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>
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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. 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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 pathways26. Unique capabilities of the RapR approach allowed researchers to identify transient events induced by activation of Shp2 and dissect specific downstream signaling pathways regulating individual morphodynamic processes. 
Several critical factors influence the design of a RapR phosphatase. A well-resolved crystal structure of the catalytic domain of the phosphatase of interest is very helpful in guiding the selection of the insertion site for iFKBP. However, due to high structural similarity among tyrosine phosphatases, alignment of the amino acid sequences of the catalytic domains could provide sufficient information for the identification of the insertion site as illustrated by the alignment of Shp2 to PTP1B and PTPPest (Figure 2). For RapR-Shp2, Val406, located in the site coupled to the critical catalytic WPD loop through a &#946;-strand, was chosen as the most optimal insertion site. The same insertion site may result in successful regulation of other PTPases, as was demonstrated for PTP1B and PTP-PEST12 (Figure 2A).
If the phosphatase of interest is a multidomain protein, structural information about the organization of the domains will help prevent interfering with other functions of the PTPase. Another factor influencing the design of optimal RapR-PTPase is how many amino acids should be replaced by inserted iFKBP. Larger and more flexible insertion loops in the PTPase may require additional shortening to ensure tight regulation of catalytic activity by iFKBP. Similarly, the length and the composition of the linkers connecting iFKBP to the PTPase will affect the efficiency of regulation. Short linkers composed of a single Gly residue will provide tighter regulation but may reduce the maximum activity of the enzyme if they introduce persistent structural distortion even when iFKBP is bound to rapamycin and FRB. Medium length Gly-Ser-Gly linkers provide more flexibility but may not be rigid enough for some PTPases. Long linkers composed of Gly-Ser-Gly-Gly-Pro-Gly will help prevent the formation of unintended secondary structures that may influence PTPase regulation. RapR-Shp2 was tested with all three types of linkers; a Gly-Ser-Gly linker was found to be the most optimal. 
A critical aspect determining the successful development of RapR tools is the establishment of a robust phosphatase assay and the presence of appropriate controls. Comparison of RapR-phosphatase activity to the activity of dominant-negative and constitutively active versions of the POI provides an assessment of the leakiness of the RapR-construct and the extent of activation. Furthermore, the lack of dephosphorylation by the constitutively active phosphatase or reduced phosphorylation by the dominant negative mutant will indicate improper reaction conditions. 
For the phosphatase assay, the reaction conditions will require optimization for each PTPase. Adjusting the temperature, reaction time, and buffer conditions will depend on the PTPase of interest. Vigorous agitation of samples is critical to ensure sufficient mixing of the Sepharose-bound PTPase and its substrate. Lastly, if the phosphatase assay described is not optimal for the particular PTPase of interest, then a phosphatase reaction using p-nitrophenyl phosphate as a substrate can be used as an alternative assay27. 
In live cell imaging experiments, several criteria should be taken into account when preparing a sample. The outlined protocol uses L-15 media based on HEPES buffer, which is not sensitive to CO2 concentration. If the use of a bicarbonate-based media is desired, then HEPES should be supplemented to maintain the pH of the sample or an environmental imaging chamber supplementing CO2 should be used. The protocol recommends applying a layer of mineral oil on top of the sample to prevent evaporation and simplify the addition of rapamycin. Other setups using an environmental imaging chamber or a closed chamber can be used, but the addition of rapamycin could be more challenging. When adding rapamycin to the cells while imaging, ensure that the stage does not shift and the cells are not disturbed. Any additional movements of the sample during the imaging process will obstruct data collection. Illumination intensity and exposure time should be kept low to reduce phototoxicity, especially for long-term imaging. Adequate transfection efficiency of the cells is also critical. A 1:1 ratio of FRB to RapR-Shp2 DNA provides adequate expression, but for new constructs, this ratio will require adjustment. To ensure efficient activation, FRB should be expressed at a higher level than the RapR phosphatase.
A limitation of RapR-based tools is the inability to quickly deactivate. Changing media removes extracellular rapamycin, but deactivation of RapR constructs can take hours due to the very tight binding of rapamycin to the RapR construct. Fast inactivation can be achieved by the addition of an active site inhibitor of the PTPase. However, potential off-target effects of the inhibitor could make interpretation of the results challenging. Furthermore, even in combination with a PTPase inhibitor, the RapR approach cannot be used for cyclical activation/inactivation experiments. Another limitation of the RapR system is the lack of spatial control. RapR constructs are globally expressed and therefore activated everywhere in the cell. One potential solution is the application of caged rapamycin that can be released by near UV light locally in the cell28,29. Further, the RapR tool can be modified to achieve activation of the phosphatase of interest in a specific protein complex or at a specific subcellular location. By attaching a binding partner or a subcellular tag to FRB, RapR-PTPase will be targeted to the specific protein or location in the cell. Lastly, rapamycin is a well-characterized immunosuppressor via mTOR inhibition and can influence cellular signaling, potentially disrupting the signaling of the PTPase of interest. To overcome this concern, non-immunosuppressive analogs of rapamycin (iRap and AP21967) represent a good alternative. Both compounds have been shown to regulate RapR constructs14.

