Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

Summary

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

Abstract

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

Introduction

The RAF family kinases are a key component of RAS/RAF/MEK/ERK signaling cascade, which transmit a signal from RAS to activate mitogen-activated protein kinase (MEK)^1,2,3,4. This family of kinases plays a crucial role in cell growth, survival and differentiation, and their alterations induce many diseases, notably cancer^5,6,7,8. Recently, genomic sequencings have identified many disease-related RAF mutants that exhi....

Protocol

1. In Vitro Kinase Assay for Measuring the Catalytic Activity of RAF Mutants

1. Construct vectors encoding RAF mutants (Figure 1A) with FLAG(DYKDDDDK) tag at C-terminus by using Gibson Assembly or traditional molecular cloning methods.
   1. Introduce the FLAG tag and mutations into the RAF coding sequences by PCRs, and then insert whole sequences into pCDNA3.1(+) vector by using Gibson assembly or T4 DNA ligation and following the manufacture’s protocols. Use the following conditions for PCR reactions: (1) 95 °C, 2 min; (2) 95 °C, 30 s; (3) 59 °C, 30 s; (4) 68 °C, 3 min; (5) 20 cy....

Results

The RAF family kinases have both catalytic and allosteric activities, which enable their disease-related mutants to turn on the downstream signaling through different mechanisms^{13,14,16,17,18}. The constitutively active RAF mutants directly phosphorylate their substrates, while the kinase-dead RAF mutants fulfill their function through transactivating wild-type.......

Discussion

In this article, we presented three methods for characterizing disease-related RAF mutants, which include in vitro kinase assay, RAF co-activation assay, and complimentary split luciferase assay. Since RAF kinases have both catalytic activity and allosteric activity, various RAF mutants can activate the downstream signaling through two distinct mechanisms^13,14,16,17,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-phosphoERK1/2	Cell Signaling Technologies	4370
anti-phosphoMEK1/2	Cell Signaling Technologies	9154
anti-ERK1/2	AB clonal	A0229
anti-MEK1/2	Cell Signaling Technologies	9124
anti-FLAG(mouse)	Sigma-Aldrich	F3165
anti-HA	Novus Biologicals	MAB6875
anti-FLAG(Rabbit)	Cell Signaling Technologies	14793
anti-β-actin	Sigma-Aldrich	A2228
anti-FLAG beads(M2)	Sigma-Aldrich	A4596
HRP-conjugated anti-mouse IgG	Jackson Laboratories	115-035-003
HRP-conjugated anti-Rabbit IgG	Jackson Laboratories	111-035-144
pcDNA3.1(+)	In vitrogen	V79020
Gibson Assembly Cloning  Kit	New England Biolabs	E5510
T4 DNA ligase	New England Biolabs	M0202
Lipofectamine 2000	Invitrogen	11668019
Fugene 6	Roche	11 814 443 001
DMEM w/o phenol red	Invitrogen	21063-029
D-luciferin	GoldBio	LUCK-100
6xhis-tagged MEK1 (K97A)	prepared in our previous studies	N.A.	Reference 15.
GloMax-Multi Detection System.	Promega	E7041