General Information

This is the HTML version of the paper. Click here to see a PDF file of the paper as it was published in 2007.

To view this article at the publisher's website, click here.

Publication Information

Protein Science
Vol. 16, pp. 550-556, March 2007

Semisynthesis of unnatural amino acid mutants of paxillin

Abstract

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

Caged phosphopeptides and phosphoproteins are valuable tools for dissecting the dynamic role of phosphorylation in complex signaling networks with temporal and spatial control. Demonstrating the broad scope of phosphoamino acid caging for studying signaling events, we report here the semisynthesis of a photolabile precursor to the cellular migration protein paxillin, which is a complex, multidomain phosphoprotein. This semisynthetic construct provides a powerful probe for investigating the influence that phosphorylation of paxillin at a single site has on cellular migration. The 61-kDa paxillin construct was assembled using native chemical ligation to install a caged phosphotyrosine residue at position 31 of the 557-residue protein, and the probe includes all other binding and localization determinants in the paxillin macromolecule, which are essential for creating a native environment to investigate phosphorylation. Following semisynthesis, paxillin variants were characterized through detailed biochemical analyses and by quantitative uncaging studies.

Keywords: paxillin; native chemical ligation; caging; phosphoprotein; phosphorylation; semisynthesis

Introduction

The control of cellular adhesion and migration is essential for the regulation of biological processes, including embryogenesis, wound repair, and metastasis. Paxillin is a multidomain protein that orchestrates a pivotal role in these processes by acting as a dynamic scaffold for signaling and structural proteins. The phosphorylation of paxillin at specific residues spanning the macromolecule creates distinct protein-binding sites and thereby directs paxillin localization to focal adhesions, sites of cell contact with the extracellular matrix, and influences the controlled assembly and dissolution of signaling cascades . For investigating proceses such as cell migration, there is a need for tools that enable researchers to dissect the dynamic roles of protein phosphorylation within complex signaling networks.

The essentiality of specific protein phosphorylation events can be assessed by a number of approaches, including gene knockout, RNA interference, and site-directed mutagenesis. One limitation with these strategies is that they cannot afford information on phosphorylation in "real time." As a complement to these approaches, the synthesis of caged phosphopeptides, which enable the controlled release of specific phosphorylated species upon photolysis, was introduced. Recently, a general method for the synthesis of these probes has facilitated the application of caged phosphopeptides in cellular studies . Expanding on this work, and as part of an initiative to develop generalizable approaches for the preparation of full-length caged phosphoproteins, we report the semisynthesis and biochemical characterization of a mutant of the 61-kDa protein paxillin that includes a caged phosphotyrosine (cpTyr) residue at position 31 within the N terminus. We similarly report related paxillin variants with a Tyr or phosphotyrosine (pTyr) at residue 31, which were constructed through a parallel approach to furnish the nonphosphorylated and discretely phosphorylated species as biological controls.

Following semisynthesis, the paxillin variants were characterized by in vitro binding to a selection of cognate proteins, by phosphorylation using partner kinases and probed with phosphoprotein-specific antibodies, and by quantitative uncaging studies. The semisynthetic caged phosphoTyr31 paxillin permits the time-sensitive investigation of a single phosphorylation event and will serve as a valuable tool to probe the role of Tyr31-paxillin phosphorylation in cellular migration.

Results and Discussions

For the C-terminal fragment of paxillin, residues 37-557 were expressed as a GST-fusion construct with a FLAG tag (DYKDDDDK) included at the C terminus to provide a second handle for purification. A TEV protease cleavage sequence, ENLYFQC, was incorporated immediately preceding the paxillin insert. TEV protease is a highly selective cysteine protease that typically recognizes a serine or glycine in the P1' site, but also will accept a cysteine residue at that position. Therefore, treatment of the GST-paxillin(38-557)-FLAG protein (2) with TEV protease concurrently removed the GST tag and revealed an N-terminal cysteine residue to afford Cys37-paxillin(38-557)-FLAG (3) for subsequent ligation. Paxillin is known to be a challenging protein to express in Escherichia coli, in part because of a significant number of rare codons, including 27 rare CCC (Pro) codons. Therefore, to access sufficient quantities of protein and overcome expression and truncation difficulties, the protein was fermented in 10-L batches using codon-enhanced cells and purified via both the N-terminal GST and C-terminal FLAG tags. The FLAG tag was essential for separating any truncation products from the full-length protein.

