The authors acknowledge Dr. Jordan Fauser for her contribution to the development of RapR-Shp2 and associated protocols. The work was supported by a 5R35GM145318 award from NIGMS, an R33CA258012 award from NCI, and a P01HL151327 award from NHLBI.

Temporal Regulation of Phosphatase Activity with the Rapamycin-Regulated System

<ol>
	<li>Amin, A. R. M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHP-2+tyrosine+phosphatase+inhibits+p73-dependent+apoptosis+and+expression+of+a+subset+of+p53+target+genes+induced+by+EGCG.">SHP-2 tyrosine phosphatase inhibits p73-dependent apoptosis and expression of a subset of p53 target genes induced by EGCG.</a> Proc Natl Acad Sci U S A. 104 (13), 5419-5424 (2007).</li><li>Niogret, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shp-2+is+critical+for+ERK+and+metabolic+engagement+downstream+of+IL-15+receptor+in+NK+cells.">Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells.</a> Nat Commun. 10, 1-14 (2019).</li><li>Wang, Y. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+infection+activates+Src+homology-2+domain-containing+phosphatase+2+to+suppress+IFN-&#947;+signaling.">Helicobacter pylori infection activates Src homology-2 domain-containing phosphatase 2 to suppress IFN-&#947; signaling.</a> J Immunol. 193 (8), 4149-4158 (2014).</li><li>Yu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tyrosine+phosphatase+SHP2+promotes+proliferation+and+oxaliplatin+resistance+of+colon+cancer+cells+through+AKT+and+ERK.">The tyrosine phosphatase SHP2 promotes proliferation and oxaliplatin resistance of colon cancer cells through AKT and ERK.</a> Biochem Biophys Res Commun. 563, 1-7 (2021).</li><li>Lauriol, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+SHP2+dysfunction+underlies+cardiac+hypertrophy+in+Noonan+syndrome+with+multiple+lentigines.">Developmental SHP2 dysfunction underlies cardiac hypertrophy in Noonan syndrome with multiple lentigines.</a> J Clin Invest. 126 (8), 2989-3005 (2016).</li><li>Tartaglia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+PTPN11,+encoding+the+protein+tyrosine+phosphatase+SHP-2,+cause+Noonan+syndrome.">Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome.</a> Nat Genet. 29, 465-468 (2001).</li><li>Xu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Shp2+tyrosine+phosphatase+is+implicated+in+leukemogenesis+in+adult+human+leukemia.">Overexpression of Shp2 tyrosine phosphatase is implicated in leukemogenesis in adult human leukemia.</a> Blood. 106 (9), 3142-3149 (2005).</li><li>Mustelin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+tyrosine+phosphatases.">Protein tyrosine phosphatases.</a> Front Biosci. 7 (4), d85-d142 (2002).</li><li>Stanford, S. M., Bottini, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+tyrosine+phosphatases:+time+to+end+the+stigma.">Targeting tyrosine phosphatases: time to end the stigma.</a> Trends Pharmacol Sci. 38 (6), 524-540 (2017).</li><li>Guo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+phosphatases:+From+molecule+design+to+clinical+trials.">Targeting phosphatases: From molecule design to clinical trials.</a> Eur J Med Chem. 264, 116031 (2024).</li><li>Weiser, D. C., Shenolikar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+protein+phosphatase+inhibitors.">Use of protein phosphatase inhibitors.</a> Curr Protoc Mol Biol. 62, 18.10.1-18.10.13 (2003).</li><li>Fauser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+protein+tyrosine+phosphatase+signaling+by+engineered+chemogenetic+control+of+its+activity.">Dissecting protein tyrosine phosphatase signaling by engineered chemogenetic control of its activity.