 2° anti-rabbit IgG at a 1:10,000 concentration. All samples were produced and tested according to the developed screening method protocol as described in materials and methods section.
Using this method, the three strains containing Abl, Csk and Src co-expressed individually with crk-l substrate were used to prepare a membrane as shown in figure   10a. Once prepared, the membrane was blotted using the Phospho-crk-l (Tyr207) antibody which detects phosphorylation on tyrosine residue 207 of crk-l, Abl's preferred substrate. In contrast, Src minimally phosphorylates this residue while Csk has not been shown to act on this residue. Once the prepared membrane was blotted, Abl did display a much higher phosphorylation activity when compared to both Csk and Src as shown in figure 10b.
Although the correct substrate specificity was demonstrated with the protocol used to generate samples used in figure 10b, the signals tended to overlay and transfer to other sections of the membrane while exposing films. Therefore, double sets of identical signals would be observed either below or above where the colonies were toothpicked onto the membrane. Another issue with this protocol was that colonies would wash off occasionally during the induction and lysing process and therefore, no signal would be observed. In an effort to overcome this, the protocol was altered so that the strains tested were to be grown and induced in liquid culture, rather than grown on an LB plate and induced on the sample-containing membrane as used to generate samples in figure 10b. Samples would then be pipetted directly onto them membrane, lysed and blotted. This optimization was done in hopes to limit the problems of transferred colonies washing off the membrane, or signals being transferred over to nearby sections of membrane. Although the prior protocol did demonstrate the ability to screen PTKs for substrate specificity when phosphorylation activity of PTKs Abl, Csk, and Src was tested at tyrosine residue 207 of crk-l substrate, its optimization did indeed lead to better and cleaner results.
Use of optimized sample preparation protocol for colony screening substrate specificity of kdSrc  Figure 11. Therefore, the amount of each culture pipetted directly onto the membrane was reduced to approximately 0.5µl. At this low volume, samples were shown to demonstrate proper substrate specificity.   figure 11 below.
As Figure 12 demonstrates, proper substrate specificity by the tested PTKs was achieved in relation to phosphorylation activity at tyrosine residue 416 using this lesser volume of sample. Therefore, this identical 0.5µl sample volume was used in the last blot due to its success.  Membrane was blotted with primary antibody Phospho-Src (Tyr527) at a 1:5,000 concentration and secondary anti-rabbit IgG antibody at a 1:10,000 concentration.

Discussion
Overall, the goal when developing this novel system to screen PTKs for substrate specificity was to first optimize the protocol to make it very easy to screen for substrate specificity when testing different PTKs. produced, and these samples are tested and blotted for phosphorylation of kdSrc at tyrosine residue 416, then a difference in substrate specificity will not be seen. The researcher will observe that kdSrc at tyrosine residue 416 has been phosphorylated equally by both Csk and Src even though this is not what occurs in nature. Therefore, to overcome this problem, the genes of the substrates tested were cloned and expressed in high-copy number plasmids pRSET-A (kdSrc) or pGEX-4t-1(crk-l) and genes of PTKs in low-copy number plasmid pCDF-1b. By limiting the amount of PTKs expressed in contrast to the number of substrate being produced, proper difference in substrate specificity could be observed and achieved with this developed system. These were the two biggest optimizations made to the developed system in an effort to make it as accurate as possible when analyzing substrate specificity. The second goal of this research was to use this optimized system in combination with testing three important PTKs Abl, Csk and Src along with two substrates, crk-l and kdSrc, in an effort to demonstrate that it can achieve proper substrate specificity. With the optimizations made, the results seen did indeed show that the system could demonstrate a difference in substrate specificity by different PTKs when tested against chosen substrates.