The ligations between 1a, 1b, or 1c and 3 were conducted in nondenaturing conditions to access Y31Pax (4a), pY31Pax (4b), and cpY31Pax (4c) in multimilligram quantities. Exchange of the semisynthetic proteins into PBS using 50-kDa MWCO dialysis membrane concurrently removed unreacted peptide thioester (MWt 4.8-5.1 kDa).

In vitro characterization of semisynthetic paxillin
Since paxillin is a molecular adaptor protein with no known enzymatic activity, the function of the reconstituted paxillin was validated in vitro by analyzing binding to known paxillin-binding partners and by assessing activity with kinases that are documented to phosphorylate paxillin. The paxillin-binding partners focal adhesion kinase (FAK), GRK interactor 1 (GIT1), and PTP-PEST were selected to probe binding along the entire length of the protein, while the phosphorylation studies were focused on the NCL-introduced N terminus, which includes the Tyr31 site.

Phosphorylation by upstream kinases
Next, to demonstrate that the semisynthetic paxillin analog functions as a substrate for selected kinases that natively phosphorylate the protein, phosphorylation of 4a was attempted with Src, ERK, and JNK and probed with phosphorylation-specific antibodies to four sites along paxillin. Src is a tyrosine kinase that is known to phosphorylate paxillin at Tyr31 and Tyr118, while ERK and JNK are serine/threonine kinases that phosphorylate residues Ser126 and Ser178, respectively. In the phosphorylation assays with semisynthetic 4a, residue Tyr31, a site introduced by ligation, and residue Tyr118 were phosphorylated exclusively by Src; residue Ser126 was phosphorylated only by ERK; and residue Ser178 was phosphorylated only by JNK. The general anti-paxillin antibody recognized protein in all reactions. Importantly, detection of Src-treated 4a by the pTyr31 phosphorylation-specific antibody validated both the successful ligation to install the Tyr31 site and the recognition of that site by an upstream kinase. As a further control, a full-length paxillin construct (GST-paxillin[1-557]-FLAG) was expressed, purified, and subjected to the kinase treatment, and the expressed paxillin responded identically to the semisynthetic version (data not shown). The binding and phosphorylation experiments confirm that the native interactions of the semisynthetic reconstituted control 4a, including those of the phosphorylation site of interest, Tyr31, were not compromised by the expression and ligation procedures.

Uncaging of cpY31Pax (4c)
The extent of cpY31Pax (4c) uncaging was quantified following irradiation with long-wavelength UV light centered at 365 nm. Importantly for cellular applications, the photo-byproduct of NPE-caged species, o-nitrosoacetophenone, has previously been shown to be cellularly inert. This byproduct is less reactive than the analogous o-nitrosobenzaldehyde produced by irradiation of widely used ortho-nitrobenzyl-derived caging groups. For uncaging, 4c was irradiated for 90 sec on a standard DNA transilluminator. The amount of phosphorylated paxillin (pY31Pax) present at t = 0 and t = 90 sec post-photolysis was quantified by chemiluminescent detection of the phosphorylated protein using a paxillin [pY31] phosphorylation-specific antibody. The total amount of protein loaded per blot was detected using a general anti-paxillin antibody. In addition, semisynthetic pY31 (4b) was used as a photochemically inert internal standard in the phosphorylation-specific and general paxillin Western blots. The relative intensity of each sample was determined as a fraction of total pixel intensity compared to the internal pY31Pax (4b) standard. The ratio of uncaged phosphopaxillin to the total protein amount indicated 83%-91% conversion of 4c to phosphoTyr31-paxillin after irradiation. A minimal amount of uncaged phosphoprotein, typically <6% of the total protein, was detected in the absence of intentional uncaging.

