Activation of Epidermal Growth Factor Receptor Signaling Pathway with SDS-PAGE and Western Blotting - Essay Example

?Studies on the activation of epidermal growth factor receptor signaling pathways using SDS-PAGE and Western blotting Introduction Protein molecules can be isolated through use of gel electrophoresis. Electrophoresis is the process in which a particle with a net charge travels in an electric field as a way of assorting proteins and other macromolecules, like the DNA or RNA. Electrophoretic separations are almost always applied with use of gels since they can function as sieve for molecules which betters protein separation. Smaller protein molecules compared with the pores present in the gel promptly travel through the distance of the gel, while larger protein molecules relative to the size of the gel pores are rendered nearly fixed. Average-sized molecules travel through the gel with different degrees of facility. Electrophoresis is done in a thin vertical slab of polyacrylamide. The course of protein movement is from top to bottom. Protein molecules can be isolated on the basis of their individual mass through use of electrophoresis in a polyacrylamide gel under protein denaturing process. The mixture of protein macromolecules is initially denatured in a homogeneous mixture of sodium dodecyl sulfate (SDS), which is an anionic emulsifier with the ability of disrupting almost all noncovalent interactions in native proteins macromolecules. SDS forms complexes with the denatured proteins which are then subjected to electrophoresis. After the process of electrophoresis, the proteins in the gel are imaged after silver or dye stains are applied. The product is visualized as series of bands. Tiny proteins molecules travel faster through the gel, while bigger molecules of proteins remain at the top where the samples are applied or put. Mobility of most polypeptide chains under these settings is linearly proportional to the logarithm of their mass. SDS polyacrylamide gel electrophoresis (SDS-PAGE) is very fast, sensitive, and has capacity of a high degree of resolution. As tiny as 0.1 µg which is equivalent to 2 pmol of a protein produces a discrete band when it is stained with Coomasie blue or less (~0.02 µg) can be determined with the use of a silver stain. Protein molecules which vary in mass by about 2% are commonly detected (Berg, et al., 2002). It is essential to detect minute quantities of a specific protein molecule in the presence of several other protein molecules, like for instance the presence of viral proteins in blood circulation. Very minute quantities of a protein molecule of interest concern in a cell or in the human blood can be determined by an immunoassay procedure which is called Western blotting. A sample is subjected to electrophoresis on an SDS-polyacrylamide gel. Blotting or more commonly called electroblotting moves the resolved protein molecules on the SDS-polyacrylamide gel to the surface of a polymer sheet in order to have the proteins more obtainable during the reaction. A specific antibody for the protein of concern is incorporated to the polymer sheet containing the resolved protein which then forms complexes with the specific antibody. The antibody-antigen complex on the polymer sheet can be determined via rinsing the sheet with the use of a second specific antibody for the first antibody. A radioactive label that is located on the second antibody causes an illumination of dark band on an x-ray film. An alternative procedure using the ELISA method is through the use of an enzyme that is also located on the second antibody which produces a colored product. The use of Western blotting has been a breakthrough in finding a specific protein molecule in a complex mixture of different proteins. Currently, it is not only used as the basis for testing infection of hepatitis C, wherein it is utilized to determine a core protein of the virus but the technique is also now very purposeful in genetic cloning (Berg, et al., 2002). The objective of this activity is to demonstrate stimulation of the epidermal growth factor receptor-signaling pathway by virtue of SDS-PAGE and Western blotting experimental methods. Through this researchers are able to understand the biomolecular level of how a specific signaling pathway resumes. Furthermore, specific proteins are identified by use of specific antibodies in the practical. Results The figure above shows the calibration graph of the given marker proteins in consideration with the logarithm of their molecular weights versus the distance traveled in the blot. This is utilized to determine the molecular weights of the EGF receptor as well as the MAP kinase. Figure 1 on appendix shows the digital image of the Western blotting technique. The presence of blots in between the predetermined proteins, the