preparing cells expressing rapamycin-regulated phosphatase for immunoprecipitation to assess temporal control of tyrosine phosphatases

Temporal Regulation of Phosphatase Activity with the Rapamycin-Regulated System

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.

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

Development and Application of Rapamycin-regulated Tyrosine Phosphatases

Our work focuses on the development of tools that regulate the activity of tyrosine phosphatases. We've applied the rapamycin-regulated system to several phosphatases and shown that it imparts specific and temporal regulation of our protein of interest. We've aimed to determine the morphological changes induced by the activation of SHIP-2 phosphatase.The RapR tool has allowed us to determine transient effects of phosphatase activation in cells, and we've determined complex specific downstream effects of a phosphatase involved in cell spreading and migration. Additionally, we've determined domain-driven behaviors of the same phosphatase. This has contributed to our understanding of phosphatase mechanisms in regulating cell morphodynamics and downstream signaling.Our RapR tool offers the ability to specifically and temporally regulate a phosphatase of interest. Once inserted into the phosphatase, the iFKBP domain acts as an allosteric switch that, once bound to rapamycin, restores activity to the phosphatase, acting as an on-switch. We can also use the rapamycin-regulated system to reconstruct signaling complexes.Our findings using the RapR tool have opened the door to determining the effects of SHIP-2's interactome on its ability to regulate cell activity. We found that SHIP-2s in SH2 domain binding is crucial in cell retraction. Using the RapR tool, we can further investigate what binding partners may be responsible for this regulation.

Preparing Cells Expressing Rapamycin-Regulated Phosphatase for Immunoprecipitation to Assess Temporal Control of Tyrosine Phosphatases

To begin, obtain HEK 293T cells and plate 800, 000 cells per well in a 6-well tissue culture plate in supplemented DMEM media. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide to achieve 60 to 80%confluency. Next, mix one microgram of each plasmid DNA with 200 microliters of serum-free DMEM and six microliters of transfection reagent.After 15 minutes of incubation, dispense 100 microliters of transfection mixture per well of cells. Incubate overnight in a 37 degrees Celsius and 5%carbon dioxide incubator. On day three, add an appropriate amount of either rapamycin or ethanol separately to the cells and incubate them for one hour in a 37 degrees Celsius and 5%carbon dioxide incubator.Then place the plate on ice and gently wash the cells with three milliliters of cold PBS. After removing the PBS add 300 microliters of lysis buffer containing either rapamycin or ethanol, and scrape the cells with a cell scraper. Transfer the sample to a 1.5 milliliter tube and microcentrifuge it for 10 minutes at 1, 000 g at four degrees Celsius.To check for expression levels, aliquot 20 microliters of the supernatant into a new 1.5 milliliter tube. Incubate the sample with 20 microliters of 2x Laemmli buffer containing 5%beta mercaptoethanol at 95 to 100 degrees Celsius for five minutes.

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18 Views. To begin, obtain HEK 293T cells and plate 800, 000 cells per well in a 6-well tissue culture plate in supplemented DMEM media. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide to achieve 60 to 80%confluency. Next, mix one microgram of each plasmid DNA with 200 microliters of serum-free DMEM and six microliters of transfection reagent.After 15 minutes of incubation, dispense 100 microliters of transfection mixture per well of cells. Incubate overnight in a 37 deg...