To date, caged phosphorylated amino acids have been systematically incorporated into increasingly complex targets, including peptides and a protein domain. Herein, we have described the culmination of this progress with the assembly of a full-length, eukaryotic protein target. Ongoing cellular experiments using microinjected cpY31Pax (4c) should yield important information on the effect of Tyr31 phosphorylation on cellular migration.

Materials and Methods

Additional methods are available in Supplemental Methods online.

Thioester synthesis
Peptides were synthesized manually and on an automated (Advanced ChemTech 396) peptide synthesizer using standard (fluorenylmethoxy)carbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocols on preloaded Fmoc-Gly-Novasyn TGT resin (Novabiochem). Phosphotyrosine was introduced as Fmoc-Tyr(PO(OBzl)OH)-OH, and caged phosphotyrosine was introduced as Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-tyrosine. For acetylation of the amino terminus of each peptide, 120 μmol of peptide on resin was treated with acetic anhydride (113 μL, 1.2 mmol) and pyridine (97 μL, 1.2 mmol) in 10 mL of DMF for 30 min. The peptides were individually cleaved from the resin with side-chain protection intact by agitating with 0.5% TFA in DCM for 1.5 h. The resin was removed by filtration and rinsed with DCM, the solvent was mostly evaporated under a stream of nitrogen, and the peptide was triturated with cold hexanes. The hexanes were removed in vacuo, and the resulting white powder was dissolved in anhydrous THF (50 mL) and treated with HBTU (180 mg, 480 μmol), DIPEA (165 μL, 960 μmol), and benzylmercaptan (54 μL, 480 μmol) under nitrogen overnight. The THF was removed in vacuo and the peptide was deprotected with 95% (vol/vol) TFA, 2.5% (vol/vol) TIS, and 2.5% (vol/vol) water for 2 h. Peptides were triturated with cold diethylether and purified by reverse-phase HPLC on a Waters 600 instrument with a YMC C₁₈ preparative column using an elution gradient of water/acetonitrile with 0.1% TFA. The identities of the peptides as free acids and of the final peptide thioester products were confirmed by electrospray ionization (ESI) mass spectrometry on a Perspective Biosystems Mariner Biospectrometry Workstation (turbo ion source).

Peptide characterization

Ac-HHHHHHDDLDALLADLESTTSHISKRPVFLSEETP-Y-SYPTG-COOH, Reverse-phase HPLC (t_R = 25.4 min). Exact mass calculated for C₂₀₉H₃₀₇N₅₉O₆₈, 4731.2; found by MS(ESI), 947.8 [MH₅]⁵⁺, 790.0 [MH₆]⁶⁺.

Ac-HHHHHHDDLDALLADLESTTSHISKRPVFLSEETP-Y-SYPTG-COSBn (1a), Reverse-phase HPLC (t_R = 25.7 min). Exact mass calculated for C₂₁₆H₃₁₃N₅₉O₆₇S, 4837.3; found by MS(ESI), 968.7 [MH₅]⁵⁺.

Ac-HHHHHHDDLDALLADLESTTSHISKRPVFLSEETP-pY-SYPTG-COOH, Reverse-phase HPLC (t_R = 25.4 min). Exact mass calculated for C₂₀₉H₃₀₈N₅₉O₇₁P, 4811.2; found by MS(ESI), 1204.6 [MH₄]⁴⁺, 963.9 [MH₅]⁵⁺.

Ac-HHHHHHDDLDALLADLESTTSHISKRPVFLSEETP-pY-SYPTG-COSBn (1b), Reverse-phase HPLC (t_R = 25.6 min). Exact mass calculated for C₂₁₆H₃₁₄N₅₉O₇₀PS, 4917.2; found by MS(ESI), 1231.9 [MH₄]⁴⁺, 985.7 [MH₅]⁵⁺.

