Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates

Transcription

1 McNair Scholars Journal Volume 9 Issue 1 Article Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates Derrick Kroodsma Grand Valley State University Follow this and additional works at: Recommended Citation Kroodsma, Derrick (2005) "Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates," McNair Scholars Journal: Vol. 9: Iss. 1, Article 11. Available at: Copyright 2005 by the authors. McNair Scholars Journal is reproduced electronically by ScholarWorks@GVSU. mcnair?utm_source=scholarworks.gvsu.edu%2fmcnair%2fvol9%2fiss1%2f11&utm_medium=pdf&utm_campaign=pdfcoverpages

2 Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates Derrick Kroodsma McNair Scholar Laurie A. Witucki, Ph.D. Faculty Mentor Abstract This research study examines the synthesis of tyrosine containing peptides based on the protein paxillin. Paxillin is a proposed substrate of the Focal Adhesion Kinase (FAK) protein tyrosine kinase. FAK is overexpressed in tumor cells; therefore, its abnormal function is associated with several cancer types. The peptides designed here are based on the substrate sequence FAK preferably binds to and phosphorylates. Solid Phase Peptide Synthesis (SPPS) provided substrate compounds which were then tested via biological assays with FAK. The synthesis of large peptides provides an accurate method to determine kinase amino acid phosphorylation preference to confirm paxillin as a FAK substrate. Introduction Cancer is the second leading cause of death in the United States. About 570,280 Americans are expected to die of cancer in 2005, more than 1,500 people per day. 1 There are numerous factors which contribute to the oncogenesis of cells and a better understanding of these variables may one day lead to improved cancer treatments. One such factor is abnormal, over expression of certain types of enzymes called protein tyrosine kinases (PTK). A kinase may be thought of as a chemical switch since it chemically alters its substrates by covalently attaching a phosphate (PO3-2 ) group to them. Once phosphorylated, these substrate enzymes may be turned on or turned off. A specific kinase is FAK 2 (See Figure 1.) FAK, located in the mammalian cell membrane, has been associated with several cancer types, including lung and breast cancer 3-4 (See Figure 2.) A proposed substrate for FAK is paxillin. A substrate can be thought of as a key which fits a kinase lock. The protein paxillin is a ligand that binds to membrane bound receptors that trigger cellular transduction pathways. FAK functions in regulation of cell adhesion and that trigger cellular transduction pathways. FAK functions in regulation of cell adhesion and migration by phosphorylating a tyrosine residue () in paxillin (See Figure 3.) Within the amino acid sequence of paxillin, there are many possible tyrosines, which can be phosphorylated by FAK (See Figure 4.) The aim of this project is to focus on the tyrosine in paxillin and synthesize short versions of the paxillin protein around this targeted residue. While proteins are larger (longer) chains of amino acids, peptides are short chains of amino acids (See Figure 5.) The peptides for this research project include a specific Abbreviations: FAK, focal adhesion kinase; PTK, protein tyrosine kinase, ATP, adenosine triphosphate; SPPS, solid phase peptide synthesis; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, hydroxybenzotriazole hydride; HBTU, 2-(1 H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; TFA, trifluroacetic acid; TIS, triisopropylsaline GVSU McNair Scholars Journal VOLUME 9,

3 tyrosine from the paxillin sequence, 118 (Residue number 118). This was chosen based on previous Western blots using anti-paxillin antibody indicating that phosphorylation may occur at this tyrosine location. 5-7 This study synthesized, via SPPS, tyrosine containing paxillin peptides. 8 A general tyrosine peptide sequence is: H 2 N-aa 7 -aa 6 -aa 5 --aa 3 -aa 2 -aa 1 -COOH. H 2 N = N-terminal COOH = C-terminal Testing a synthesized peptide with the kinase FAK will provide a more accurate quantitative determination of the extent of phosphorylation instead of the qualitative Western blot analysis. This study will also investigate varying lengths of peptides surrounding tyrosine 118; the range of sizes is from amino acids in length. Testing with FAK will determine if it is possible to have 12 amino acid peptides act as a substrate for this kinase. Methods Solid Phase Peptide Synthesis (SPPS) The peptide synthesis was automated with an Advanced Chemtech peptide synthesizer. Table 1 shows the target peptides for this study. Polylysine (K) tails were attached to the C-terminal end of each peptide for adhesion to the negatively charged phosphocellulose paper disks used during assaying. 9 The γ-phosphate is transferred to the tyrosine residue from ATP to produce the phosphorylated tyrosine P, and ADP. This is measured quantitatively with γ-p 32 ATP. The C- terminal of each peptide is bonded to a 90µm diameter polymer resin bead called Wang ( mesh). 10 Each amino acid attached to the peptide initially contains an N-α-Fmoc protection group and amino acids may also contain side chain protecting groups (See Figure 6 for chemical structures of reagents) and (See Figure 7 for steps of SPPS.) To ensure successive amino acid coupling before Fmoc deprotection, a three-fold excess of each amino acid was used. The Fmoc group was deprotected or removed with piperidine before each coupling step. Activation of the amino acid s carboxy group was done with HOBt and HBTU. A ninhydrin test was performed to confirm complete lysine coupling to the resin. 13 Each peptide used 0.25 g of the polylysine resin. All of the other amino acids were attached as described above by the automated Advanced Chemtech peptide synthesizer. The continuous, computerized process handles larger peptides without compromising either purity or the product yield. Upon completion, the Wang resin and any amino acid side chain protecting groups were cleaved and deprotected by TFA and scavengers Once cleaved, the solid peptides were obtained, rinsed, and dried. The amounts and percent yield are shown in Table 2. Table 1. Description of Target Peptides Target Peptide Sequence Molecular Weight Length Peptide #1 E 114 EHV 118 SFPN 122 KKK MW: g/mol 12-mer Peptide #2 V 111 GEEEHV 118 SFPNKQK 125 KK MW: g/mol 17-mer Peptide #3 G 105 SPCSRVGEEEHV 118 SFPNKQKSAEPSP 131 KKK MW: g/mol 30-mer Results Table 2. Analysis of Peptides Target Peptide Grams Obtained Percent ield Peptide # % Peptide # % Peptide # % 94 Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates

4 Discussion Three peptides were successfully synthesized by SPPS on an automated peptide synthesizer. The peptides are in 3 successively increasing lengths that are identical to the structure in paxillin around 118. The purpose is to determine, through biological assaying, which peptide best represents an actual substitution for paxillin. If the rates of phosphorylation on 118 are indistinguishable between the 12-mer and 30-mer peptides, then it is reasonable to synthesize smaller peptides for future research. Future research using these peptides will be a confirmation of the peptide structure by Edman sequencing and mass spectrometry. Biological assaying against FAK, using γ-atp 32, will quantify the phosphorylation rate on 118. The numerical results would help determine if 118 is the tyrosine FAK preferably binds to within paxillin. Knowledge gained through research such as this would assist the pharmaceutical industry by providing a tool that could be used to screen thousands of possible drug molecules. Although these peptides are neither drugs nor treatments, therapeutic agents could be developed for FAK-related cancers. GVSU McNair Scholars Journal VOLUME 9,

5 References 1 American Cancer Society. Cancer facts and figures; Atlanta, Georgia, Hardie G.; Hanks, S. The protein kinase facts book: Protein-tyrosine kinases; Academic Press Inc.: San Diego, Mukhopadhyay N.K.; Gordon G.J.; Chen C.J.; Bueno R.; Sugarbaker D.J.; Jaklitsch M.T. Activation of focal adhesion kinase in human lung cancer cells involves multiple and potentially parallel signaling events. J. Cell. Mol. Med. 2005, 2, Schmitz K.J.; Grabellus F.; Callies R.; Otterbach F.; Wohlschlaeger J.; Levkau B.; Kimmig R.; Schmid K.W.; Baba H.A. High expression of focal adhesion kinase (p125fak) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome. Breast Cancer Res. 2005, 2, Bellis S.L.; Miller J.T.; Turner C.E. Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase. J. Biol. Chem. 1995, 29, Iwasaki T.; Nakata A.; Mukai M.; Shinkai K.; ano H.; Sabe H.; Schaefer E.; Tatsuta M.; Tsujimura T.; Terada N.; Kakishita E.; Akedo H. Involvement of Phosphorylation of TR-31 and TR-118 Of Paxillin in MM1 Cancer Cell Migration. Int. H. Cancer 2002, 97, Romanova L..; Hashimoto S.; Chay K.; Blagosklonny M.V.; Sabe H.; Mushinski J.F. Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive J. Cell Sci. 2004, 117, Merrifield R.B. Solid phase peptide synthesis: The synthesis of a tetra-peptide J. Am. Chem. Soc. 1963, 85, Rininsland F.; Stankewicz C.; Weatherford W.; McBranch D. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching. BMC Biotechnol. 2005, Wang S. p-alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments. J. Am. Chem. Soc., 1973, 4, Bodanszky M.; Deshmane S.S.; Martinez J. Side reactions in peptide synthesis: Possible removal of the 9-fluorenylmethoxycarbonyl group by the amino acid components during coupling. J. Org. Chem. 1979, 10, Carpino L.A.; Han G.. 9-fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group J. Org. Chem. 1972, 22, Ruhemann S. Triketohydrindene Hydrate Trans. Chem. Soc. 1910, 1438, King D.S.; Fields C.G.; Fields G.B. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int. Peptide Protein Res. 1990, 36, Chang C.; Waki M.; Ahmad M.; Meienhofer J.; Lundell E.O.; Haug J.D. Preparation and properties of Nalpha-9-fluorenylmethyloxycarbonylamino acids bearing tert.-butyl side chain protection. Int. J. Peptide Protein Res. 1980, 15, Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates

6 Figure 1. A 3-dimensional crystal structure of FAK in ribbon structure. The peptide substrate is absent but ATP is shown in the center in ball & stick notation. GVSU McNair Scholars Journal VOLUME 9,

7 Figure 2. Location of FAK within a cell and a cellular process it participates in. Signal Trans- Membrane Receptor Intracellular Target Nucleus Receptor Protein Tyrosine Kinase Pathways GF GF Growth Factor or Hormone PI-3 Kinase PLC-γ Receptor PTK SHC SRC Grb-2 SOS Ras Plasma Membrane MEK FAK PKC Raf MAPK Nuclear Membrane Paxillin? RSK Transcriptional Regulation Differentiation Differentiation/Growth 98 Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates

8 Figure 3. Cartoon representation of a tyrosine kinase phosphorylation reaction. Figure 4. This full amino acid sequence of the paxillin protein was used to derive the synthesized peptides. The 30 amino acids underlined above correspond to peptide #3 in Table 1. GVSU McNair Scholars Journal VOLUME 9,

9 Figure 5. A general amino acid structure is shown on the left and a dipeptide structure is shown on the right. Peptides are chains of repeating units (amino acids) connected together by amide bonds (circled). Figure 6. Chemical structures of reagents used during peptide synthesis via SPPS. 100 Solid Phase Peptide Synthesis: Analysis and Identification of Protein Kinase Substrates

10 Figure 7. Chemical structures of reagents used during peptide synthesis via SPPS. GVSU McNair Scholars Journal VOLUME 9,