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Identification of Csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure

Joukov, V; Vihinen, Mauno LU ; Vainikka, S; Sowadski, JM; Alitalo, K and Bergman, M (1997) In Biochemical Journal 322. p.927-935
Abstract
The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the... (More)
The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity. Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain. Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins. Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain. Taken together these results indicate that Csk can be phosphorylated in vitro at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro. Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemical Journal
volume
322
pages
927 - 935
publisher
Portland Press Limited
external identifiers
  • wos:A1997WQ35500034
  • scopus:0031003439
ISSN
0264-6021
language
English
LU publication?
no
id
46f40690-901c-4c90-9082-89d7953b9931 (old id 3852853)
date added to LUP
2013-06-28 14:32:53
date last changed
2017-06-18 04:28:30
@article{46f40690-901c-4c90-9082-89d7953b9931,
  abstract     = {The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity. Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain. Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins. Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain. Taken together these results indicate that Csk can be phosphorylated in vitro at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro. Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain.},
  author       = {Joukov, V and Vihinen, Mauno and Vainikka, S and Sowadski, JM and Alitalo, K and Bergman, M},
  issn         = {0264-6021},
  language     = {eng},
  pages        = {927--935},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Identification of Csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure},
  volume       = {322},
  year         = {1997},
}