Ac-HHHHHHDDLDALLADLESTTSHISKRPVFLSEETP-cpY-SYPTG-COOH, Reverse-phase HPLC (t_R = 25.4 min). Exact mass calculated for C₂₁₇H₃₁₅N₆₀O₇₃P, 4960.3; found by MS(ESI), 993.1 [MH₅]⁵⁺.

Ac-HHHHHHDDLDALLADLESTTSHISKRPVFLSEETP-cpY-SYPTG-COSBn (1c), Reverse-phase HPLC (t_R = 25.7 min). Exact mass calculated for C₂₂₄H₃₂₁N₆₀O₇₂PS, 5066.3; found by MS(ESI), 1014.9 [MH₅]⁵⁺, 845.9 [MH₆]⁶⁺.

Plasmid construction for GST-paxillin(38-557)-FLAG
The gene fragment encoding residues 38-557 of paxillin (isoform ) was amplified from a paxillin plasmid (supplied by Martin Schwartz) with primers to insert 5'-EcoRI and 3'-NotI restriction sites. The primers also encoded an N-terminal TEV protease recognition site (ENLYFQC) and a C-terminal FLAG tag. For this amplification the following PCR primers were used: 5'-GCCGGAATTCGTGAAAACCTGTATTTTCAGTGCCACACATACCAGGAGATT-3' and 5'-GCCCCCTTTTGCGGCCGCCTACTTATCGTCA...
^....TCGTCTTTGTAGTCGCAGAAGAGCTTGAGGAA-3'. The PCR-amplified fragments were digested with NotI and EcoRI and ligated to NotI/EcoRI-digested and CIP-treated pGEX-4T-2 (GE Health Sciences). The ligation mixture was transformed into DH5 cells and grown on carbenicillin-resistant plates. Plasmid DNA was isolated from selected colonies and confirmed by sequencing.

GST-paxillin(38-557)-FLAG expression and purification
The paxillin plasmid was transformed into BL21-CodonPlus-RP competent cells (Stratagene) and grown with fermentation at 37°C to midlog phase in 10 L of LB media with carbenicillin and chloramphenicol. The culture was cooled to 16°C, and the cells were induced with 0.1 mM IPTG and fermented for 16 h. Cells were harvested by centrifugation and frozen at -80°C. For cell lysis, pellets were thawed and resuspended in 350 mL of lysis buffer (PBS, 1 mM ZnCl₂, 1 mg/mL lysozyme, 1 mM DTT, and Calbiochem protease cocktail III [100 μM AEBSF, 80 nM aprotinin, 5 μM bestatin, 1.5 μM E-64, 2 μM leupeptin, 1 μM pepstatin A]) and incubated for 20 min at 4°C. The cells were lysed with 1% NP-40 Alternative, then sonicated and subjected to centrifugation at 13, 000 rpm for 30 min, and at 35, 000 rpm for 30 min. The soluble fraction was purified using 8 mL of Glutathione Sepharose 4 Fast Flow resin following the manufacturer's protocol. Protein was dialyzed into TBS and then purified via the carboxy-terminal tag with 3 mL of anti-FLAG M2 affinity resin (Sigma). Typical yields for the doubly purified protein were 4-6 mg per 10 L fermentation, as quantified using a Biorad protein assay. The purified protein was stored at 4°C and used for all in vitro and cellular studies within 2 wk of lysis and purification.

TEV protease cleavage
The purified protein 2 was diluted to 0.5 or 1 mg/mL into a TEV cleavage buffer with a final concentration of 50 mM Tris pH 8.0, 500 μM EDTA, and 5 mM BME. TEV protease (US Biological) was added (35 μL of protease per mg of target protein), and the resulting solution was incubated at 28°C for 3 h. The protein was dialyzed into TBS (to remove glycine present from the FLAG-affinity elution) and incubated with Ni/NTA resin and glutathione sepharose beads to remove the hexahistidine-tagged TEV protease and the cleaved GST tag. The protein solution was concentrated using 50-kDa MWCO centrifugal filters (Millipore) and used immediately in NCL.

