IdentificationofaMutantKinase／ATPAnalogPair1

发布时间：2019-04-24 16:50 原文链接： IdentificationofaMutantKinase／ATPAnalogPair1

Identification of a Mutant Kinase/ATP Analog Pair

Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, VA 22908 Excerpted from Protein: Protein Interactions, Second Edition Edited by Erica A. Golemis and Peter D. Adams

ABSTRACT

Protein Kinase cascades regulate many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effects on cellular processes. A recent development in this area takes advantage of the similarity in the ATP-binding domains of protein kinases. Conserved residues in the ATP-binding site contain large side chains that come into close contact with the N6 position of bound ATP. Mutation of one or more of these large residues to alanine or glycine generates a "pocket" in the ATP-binding site that allows the mutant kinase, but not the wild-type kinase or other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N6 position. Kinase reactions performed with a mutated kinase and radiolabeled ATP analogs in a cellular lysate allow specific labeling of direct substrates of the mutant kinase, which can later be identified by mass spectrometry or other means.

Once pocket mutations have been generated in the kinase, it is necessary to screen ATP analogs for their compatibility with the kinase mutant. This step requires an active form of the wild-type and mutant kinase, either as recombinant activated protein, or as protein that has been immunoprecipitated from transfected, stimulated cells. In addition, a known in vitro substrate for the kinase is also required. Screening the mutant kinases with ATP analogs is best performed in a two-step process. The first step involves assaying the ability of an ATP analog to inhibit the incorporation of radioactive phosphate from normal [y-³²P]ATP into a known substrate in an in vitro kinase reaction. This method allows a large number of ATP analogs to be screened easily, without the need for creating numerous radiolabeled ATP analogs, saving time and bypassing the need for unnecessary radioactive work. Moreover, quantitative competition studies can be performed to identify analog-mutant pairs with high affinity.

It is important to keep in mind that there are two reasons an ATP analog can inhibit incorporation of labeled phosphate into substrate. The first is that the ATP analog is a good ATP source for use by the kinase and competes with the [y-³²P]ATP as a substrate. The second is that the ATP analog is small enough to interact with the ATP-binding site of the kinase but is unable to be used by the kinase as an ATP source and is therefore simply blocking [y-³²P]ATP from the ATP-binding site. To distinguish between these, it is necessary to directly test the ability of the mutant kinase to phosphorylate a substrate with analog ATP. Doing this requires either radiolabeled ATP analogs or, preferably, a phospho-specific antibody to the phosphorylation site on the known substrate. In our experiments, we used a commercially available phospho-specific antibody to the ERK2 substrate Elk1. A phospho-specific antibody allows a large number of ATP analogs to be screened without their having to be made radioactive. In this assay, active wild-type or mutant kinase is mixed with ATP analog and substrate in an in vitro kinase reaction. The reaction is then analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and either stained with Ponceau S and counted by Cerenkov counting (if [y-³²P]ATP analog is used) or immunoblotted with a phosphospecific antibody (if available) to determine the extent of substrate phosphorylation. The kinase/ATP analog pair that provides the best phosphorylation of the substrate is then used for future experiments. Only those analogs that cannot be used by the wild-type kinase and other cellular kinases are chosen.

MATERIALS

Buffers, Solutions, and Reagents

M2 Lysis buffer (cold) [II], freshly prepared
50 mM Tris-base (pH 7.4)
150 mM NaCl
10% glycerol
1% Triton X-100
0.5 mM EDTA
0.5 mM EGTA
50 mM NaF
40 mM β-glycerophosphate
5 mM tetrasodium pyrophosphate
0.1 mM sodium vanadate
10 µg/ml aprotinin
5 µg/ml leupeptin
2 mM phenylmethylsulfonyl fluoride (PMSF), fresh
Acetic acid (1%)
10x Kinase buffer
250 mM HEPES (pH 7.4)
100 mM magnesium acetate
10 mM dithiothreitol (DTT)
Kinase reaction buffer A (for the analog inhibition assay)
1x kinase buffer
10 µM ATP
100 µM ATP analog
10 µCi/reaction [y-³²P]ATP
1-5 µg of a known substrate/reaction
Kinase reaction buffer B (for the substrate phosphorylation assay)
1x kinase buffer
100 µM ATP analog
1-5 µg of a known substrate/reaction
10 µCi/reaction [y-³²P]ATP analog (required only if a phospho-specific antibody to the substrate is not available)
1x PBS (cold)[I]
25.6 g of Na₂HPO₄ &dot;7H₂O
80 g of NaCl
2 g of KCl
2 g of KH2PO₄
Bring to 1 liter with distilled, deionized water; autoclave for 40 min at 121°C.
Ponceau S solution
0.5% (w/v) in 1% acetic acid
2x Laemmli sample buffer
100 mM Tris (pH 6.8)
2% SDS
20% glycerol
4% β-mercaptoethanol (added fresh)
Dulbecco's modified Eagle medium (DMEM)
Fetal bovine serum (FBS)

Cells and Antibodies

COS-1 African green monkey kidney epithelial cells (ATCC)
Antibodies to the epitope tag on the protein kinase of interest
A phospho-specific antibody to a known substrate of your kinase (if available)

Gels

SDS-PAGE gel

Special Equipment

Microcentrifuge with cooling facility
Transfer tank for western blotting
30°C water bath
Scintillation counter
Heating block, preset to 100°C (I)

Additional Reagents

[y-³²P]ATP (6000 Ci/mmole)
Agonist (I); e.g., epidermal growth factor, serum, platelet-derived growth factor Lipofectamine
M2 agarose (for FLAG-tagged protein kinases)
Protein A- or protein G-conjugated agarose or Sepharose CL-6 beads (for preparation of antibody, see step 7)
BCA protein assay kit
Hypodermic needle 1", 27 gauge
Syringe, 1 cc
Nitrocellulose membrane
Vacuum flask
Shaker, preset to 4°C
Incubator, preset to 37°C [I]
60-mm tissue culture dishes [I]

Plasmids

Expression plasmid of choice

METHOD

IdentificationofaMutantKinase／ATPAnalogPair1

其他网友还关注过

科学家将人工智能技术成功用于蛋白质复合物结构预测

发力癌症分子病理诊断，无锡臻和全资收购TissueofOrigin®

这3个杂志撤回中国学者249篇文章，包含上交、中山等名校

肿瘤治疗的强心剂，中国学者开发肿瘤治疗新策略

llumina宣布推出新型基因分型芯片|支持AllofUs研究计划

SDSPAGE异常电泳现象及分析SDSPAGEHallofShame

Preparationofdenaturing6％