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.bdigitaluss.remotexs.co/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><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 &amp;micro;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&#039;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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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>&amp;beta;-Mercaptoethanol</td><td>Fisher Chemical</td><td>O3446I-100</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</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&amp;gamma; Y783 Antibody</td><td>Cell Signaling</td><td>14008</td><td></td></tr><tr><td>Anti-PLC&amp;gamma; 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, indicating that the active RapR-Shp2 had not been affected negatively by the introduction of the RapR domain and it retained full activity once activated. Dominant negative Shp2 and inactive RapR-Shp2 were also similar, indicating that the RapR-Shp2 construct does not have activity when inactive. This indicates the successful design of a RapR-phosphatase. The phospho-substrate used for this assay may differ based on the phosphatase of interest.
Figure 5, similar to Figure 4, compares constitutively active, dominant negative, and active and inactive RapR-Shp2. This experiment was conducted without immunoprecipitation of the constructs. Instead, it was designed to characterize the downstream signaling effects of the RapR-Shp2 construct within cells. Here, the Shp2 constructs were expressed in HEK293T cells. Downstream effector ERK1/2 was shown to increase in phosphorylation in response to RapR-Shp2 activation, just as in the constitutively active sample. The inactive RapR-Shp2 did not induce this change. This indicates that downstream signaling of Shp2 is preserved with the incorporation of the RapR-domain. Similarly, known phospho-substrates EGFR and PLC&#947; were dephosphorylated in response to RapR-Shp2 activation in A431 cells.
Finally, Figure 6 presents the results of RapR-Shp2 activation on the morphodynamics of HeLa cells. Once RapR-Shp2 was activated, the cells showed an increase in protrusive activity as well as cell area. This indicates that RapR-Shp2 activation is sufficient to induce morphodynamic changes in HeLa cells and may allow researchers to probe specific downstream signaling pathways responsible for this effect.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67142/67142fig04.jpg" /> 
Figure 4:&#160;Results from the paxillin-based activity assay: Constitutively active, dominant negative, and RapR-Shp2 were immunoprecipitated and subject to the activity assay using the outlined protocol. The levels of pY31 paxillin in both CA and RapR-Shp2 were similar, indicating that the RapR-Shp2 construct had similar activity compared to CA Shp2 once activated. The non-activated RapR-Shp2 sample had no activity, similar to the DN sample, indicating that it was not &#34;leaky&#34;. This figure was modified from Fauser et al.12. Abbreviations: RapR = Rapamycin-regulated; Shp2 = Src homology-2 domain-containing protein tyrosine phosphatase; DN = dominant negative; CA = constitutively active; FRB = FKBP-rapamycin-binding domain. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67142/67142fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67142/67142fig05.jpg" /> 
Figure 5:&#160;Results from the whole-cell lysate assay: Constitutively active, dominant negative, and RapR-Shp2 were expressed in HEK293T cells and the whole-cell lysate protocol was completed. When inactive, RapR-Shp2 did not activate ERK1/2 by phosphorylation, similar to the DN Shp2 sample. This indicates that RapR-Shp2 does not have activity when inactive. Once activated, the level of ERK1/2 phosphorylation was similar to that of the CA Shp2 sample, indicating successful activation and downstream signaling of Shp2. Similarly, A431 cells expressing RapR-Shp2 showed decreases in phosphorylation of both EGFR and PLC&#947;, both known substrates of Shp2. This figure was modified from Fauser et al.12. Abbreviations: RapR = Rapamycin-regulated; Shp2 = Src homology-2 domain-containing protein tyrosine phosphatase; DN = dominant negative; CA = constitutively active; ERK = extracellular signal-regulated kinase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; EGFR = epidermal growth factor receptor; PLC&#947; = phospholipase C gamma; FRB = FKBP-rapamycin-binding domain. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67142/67142fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67142/67142fig06.jpg" /> 
Figure 6:&#160;Cell area and protrusive activity data analysis based on live imaging: HeLa cells were transfected with RapR-Shp2 and subjected to the outlined live imaging protocol. Data were analyzed as outlined in the data analysis protocol. Once activated (indicated by the vertical grey line), HeLa cells showed increases in both cell protrusive activity as well as cell area. In the negative samples that were only expressing FRB, this activity was not present. This indicates that RapR-Shp2 activation induces fast morphodynamic changes within HeLa cells and encourages cell spreading and protrusion. This figure was modified from Fauser et al.12. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67142/67142fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Universidad San Sebastian