</a> J Cell Biol. 221 (8), e202111066 (2022).</li><li>Karginov, A. V., Hahn, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+activation+of+kinases:+design+and+application+of+RapR+kinases:+design+and+application+of+RapR+kinases.">Allosteric activation of kinases: design and application of RapR kinases: design and application of RapR kinases.</a> Curr Protoc Cell Biol. Chapter 14 (Unit 14.13), (2011).</li><li>Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V., Hahn, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+allosteric+activation+of+kinases+in+living+cells.">Engineered allosteric activation of kinases in living cells.</a> Nat Biotechnol. 28, 743-747 (2010).</li><li>Dagliyan, O., Dokholyan, N. V., Hahn, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+proteins+for+allosteric+control+by+light+or+ligands.">Engineering proteins for allosteric control by light or ligands.</a> Nat Protoc. 14, 1863-1883 (2019).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+SHP2+phosphatase+promotes+vascular+damage+and+inhibition+of+tumor+growth.">Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth.</a> EMBO Mol Med. 13, e14089 (2021).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHP2+regulates+the+development+of+intestinal+epithelium+by+modifying+OSTERIX++crypt+stem+cell+self-renewal+and+proliferation.">SHP2 regulates the development of intestinal epithelium by modifying OSTERIX+ crypt stem cell self-renewal and proliferation.</a> FASEB J. 35, e21106 (2021).</li><li>Zhang, E. E., Chapeau, E., Hagihara, K., Feng, G. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Shp2+tyrosine+phosphatase+controls+energy+balance+and+metabolism.">Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism.</a> Proc Natl Acad Sci U S A. 101 (45), 16064-16069 (2004).</li><li>Song, Y., Zhao, M., Zhang, H., Yu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-edged+roles+of+protein+tyrosine+phosphatase+SHP2+in+cancer+and+its+inhibitors+in+clinical+trials.">Double-edged roles of protein tyrosine phosphatase SHP2 in cancer and its inhibitors in clinical trials.</a> Pharmacol Ther. 230, 107966 (2022).</li><li>Hartman, Z. R., Schaller, M. D., Agazie, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tyrosine+phosphatase+SHP2+regulates+focal+adhesion+kinase+to+promote+EGF-induced+lamellipodia+persistence+and+cell+migration.">The tyrosine phosphatase SHP2 regulates focal adhesion kinase to promote EGF-induced lamellipodia persistence and cell migration.</a> Mol Cancer Res. 11 (6), 651-664 (2013).</li><li>Yu, D. H., Qu, C. K., Henegariu, O., Lu, X., Feng, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-tyrosine+phosphatase+Shp-2+regulates+cell+spreading,+migration,+and+focal+adhesion.">Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion.</a> J Biol Chem. 273 (33), 21125-21131 (1998).</li><li>Kramer, M. F., Coen, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+tyrosine+phosphatases+and+substrates.">Analysis of protein tyrosine phosphatases and substrates.</a> Curr Protoc Mol Biol. 18 (Unit-18.16), (2010).</li><li>Karginov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+regulation+of+protein+dimerization+and+kinase+activity+in+living+cells+using+photocaged+rapamycin+and+engineered+FKBP.">Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP.</a> J Am Chem Soc. 133 (3), 420-423 (2011).</li><li>Karginov, A. V., Hahn, K. 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The authors have no conflicts of interest to disclose.