Ligations
In general, reactions were carried out with 50 μM protein, 0.8 mM peptide, and 100 mM MESNA in TBS at pH 8.0. Accordingly, to a solution of Cys-Pax(38-557)-FLAG (3) (600 μg, 10.7 nmol) in TBS (150 μL) was added Ac-HHHHHH-DDLDALLADLESTTSHISKRPVFLSEETP-X-SYPTG-COSBn (lyophilized, then dissolved into 20 μL of water for transfer; 800 μg, 169 nmol for X = Tyr [1a], 163 nmol for X = pTyr [1b], and 158 nmol for X = cpTyr [1c]), 10 μL of 2 M MESNA, and 20 μL of 500 mM Tris pH 8.0. The reaction was incubated for 16 h at 25°C, and then dialyzed into PBS using a 50-kDa MWCO dialysis membrane to remove excess (4.8-5.1 kDa) peptide. Protein was either used directly for assays without a final purification or purified using a Ni/NTA spin column to isolate ligation product via the ligation-introduced N-terminal hexahistidine tag. The protein was analyzed by 10% SDS-PAGE gels and visualized with Coomassie blue dye, and by Western blot with a mouse anti-hexahistidine primary antibody. For ligations using 1b, a mouse anti-pY31 antibody was also used for visualization.

Sample uncaging calculation for cpY31Pax
For uncaging, a 1.2 mg/mL solution of cpY31Pax (4c) in TBS with 2.5 mM DTT was irradiated for 90 sec in a glass cell (pathlength 1 mm) with light centered at 365 nm with an intensity of 7330 μW/cm² on a DNA transilluminator. Semisynthetic pY31Pax (4b) and equal amounts of caged and uncaged protein were run on 10% SDS gels and transferred to nitrocellulose. Western blots were developed with mouse (monoclonal) anti-human paxillin and phosphospecific rabbit (polyclonal) anti-paxillin[pY31] primary antibodies and visualized by chemiluminescence. Pixel counting was used to calculate the relative intensity, compared to a photochemically inert standard (4b), of phosphoprotein at t = 0 and t = 90 sec after uncaging of cpY31Pax (4c). A sample calculation is shown in Table 1 with data from the Western blot shown in.

Footnotes

Reprint requests to: Barbara Imperiali, Department of Chemistry, 77 Massachusetts Ave., 18-590, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; e-mail: imper@mit.edu; fax: (617) 452-2419.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062549407.

Supplemental material: see www.proteinscience.org

Acknowledgements

This research was supported by an NIH Glue grant (GM64346 Cell Migration Consortium). Support for E.M.V. was provided by the Merck MIT CSBI fellowship and by the Charles Krakauer Graduate Fellowship. We thank Professors Rick Horwitz (Univ. of Virginia), Martin Schwartz (Univ. of Virginia), Tom Parsons (Univ. of Virginia), and Mike Schaller (Univ. of North Carolina, Chapel Hill) for the generous donation of GIT, paxillin, FAK, and PTP-PEST constructs, respectively, and Dr. Erik Schaefer (Invitrogen Corp.) for the donation of paxillin antibodies.

References

Brown, M.C. and Turner, C.E. 2004. Paxillin: Adapting to change. Physiol. Rev. 84: 1315-1339.

Dawson, P.E., Muir, T.W., Clark-Lewis, I., and Kent, S.B.H. 1994. Synthesis of proteins by native chemical ligation. Science 266: 776-779.

Futaki, S., Sogawa, K., Maruyama, J., Asahara, T., and Niwa, M. 1997. Preparation of peptide thioesters using Fmoc-solid-phase peptide synthesis and its application to the construction of a template-assembled synthetic protein (TASP). Tetrahedron Lett. 38: 6237-6240.