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The first important result demonstrated was when testing phosphorylation activity at tyrosine residue 207 of substrate crk-l when co-expressed individually with Abl, Csk and Src. Since crk-l is one of Abl's preferred substrates and is minimally phosphorylated by both Csk and Src, this difference in substrate specificity should be and was demonstrated by this system. The second result was testing phosphorylation activity of kdSrc at tyrosine residue 416 when co-expressed individually with the same three PTKs as above. Since Src phosphorylates this residue heavily in comparison to both Abl and Csk, this should be demonstrated when tested using the developed system. When tested, this expected result was produced and repeated. Finally, phosphorylation activity of kdSrc at tyrosine 527 by Abl, Csk and Src was tested using this optimized system. Csk shows much greater phosphorylation activity at this residue when compared to Abl and Src. It was indeed shown that Csk phosphorylation activity on tyrosine residue 527 on kdSrc was much greater in comparison to the Abl and Src as expected. Once substrate specificity was demonstrated using the three chosen model PTKs Abl, Csk, and Src, the final step taken was to validate the use of this developed method with mutant PTKs and substrates.
The first mutant tested, Csk (DM) R281A + R283A, has specific amino acids arginine residues replaced with alanine in its peptide binding lobe region. These residues, when mutated, cause a dramatic loss in this mutant's phosphorylation activity on tyrosine residue 527 of kdSrc. [6] When tested using the developed system, this loss of activity on tyrosine 527 of kdSrc was demonstrated and highly reduced when compared to Csk's (wt) activity on this same substrate.

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The second mutant tested, Src RSRGRS +K458F, has had amino acids 354-359 (KGETGK) replaced with RSRGRS amino acid sequence and a K458F replacement, giving this mutant the ability to phosphorylate kdSrc substrate on tyrosine residue 527. [6] This mutant should demonstrate this altered phosphorylation activity when implemented in the developed system if the system is to be further validated. It was shown that the Src RSRGRS + K458F mutant phosphorylated kdSrc on tyrosine residue 527 much more when compared to Src (wt)'s activity on this residue as expected. Once this was accomplished, the final validation for this developed method was to test two specific mutant substrates, kdSrc (Y416F) and kdSrc (Y527F). The first mutant, kdSrc (Y416F) has had the tyrosine residue at its auto-phosphorylation site replaced with a phenylalanine residue. The result of this mutation is that this site can no longer be phosphorylated by PTKs and therefore this should be demonstrated when tested using the method developed to screen PTKs for substrate specificity.
Once tested, the kdSrc (Y416F) mutant when co-expressed with either Csk or Src shows little to no phosphorylation activity. In contrast, kdSrc (wt) is phosphorylated heavily by Src as shown by testing. Finally, mutant kdSrc (Y527F) which has had the tyrosine residue 527 at its inactivation site replaced with a phenylalanine residue should demonstrate a lack of phosphorylation activity by both Csk and Src when tested on this residue. As expected, Csk which heavily phosphorylates kdSrc (wt) on tyrosine 527 showed a lack of phosphorylation activity when co-expressed with kdSrc (Y527F) in this system. Src which minimally phosphorylated kdSrc(wt) at tyrosine residue 527 also showed a loss of activity when co-expressed with this mutant.

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Overall, after optimization and testing of both wild type and mutant PTKs/substrates using this developed system, its effectiveness at displaying substrate specificity of different wild type and mutant model PTKs and substrates was demonstrated through repeated testing. Concluding, there are many beneficial reasons to use this developed method to screen protein tyrosine kinases for substrate specificity. For one, it negates most of the drawbacks that other in vivo and in vitro methods used for analyzing PTK substrate specificity, present. Second to this, once setup of system is established, results can be produced within 1-2 days, which is rapid compared to other methods.
Finally, with the established protocol, using this system is fairly convenient and employs basic laboratory techniques in which any researcher can execute.
Once the method above was established in being effective for screening PTKs for substrate specificity, the protocol was optimized to allow a researcher to rapidly screen a large number of PTK mutants generated through current techniques such as DNA shuffling. [7,8] After a few changes, a developed colony screening method for rapidly screening PTKs was established and proven effective by using model PTKs Abl, Csk and Src and the preferred substrates. Although no mutant PTKs were tested during this study, by demonstrating the proper substrate specificity of model PTKs, the goal was to provide convincing evidence that this method can be used to screen mutant PTKs that can be generated through DNA shuffling or other methods.
Concluding, this novel colony screening method provides a rapid way to screen mutant PTKs, once generated, for chosen substrate specificity in a cost-effective, replicative manner.

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One drawback of both systems is that the observed specificity is not quantitative and thus once substrate specificity is demonstrated using either method, it needs to be further quantified by other methods, such as radioactive analysis.