This protocol provides detailed steps for the development, characterization, and application of chemogenetically controlled phosphatases. The RapR-Shp2 tool relies on a rapamycin-regulated switch inserted in the Shp2 catalytic domain. The strength of this tool is the specificity and tight temporal control of phosphatase activity. The tool is applicable to other phosphatases and, in combination with previously described RapR-TAP technology, allows for the reconstruction of individual downstream signaling pathways<sup clas...

Protein tyrosine phosphatases are a critical family of proteins involved in a plethora of cell signaling events. They have been shown to play a key role in the regulation of cell proliferation, migration, and apoptosis1,2,3. Consequently, the dysregulation of protein tyrosine phosphatases leads to a variety of debilitating diseases and disorders4,5,6,7. Studying the physiological function of tyrosine phosphatases and their role in the development of these pathologies has been historically hindered by a lack of tools needed for probing the intricacies of phosphatase signaling8.
Traditionally, phosphatases are studied using methods that do not have the desired specificity and/or do not provide temporal control of their activity. These critical limitations of available tools make it challenging to dissect specific roles of phosphatases in signaling pathways. Overexpression of constitutively active and dominant negative mutants or downregulation of expression of the phosphatase provide specificity but lack temporal control and often can trigger compensatory mechanisms that will mask the true function of the enzyme.
Pharmacological inhibitors allow for the temporal regulation of phosphatases. However, many phosphatase inhibitors are notoriously nonspecific due to the well-conserved composition of the active site in tyrosine phosphatases9. Additionally, due to design constraints, inhibitors targeting the catalytic site exhibit poor cell membrane permeability10. Another limitation of inhibitors is that they only allow for the examination of the effects of phosphatase inactivation11. Thus, there is a need for tools that enable specific, temporally regulatable activation of phosphatases. These tools will allow researchers to identify the direct effects of phosphatase activation, separating them from multiple parallel signaling cascades often activated by biological stimuli. Importantly, tight temporal control of activity enables the identification of transient events induced by a phosphatase and separates the effects of acute and prolonged phosphatase activity. Combining temporal regulation with mutational analysis will allow for a detailed dissection of specific roles of individual domains of the phosphatase and interrogation of its downstream signaling12.
To address the lack of desired capabilities in the existing tools, the Karginov group has developed the Rapamycin Regulated (RapR) system13,14,15. The RapR system utilizes an engineered switch domain, iFKBP, that allows for allosteric regulation of the protein of interest (POI). The insertion of the iFKBP domain at a position allosterically coupled to the catalytic site of the POI renders it susceptible to regulation by rapamycin. In the absence of rapamycin, iFKBP disrupts the catalytic site due to the intrinsically high structural dynamics of iFKBP and thus inactivates the POI. The addition of rapamycin induces the interaction of iFKBP with the co-expressed protein FRB (Figure 1). This causes stabilization of the switch domain, which consequently restores the structure and function of the POI&#39;s catalytic domain. As such, the tool allows for specific and temporally regulatable activation of the POI.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67142/67142fig01.jpg" /> 
Figure 1: Schematic of the RapR-Shp2 rapamycin-regulated system. RapR allows for allosteric activation of the protein of interest with the addition of rapamycin. This figure was modified from Fauser et al.12. Abbreviations: iFKBP = insertable FKBP12; FRB = FKBP-rapamycin-binding domain; R = rapamycin; Shp2 = Src homology-2 domain-containing protein tyrosine phosphatase. <a href="https://www.jove.com/files/ftp_upload/67142/67142fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The RapR tool can be applied to different protein families. It can be used to regulate protein kinases as well as phosphatases12,14. This protocol will focus on the application of the RapR tool to control Shp2 phosphatase. Shp2 is a ubiquitously expressed protein tyrosine phosphatase that is involved in signaling processes such as proliferation, migration, immunomodulation, and differentiation1,16,17,18. Dysregulation of Shp2 has been associated with a number of solid cancers, myeloid leukemia, and developmental disorders5,7. However, Shp2 has fallen victim to the same tool shortcomings as described above. To combat these limitations, RapR-Shp2, a specifically and temporally regulatable Shp2 construct, was developed and characterized12.
Prior to the development of RapR-Shp2, it was known that Shp2 was involved in cell migration19,20,21. However, its specific role in the signaling involved in this process was unknown. It was also unknown on what time scale Shp2 regulates the migration of cells and what specific morphodynamic changes it induces at different time points of its activation. Further, it was unclear whether acute and prolonged activation of Shp2 will cause different effects. Using RapR-Shp2, it was found that acute activation of Shp2 induces transient cell spreading, an increase in protrusions, cell polarization, and migration. Specific pathways downstream of Shp2 that regulate distinct morphodynamic changes were also identified12. This protocol provides details for the design and characterization of RapR-Shp2, which can be used to guide the development and application of other RapR phosphatases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#1.5 Glass Coverslips 25 mm Round</td>
			<td>Warner Instruments</td>
			<td>64-0715</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Tubes</td>
			<td>USA Scientific</td>
			<td>cc7682-3394</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Buffer</td>
			<td></td>
			<td></td>
			<td>For 500 mL: 5.18 g Tris-HCL, 131.5 mL glycerol, 52.5 mL 20% SDS, 0.5 g bromophenol blue, final pH 6.8</td>
		</tr>
		<tr>