carbonic anhydrase and bovine serum albumin, implies where the unknown proteins have traveled which is in between 40 to 70 mm. From this data, it can be deduced that the molecular weights of the two unknown proteins, EGF receptor and MAP kinase range from 41-80 kDa. This is derived from the basic principle that a molecule with least molecular weight travels faster to the bottom of the gel. In contrast, relatively larger molecules stay almost completely at the top and migrate slowly. While molecules considered in between these sizes stay approximately halfway their distance. The figure above is the digital image of protein bands isolated by utilizing the technique SDS-PAGE. The size of this photograph had been reduced so that the distance traveled by each protein is only a representation of the original image. The visible bands in between the proteins with molecular weights between 81 and 40 kDa correspond to the weights and distance traveled by the unknown proteins. The unknown proteins have been revealed by the virtue of the antibodies that formed complexes with the unknown proteins. The procedure necessitated the application of primary antibodies which are mouse monoclonal anti-EGFR antibody, mouse monoclonal anti-phosphotyrosine, and rabbit polyclonal anti-active MAPK antibody which formed complexes to the “unknown” proteins of interest. Secondary antibodies which contain enzymes were then integrated, such as anti-mouse IgG-alkaline phosphatase conjugate and rabbit polyclonal anti-active MAPK antibody. From the basis that a specific antibody only forms complexes with a protein compatible to its receptor, it can be now be deduced that the proteins of interest are those protein peptides attacked by the given antibodies. Thus, the two proteins include epidermal growth factor receptor (EGFR) which was allowed to proceed to its phosphorylated form, called tyrosine phosphorylated EGFR, and the activated MAP kinase protein, an enzyme in which its activity was demonstrated to be inhibited by UO 126. Discussion Activation of the protein kinase cascade The major protein superfamily is composed of protein kinases discovered mainly from eukaryotic cells. Three subfamilies of protein kinases are identified: serine/threonine specific kinases, which comprise almost 99% of the total kinases; tyrosine specific kinases, no more than 0.1%; and kinases which is specific to tyrosine as well as with serine/threonine. The conventional protein-tyrosine kinases, other name for tyrosine specific kinase is composed of enzymes, such as Src and yes, which function to specifically phosphorylate tyrosine residues. The enzymes were initially discovered in retroviral oncoproteins (Eckhart, 1979; Hunter, 1980; Witte, 1980; Bishop, 1983) and have only been discovered in eukaryotic cells where they are distinguished to have a function growth & signal transduction (Ushiro & Cohen, 1980). Cellular signaling pathways require proteins or enzyme via phosphorylation on tyrosine residues. With this mechanism of cellular signaling, signals are produced due to interactions of protein involving series of phosphorylation, which is in contrast to other pathways that rather involve a second messenger for signal transduction (Broadbridge & Sharma, 2000). An example that utilizes this type of cell signaling mechanism are the growth factor receptors and G-protein coupled receptors which involve intrinsic tyrosine kinase activity (Fantl, et al., 1993). Growth factor receptors are the prototype of a family of receptors which contains an extracellular binding domain, a large intracellular catalytic domain and a single transmembrane portion (Fantl, et al., 1993). The main activity after the activation of growth factor is autophosphorylation within the intracellular domain along with dimerization of the receptor (Fantl, et al., 1993; Kazlauskas & Cooper, 1989). Autophosphorylation of the epidermal growth factor (EGF) receptor happens in the C-terminal end of tyrosine residues, thus termed as receptor tyrosine kinases. Meanwhile receptors containing kinase insert domain such as the platelet-derived growth factor (PDGF) receptors, the kinase insert domain is the main location for phosphorylation (Malarkey,et al., 1995). EGF together with insulin and IGF-I receptors have intrinsic protein kinase activities located in their cytoplasmic domains. This event is activated when the receptor binds ligand. The receptor is then autophosphorylated on the C-terminal of its tyrosine residues resulting to activation of a complex series of events. The phosphorylated EGF receptor subsequently phosphorylates EGF receptor substrates on their tyrosine residues after which would attach to the SH2 domains of various proteins directly associated in regulating several effects of EGF (Murray, et al., 2006). SHC is a ubiquitous protein containing