Hackeng, T.M., Griffin, J.H., and Dawson, P.E. 1999. Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology. Proc. Natl. Acad. Sci. 96: 10068-10073.

Hahn, M.E. and Muir, T.W. 2004. Bioorganic chemistry: Photocontrol of Smad2, a multiphosphorylated cell-signaling protein, through caging of activating phosphoserines. Angew. Chem. Int. Ed. Engl. 43: 5800-5803.

Hildebrand, J.D., Schaller, M.D., and Parsons, J.T. 1995. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol. Biol. Cell 6: 637-647.

Huang, C., Rajfur, Z., Borchers, C., Schaller, M.D., and Jacobson, K. 2003. JNK phosphorylates paxillin and regulates cell migration. Nature 424: 219-223.

Humphrey, D., Rajfur, Z., Vazquez, M.E., Scheswohl, D., Schaller, M.D., Jacobson, K., and Imperiali, B. 2005. In situ photoactivation of a caged phosphotyrosine peptide derived from focal adhesion kinase temporarily halts lamellar extension of single migrating tumor cells. J. Biol. Chem. 280: 22091-22101.

Kaplan, J.H., Forbush 3rd, B., and Hoffman, J.F. 1978. Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: Utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17: 1929-1935.

Lauffenburger, D.A. and Horwitz, A.F. 1996. Cell migration: A physically integrated molecular process. Cell 84: 359-369.

Lu, W., Shen, K., and Cole, P.A. 2003. Chemical dissection of the effects of tyrosine phosphorylation of SHP-2. Biochemistry 42: 5461-5468.

Manabe, R.I., Kovalenko, M., Webb, D.J., and Horwitz, A.R. 2002. GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration. J. Cell Sci. 115: 1497-1510.

Mezo, A.R., Cheng, R.P., and Imperiali, B. 2001. Oligomerization of uniquely folded mini-protein motifs: Development of a homotrimeric peptide. J. Am. Chem. Soc. 123: 3885-3891.

Muir, T.W.. 2003. Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 72: 249-289.

Nguyen, A., Rothman, D.M., Stehn, J., Imperiali, B., and Yaffe, M.B. 2004. Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function. Nat. Biotechnol. 22: 993-1000.

Rothman, D.M., Vazquez, M.E., Vogel, E.M., and Imperiali, B. 2002. General method for the synthesis of caged phosphopeptides: Tools for the exploration of signal transduction pathways. Org. Lett. 4: 2865-2868.

Rothman, D.M., Vazquez, M.E., Vogel, E.M., and Imperiali, B. 2003. Caged phospho-amino acid building blocks for solid-phase peptide synthesis. J. Org. Chem. 68: 6795-6798.

Schwarzer, D. and Cole, P.A. 2005. Protein semisynthesis and expressed protein ligation: Chasing a protein's tail. Curr. Opin. Chem. Biol. 9: 561-569.

Shen, Y., Schneider, G., Cloutier, J.F., Veillette, A., and Schaller, M.D. 1998. Direct association of protein-tyrosine phosphatase PTP-PEST with paxillin. J. Biol. Chem. 273: 6474-6481.

Tolbert, T.J. and Wong, C.H. 2002. New methods for proteomic research: Preparation of proteins with N-terminal cysteines for labeling and conjugation. Angew. Chem. Int. Ed. Engl. 41: 2171-2174.

Turner, C.E.. 2000. Paxillin and focal adhesion signalling. Nat. Cell Biol. 2: E231-E236.

Zheng, W., Schwarzer, D., LeBeau, A., Weller, J.L., Klein, D.C., and Cole, P.A. 2005. Cellular stability of serotonin N-acetyltransferase conferred by phosphonodifluoromethylene alanine (Pfa) substitution for Ser-205. J. Biol. Chem. 280: 10462-10467.