			<td>4-20% Mini-PROTEAN TGX Precast Gel</td>
			<td>Biorad</td>
			<td>4561096</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Phusion Plus Buffer</td>
			<td>Thermo Scientific</td>
			<td>F538L</td>
			<td></td>
		</tr>
		<tr>
			<td>A431 Cells</td>
			<td>ATCC</td>
			<td>CRL-1555</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarsose</td>
			<td>GoldBiotech</td>
			<td>A-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>invitrogen</td>
			<td>A7816</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchmark Fetal Bovine Serum (FBS)</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td>Heat Inactivated Triple 0.1 &#181;m sterile-filtered</td>
		</tr>
		<tr>
			<td>Brig 35,30 w/v %</td>
			<td>Acros</td>
			<td>329581000</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>GoldBiotech</td>
			<td>A-420</td>
			<td></td>
		</tr>
		<tr>
			<td>CellGeo</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Published in 10.1083/jcb.201306067</td>
		</tr>
		<tr>
			<td>CellMask Deep Red plasma membrane dye</td>
			<td>invitrogen</td>
			<td>c10046</td>
			<td></td>
		</tr>
		<tr>
			<td>Colony Screen MasterMix</td>
			<td>Genesee</td>
			<td>42-138</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5a competent cells</td>
			<td>NEB</td>
			<td>C2987H</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Thermo Scientific</td>
			<td>F-515</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Ladder</td>
			<td>GoldBio</td>
			<td>D010-500</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>NEB</td>
			<td>N04475</td>
			<td></td>
		</tr>
		<tr>
			<td>DpnI Enzyme</td>
			<td>NEB</td>
			<td>R01765</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>GoldBio</td>
			<td>DTT10</td>
			<td>DL-Dithiothreitol, Cleland&#39;s Reagents</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros</td>
			<td>409910250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE(R) 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>transfection reagent</td>
		</tr>
		<tr>
			<td>Gel extraction Kit</td>
			<td>Thermo Scientific</td>
			<td>K0692</td>
			<td>GeneJET Gel Extraction Kit</td>
		</tr>
		<tr>
			<td>Gel Green Nucleic Acid Stain</td>
			<td>GoldBio</td>
			<td>G-740-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye Purple 6x</td>
			<td>NEB</td>
			<td>B7024A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>GlutaMAX-l (100x) 100 mL</td>
		</tr>
		<tr>
			<td>HEK 293T Cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa Cells</td>
			<td>ATCC</td>
			<td>CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fischer</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Processing Software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630 (NP40)</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole Buffer</td>
			<td></td>
			<td></td>
			<td>25 mM Imidazole pH 7.2, 2.5 mM EDTA, 50 mM NaCl, 5 mM DTT</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>L-15 1x</td>
			<td>Corning</td>
			<td>10-045-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher</td>
			<td>BP1425-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis Buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 1 mM EGTA, 1% NP40</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>N/A</td>
			<td>R 2022b update was used to run CellGeo functions</td>
		</tr>
		<tr>
			<td>Metamorph Microscopy Automation and Image Analysis Software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniPrep Kit</td>
			<td>Gene Choice</td>
			<td>96-308</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Precast Gels 12 well</td>
			<td>Bio-Rad</td>
			<td>4561085</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46-000-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiband Polychroic Mirror</td>
			<td>Chroma Technology</td>
			<td>89903BS</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus UPlanSAPO 40x objective</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS w/o Ca and Mg</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Tubes</td>
			<td>labForce</td>
			<td>1149Z65</td>
			<td>0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps</td>
		</tr>
		<tr>
			<td>Phusion Plus DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F630S</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein-G Sepharose</td>
			<td>Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Membranes</td>
			<td>BioRad</td>
			<td>1620219</td>
			<td>Immun-Blot PVDF/Filter Paper Sandwiches</td>
		</tr>
		<tr>
			<td>Rapamycin</td>
			<td>Fisher</td>
			<td>AAJ62473MF</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin, 2.21 mM EDTA, 1x [-] sodium</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Acetate-EDTA (TAE) 50x</td>
			<td>Fischer</td>
			<td>BP1332-1</td>
			<td>for electrophoresis</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 100 mM NaCl, 1 mM EGTA, 1% NP40</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Fisher Chemical</td>
			<td>O3446I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EGFR Antibody</td>
			<td>Cell Signaling</td>
			<td>2232</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Erk 1/2 Antibody</td>
			<td>Cell Signaling</td>
			<td>9102</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Flag Antibody</td>
			<td>Millipore-Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GAPDH Antibody</td>
			<td>invitrogen</td>
			<td>AM4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP Antibody</td>
			<td>Clontech</td>
			<td>632380</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mCherry Antibody</td>
			<td>invitrogen</td>
			<td>M11217</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-paxillin Antibody</td>
			<td>Thermo Fischer</td>
			<td>BDB612405</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-EGFR Y992 Antibody</td>
			<td>Cell Signaling</td>
			<td>2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-Erk 1/2 T202/Y204 Antibody</td>
			<td>Cell Signaling</td>
			<td>9101</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-paxillin Y31 Antibody</td>
			<td>Millipore-Sigma</td>
			<td>05-1143</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-PLC&#947; Y783 Antibody</td>
			<td>Cell Signaling</td>
			<td>14008</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PLC&#947; Antibody</td>
			<td>Cell Signaling</td>
			<td>5690</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and application of rapamycin-regulated tyrosine phosphatases