one SH2 domain and a glycine/proline region but has no catalytic domain (Pelicci, et al., 1992). It is tyrosine phosphorylated by an activated EGF receptor (Pronk, 1994) and forms complex with other SH2 domains of other proteins. SHC may also function as a common tyrosine kinase substrate adaptor protein. Another recent signaling pathway identified in response to EGF involves activation of tyrosine kinase 2 (Tyk2) and Janus kinase (JAK) and phosphorylation of signal transducers and activators of transcription (STATs) (Malarkey,et al., 1995). The growth factor-receptor complex initiates attachment and stimulation of cytoplasmic protein kinases: Tyk 1, Jak 1 or Jak 2 which kinases phosphorylate cytoplasmic proteins and there after bind with other docking proteins via attachment to SH2 domains. Consequently, the cytosolic proteins called STATs are activated and phosphorylated. These then dimerize and translocate into the nucleus, form a complex with a particular DNA element and activate transcription. The phosphorylated tyrosine residues of the EGF receptor connect with various proteins containing SH2 domains activating the MAP kinase pathway, G-protein mediated activation of phospholipase C (PLC?) or PI-3 kinase.(Murray, et al., 2006). The Src homology 2 (SH2) domains The SH2 domain is a prototype protein complex first described as conserved, non-catalytic part in the Src family of cytoplasmic kinases (Sadowski, et al., 1986). Src homology 2 (SH2) domains are units of approximately 100 amino acid residues which are located in several intracellular signal-transduction proteins that function to bind phosphotyrosine-containing polypeptide sequences with high affinity and specificity (Koch, et al., 1991; Pawson & Gish, 1992; Couture, et al., 1996). SH2 domains are utilized by the body in signaling processes that function in cell growth and differentiation, protein ubiquitination and/or breakdown, gene transcription, and cytoskeletal rearrangement (Liu, et al., 2006; Seet, et al., 2006). One eukaryotic cell contains approximately 120 non-repeating SH2 domains located in 110 distinctive proteins such as kinases, phosphatases, structural proteins, regulators of small GTPases, or E3 ubiquitin ligases. Genetic mutations encoding several SH2 proteins are also implicated in hereditary illnesses (Liu, et al., 2006). Also, the SH2 domain is believed to be accountable for the recruitment and regulation of p56lck kinase activity. p56lck is a Src-like protein, lymphocyte-specific tyrosine kinase, which has an SH2 domain that plays a role in signaling pathways initiated upon T-lymphocytes through T-cell antigen receptor (Marth, et al., 1985; Sefton, 1991; Peri, et al., 1993; Chan, et al., 1994). They are also considered as possible factors for pharmacologic discovery (Broadbridge & Sharma, 2000). Every SH2 domains have similar structure containing a central anti-parallel ? sheet with two ? helices on its sides (Booker, et al., 1992; Overduin, et al., 1992; Waksman, et al., 1992; Eck, et al., 1993). However, few SH2 domains have arginine residue located at their ligand binding site necessary for tyrosine identification. In spite of their commonality, each sense specific protein sequences. In general, the specificity of SH2 domain is characterized by 3 to 5 residues at the C-terminal of the tyrosine (Huang, et al., 2008). Propagation of growth factor signal to the nucleus Signaling pathways have effected on the effects of several growth factors. Phosphorylation on tyrosine residues with resulting binding of growth factor which then interacts with several proteins that bind to phosphotyrosine consequently activating the binding proteins promoting a seriesof events regulated both by tyrosine and serine/threonine kinases. Eventually, proteins are modified to act in the nucleus affecting the process of transcription or DNA replicaton. Several nuclear regulatory factors are phosphorylated activating a series of events, which was stimulated by attachment of the growth factor to the cell surface. The signal is initiated when a growth factor attaches to its receptor. These receptors are structurally modified which are either monomeric that dimerize when they attach the ligand receptors as in EGF, or multimeric. There after the attachment of the ligand brings together two similar cytosolic domains of the receptor and stimulates an enzymatic activity, which is autophosphorylation of tyrosine kinase. In effect, the receptor modifies its characteristics by addition of phosphate to tyrosine. The phosphorylated proteins attach to phosphotyrosine binding proteins in the cell. These PBP have phosphotyrosine-binding (PTB) domains, and others have SH2 domains. Proteins that have phosphotyrosine-binding domains are associated with phosphotyrosine-containing proteins which have a second src-homology