Research

JoVE Journal

Biology

293 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

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Bioengineering

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

Cancer Research

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

Biochemistry

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Immunology and Infection

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Medicine

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically.
In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. ...

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin&#160; (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1&#160; (mTORC1) and mTOR complex 2&#160; (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,&#160; mTORC2 consists of a rapamycin-insensitive companion called Rictor, mammalian stress-activated protein map kinase-interacting protein 1 (mSin1 or MAPKAP1).&#160; Mammalian lethal with SEC13 protein 8 (mLST8) is a common protein of both complexes.
The binding of insulin to the insulin receptor, an RTK, initiates the mTOR pathway. The activated receptors auto-phosphorylate tyrosine 960 on its cytoplasmic domain. The phosphorylated tyrosine forms a docking site for the insulin receptor substrate (IRS) that binds the receptor via their phospho-tyrosine binding (PTB) domain. In addition, IRS contains a PH domain that helps them bind phosphoinositides on the plasma membrane.&#160; At least four types of IRS (IRS-1 to 4) assist in insulin receptor signaling. Once bound to the receptor, multiple tyrosines on the IRS are phosphorylated, which recruits the SH2 domain-containing phosphatidylinositol-3-kinase or PI-3K to the cytosolic surface of the plasma membrane. The PI-3K binds to the phosphatidylinositol (4,5)-bisphosphate&#160; (PIP2) and produces phosphatidylinositol (3,4,5)-trisphosphate or PIP3. Adjacent PIP3 functions as docking sites for protein kinase B (PKB) or AKT and 3-phosphoinositide-dependent kinase 1 or PDK1. As mTORC2 phosphorylates AKT at serine 473, it undergoes a conformational change and exposes a threonine 308 residue, which subsequently leads to phosphorylation of the AKT by PDK1.
Activated AKT dissociates from the membrane to phosphorylate a series of downstream targets to promote cell growth, proliferation, and other responses. For example, the forkhead transcription factor, FOXO3a, is a transcription factor that activates the transcription of pro-apoptotic proteins.&#160; Phosphorylation of FOXO3a by AKT induces its binding to 14-3-3 protein and its subsequent sequestration in the cytosol, thereby promoting cell survival. Activated Akt also inhibits tuberous sclerosis protein 2 or Tsc 2 and promotes Rheb activation. Activated Rheb (Rheb-GTP) further stimulates mTORC1 and induces cell growth.&#160;

Development and Application of Rapamycin-regulated Tyrosine Phosphatases

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