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, making them key targets of biological studies. Tools that enable the regulation of phosphatase activity are instrumental in the dissection of their function. Traditional approaches, such as overexpression of constitutively active or dominant negative mutants, or downregulation using siRNA, lack temporal control. Phosphatase inhibitors often have poor specificity, and they only allow researchers to determine what processes are affected by the inhibition of the phosphatase. 
We developed a chemogenetic approach, the Rapamycin-regulated (RapR) system, which allows for allosteric regulation of a phosphatase catalytic domain that enables tight temporal control of phosphatase activation. The RapR system consists of an iFKBP domain inserted into an allosteric site in the phosphatase. The intrinsic structural dynamics of the RapR domain disrupt the catalytic domain, leading to the inactivation of the enzyme. The addition of rapamycin mediates the formation of a complex between iFKBP and a co-expressed FRB protein, which stabilizes iFKBP and restores activity to the phosphatase's catalytic domain. 
This system provides high specificity and tight temporal control of phosphatase activation in living cells. The unique capabilities of this system enable the identification of transient events and interrogation of individual signaling pathways downstream of a phosphatase. This protocol describes guidelines for the development of a RapR-phosphatase, its biochemical characterization, and the analysis of its effects on downstream signaling and regulation of cell morphodynamics. It also provides a detailed description of a protein engineering strategy, in vitro assays analyzing phosphatase activity, and live cell imaging experiments identifying changes in cell morphology.

Figure 4&#160;demonstrates results that can be expected from the paxillin-based phosphatase activity assay. In this experiment, constitutively active and dominant negative Shp2 phosphatase activity was compared to that of active and inactive RapR-Shp2 using phospho-paxillin as the readout. The Shp2 constructs were immunoprecipitated and subjected to the activity assay as described in the protocol. The phospho-paxillin readouts for constitutively active Shp2 and active RapR-Shp2 were similar,...

Author Spotlight: Developing Tools to Tune the Activity of Tyrosine Phosphatases

This protocol describes the design, creation, and application of rapamycin-regulated phosphatases. This method provides high specificity and tight temporal control of phosphatase activation in living cells.

Development and Application of Rapamycin-regulated Tyrosine Phosphatases

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, ...

development-application-rapamycin-regulated-tyrosine

Research

JoVE Journal

Biology

244 Views.  University of Illinois at Chicago. This protocol describes the design, creation, and application of rapamycin-regulated phosphatases. This method provides high specificity and tight temporal control of phosphatase activation in living cells.

Video: Author Spotlight: Developing Tools to Tune the Activity of Tyrosine Phosphatases

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...