domain (SH3), which initiates further attachment to other proteins to swap GTP and GDP in which thay act as molecular bridges between receptor, and swapping protein (GRB-2), or Sos. Other discovery is that when cells were treated with mitogens like EGF, the cells develop a different kinase activity termed as mitogen-activated protein kinase (MAP kinase or called extracellular receptor-activated protein kinase/ERK). It was then documented that MAP kinase was utilized in the late process of the signaling pathway before nuclear regulatory factors were modified. MAP kinase is activated thorugh phosphorylation by a mitogen via an enzyme MEK. The activation of MEK is stimulated by Raf, which is stimulated by Ras and is stimulated by Sos. Thus MAP kinase is the one responsible for activating other proteins by phosphorylation (Reiness, 2000). Other experimental method: Northern blotting Northern blotting or Northern hybridization is an experimental method that aids in determination of particular RNA sequences. It was developed and termed by James Alwine and George Stark at Stanford University (http://www.protocol-online.org, 2011). Northern blot is the RNA counterpart of southern blot. Northern blotting is a relatively old experimental method but is still useful in detection and analysis of RNA due to the simplicity, specificity and flexibility of the procedures. RNA molecules are single stranded that can be structurally diverse which are stabilized by weak interactions. RNAs readily hybridizes with complementary nucleotide sequence. This characteristic is utilized in Northern blotting method. RNAs have significant function in cell metabolism. Monitoring the RNA types and major quantity in the cell at a designated period can be useful in determining the events happening within the cell. Northern blotting technique is the principal procedure utilized in determining and quantitating the types and levels of RNA in the cell. The procedure of Northern blot technique is initiated by extraction of RNA molecules. Usually, RNA molecule is extracted by lysing and homogenizing the sample tissue with a commercial reagent trizol. A series of purification techniques are employed until then it is subjected to electrophoresis to allow separation of the RNA molecules according to their sizes. Generally, smaller molecules travel at a faster rate through the gel. Through this procedure, it is possible to know how many types of RNA is present together with their approximated size. The sample is then ready for blotting wherein it involves the transferring of separated RNA molecules from the agarose gel to a solid matrix. The blotting techniques commonly used are capillary transfer and electro blotting. Capillary transfer makes use of a passive transfer of the RNA unto matrixes like filter paper, nitrocellulose filter or nylon membrane under the influence of capillary force. Electro blotting on the other hand utilizes electric current to transfer RNA molecules to a solid matrix, which is usually a filter paper. Then hybridization and determination of RNA molecules is performed, which is based on the principle that two single strands of DNA form base pair with each other, resulting to similar nucleotide sequences that are complementary with each other. After blotting the membrane with fixed RNA molecules are hybridized with the labeled probe. Bands are then made on x-ray films. The principal advantage of Northern blot over other experimental techniques is its simplicity and flexibility. It is usually employed in characterization of mRNA expression because it aids visualization of intact mRNA. It has also been used in comparison of abundance of a particular gene that is expressed in cells and in the study of proteins in the development and suppression of cancerous growth. Likewise, the technique is used in alternative splicing of various gene products and is utilized in researches about abnormal gene and hereditary illnesses (Umeoguaju, 2009). Appendix Table 1. Pre-stained molecular weight markers Figure 1. Western blotting digital image Figure 2. SDS-PAGE digital image References Berg, J., Tymoczko, J., & Stryer, L. 2002. Biochemistry 5th ed. W.H. Freeman and Co. Bishop, J.M. 1983. Ann. Rev. Biochem.; 52, 301-354. Booker, G. W., Breeze, A. L., Downing, A. K., Panayotou, G., Gout, I., Waterfield, M. D., and Campbell, I. D. (1992) Structure of an SH2 domain of the p85 _ subunit of phosphatidylinositol-3-OH kinase. Nature 358, 684–687 Broadbridge, R. & Sharma, R. 2000. The Src Homology-2 Domains (SH2 Domains) of the Protein Tyrosine Kinase p56lck: Structure, Mechanism and Drug Design. Current Drug Targets. Vol. 1, No. 4 Bentham Science Publishers Ltd. Chan, A. C.; Desai, D. M. and Weiss, A. (1994) Ann. Rev. Immunol.; 12, 555- 592; b, Penninger, J. 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