Development and Application of Rapamycin-regulated Tyrosine Phosphatases

Summary

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.

Abstract

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.

Introduction

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 apoptosis^1,2,3. Consequently, the dysregulation of protein tyrosine phosphatases leads to a variety of debilitating diseases and disorders^4,5,6,7. Studying the physiological function of tyrosine phosphatases and their role in the development of....

Protocol

1. Design of RapR-phosphatases

1. Planning
   NOTE: Using RapR-Shp2 as an example, this protocol details the important steps for creating a rapamycin-regulated tyrosine phosphatase. The described protocol is optimized for Shp2 and additional modifications might be needed to fit specific properties of individual phosphatases.
   1. To ensure that the catalytic activity of Shp2 is purely controlled by rapamycin and not by endogenous mechanisms, introduce a mutation into the phosphatase t.......

Discussion

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

Materials

Name	Company	Catalog Number	Comments
#1.5 Glass Coverslips 25 mm Round	Warner Instruments	64-0715
1.5 mL Tubes	USA Scientific	cc7682-3394
2x Laemmli Buffer			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
4-20% Mini-PROTEAN TGX Precast Gel	Biorad	4561096
5x Phusion Plus Buffer	Thermo Scientific	F538L
A431 Cells	ATCC	CRL-1555
Agarsose	GoldBiotech	A-201
Attofluor Cell Chamber	invitrogen	A7816
Benchmark Fetal Bovine Serum (FBS)	Gemini Bio-products	100-106	Heat Inactivated Triple 0.1 µm sterile-filtered
Brig 35,30 w/v %	Acros	329581000
BSA	GoldBiotech	A-420
CellGeo	N/A	N/A	Published in 10.1083/jcb.201306067
CellMask Deep Red plasma membrane dye	invitrogen	c10046
Colony Screen MasterMix	Genesee	42-138
DH5a competent cells	NEB	C2987H
DMEM	Corning	15-013-CV
DMSO	Thermo Scientific	F-515
DNA Ladder	GoldBio	D010-500
dNTPs	NEB	N04475
DpnI Enzyme	NEB	R01765
DTT	GoldBio	DTT10	DL-Dithiothreitol, Cleland's Reagents
EGTA	Acros	409910250
Fibronectin from bovine plasma	Sigma	F1141
FuGENE(R) 6 Transfection Reagent	Promega	E2692	transfection reagent
Gel extraction Kit	Thermo Scientific	K0692	GeneJET Gel Extraction Kit
Gel Green Nucleic Acid Stain	GoldBio	G-740-500
Gel Loading Dye Purple 6x	NEB	B7024A
Glutamax	Gibco	35050-061	GlutaMAX-l (100x) 100 mL
HEK 293T Cells	ATCC	CRL-11268
HeLa Cells	ATCC	CRM-CCL-2
HEPES	Fischer	BP310-500
ImageJ Processing Software	N/A	N/A
Igepal CA-630 (NP40)	Sigma	I3021
Imidazole Buffer			25 mM Imidazole pH 7.2, 2.5 mM EDTA, 50 mM NaCl, 5 mM DTT
KCl	Sigma	P-4504
L-15 1x	Corning	10-045-CV
LB Agar	Fisher	BP1425-2
Lysis Buffer			20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 1 mM EGTA, 1% NP40
MATLAB	MathWorks	N/A	R 2022b update was used to run CellGeo functions
Metamorph Microscopy Automation and Image Analysis Software	N/A	N/A
MgCl2	Fisher Chemical	M33-500
Mineral Oil	Sigma	M5310
MiniPrep Kit	Gene Choice	96-308
Mini-PROTEAN TGX Precast Gels 12 well	Bio-Rad	4561085
Molecular Biology Grade Water	Corning	46-000-CV
Multiband Polychroic Mirror	Chroma Technology	89903BS
NaCl	Fisher Chemical	S271-3
Olympus UPlanSAPO 40x objective	Olympus	N/A
PBS w/o Ca and Mg	Corning	21-031-CV
PCR Tubes	labForce	1149Z65	0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps
Phusion Plus DNA Polymerase	Thermo Scientific	F630S
Primers	IDT
Protein-G Sepharose	Millipore	16-266
PVDF Membranes	BioRad	1620219	Immun-Blot PVDF/Filter Paper Sandwiches
Rapamycin	Fisher	AAJ62473MF
0.25% Trypsin, 2.21 mM EDTA, 1x [-] sodium	Corning	25-053-CI
Tris-Acetate-EDTA (TAE) 50x	Fischer	BP1332-1	for electrophoresis
Wash Buffer			20 mM Hepes-KOH, pH 7.8, 50 mM KCl, 100 mM NaCl, 1 mM EGTA, 1% NP40
β-Mercaptoethanol	Fisher Chemical	O3446I-100
Antibodies
Anti-EGFR Antibody	Cell Signaling	2232
Anti-Erk 1/2 Antibody	Cell Signaling	9102
Anti-Flag Antibody	Millipore-Sigma	F3165
Anti-GAPDH Antibody	invitrogen	AM4300
Anti-GFP Antibody	Clontech	632380
Anti-mCherry Antibody	invitrogen	M11217
Anti-paxillin Antibody	Thermo Fischer	BDB612405
Anti-phospho-EGFR Y992 Antibody	Cell Signaling	2235
Anti-phospho-Erk 1/2 T202/Y204 Antibody	Cell Signaling	9101
Anti-phospho-paxillin Y31 Antibody	Millipore-Sigma	05-1143
Anti-phospho-PLCγ Y783 Antibody	Cell Signaling	14008
Anti-PLCγ Antibody	Cell Signaling	5690