Chemical rescue of a mutant protein-tyrosine kinase

Daniel M. Williams, Dongxia Wang, Philip A. Cole

Research output: Contribution to journalArticle

Abstract

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed kcat for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is ∼3000-fold smaller compared with that of wild type Csk, whereas the Km values for ATP and poly(Glu,Tyr) are only mildly affected. The kcat value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the kcat value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.

Original languageEnglish (US)
Pages (from-to)38127-38130
Number of pages4
JournalJournal of Biological Chemistry
Volume275
Issue number49
DOIs
StatePublished - Dec 8 2000

Fingerprint

Mutant Proteins
Protein-Tyrosine Kinases
Phosphorylation
Signal transduction
Molecules
Guanidine
Substrates
Viperidae
CSK tyrosine-protein kinase
Catalysis
Protein Kinases
Signal Transduction
Phosphotransferases
Copolymers
Adenosine Triphosphate
X-Rays
X rays
poly(tyrosyl-glutamic acid)
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chemical rescue of a mutant protein-tyrosine kinase. / Williams, Daniel M.; Wang, Dongxia; Cole, Philip A.

In: Journal of Biological Chemistry, Vol. 275, No. 49, 08.12.2000, p. 38127-38130.

Research output: Contribution to journalArticle

Williams, DM, Wang, D & Cole, PA 2000, 'Chemical rescue of a mutant protein-tyrosine kinase', Journal of Biological Chemistry, vol. 275, no. 49, pp. 38127-38130. https://doi.org/10.1074/jbc.C000606200
Williams, Daniel M. ; Wang, Dongxia ; Cole, Philip A. / Chemical rescue of a mutant protein-tyrosine kinase. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 49. pp. 38127-38130.
@article{09e89a824a17480bb5a22037bd827b54,
title = "Chemical rescue of a mutant protein-tyrosine kinase",
abstract = "Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed kcat for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is ∼3000-fold smaller compared with that of wild type Csk, whereas the Km values for ATP and poly(Glu,Tyr) are only mildly affected. The kcat value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the kcat value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.",
author = "Williams, {Daniel M.} and Dongxia Wang and Cole, {Philip A.}",
year = "2000",
month = "12",
day = "8",
doi = "10.1074/jbc.C000606200",
language = "English (US)",
volume = "275",
pages = "38127--38130",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "49",

}

TY - JOUR

T1 - Chemical rescue of a mutant protein-tyrosine kinase

AU - Williams, Daniel M.

AU - Wang, Dongxia

AU - Cole, Philip A.

PY - 2000/12/8

Y1 - 2000/12/8

N2 - Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed kcat for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is ∼3000-fold smaller compared with that of wild type Csk, whereas the Km values for ATP and poly(Glu,Tyr) are only mildly affected. The kcat value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the kcat value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.

AB - Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed kcat for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is ∼3000-fold smaller compared with that of wild type Csk, whereas the Km values for ATP and poly(Glu,Tyr) are only mildly affected. The kcat value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the kcat value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.

UR - http://www.scopus.com/inward/record.url?scp=0034623991&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034623991&partnerID=8YFLogxK

U2 - 10.1074/jbc.C000606200

DO - 10.1074/jbc.C000606200

M3 - Article

C2 - 11006267

AN - SCOPUS:0034623991

VL - 275

SP - 38127

EP - 38130

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 49

ER -