Rapid and efficient purification of Src homology 2 domain-containing proteins

Fyn, Csk and phosphatidylinositol 3-kinase p85

M. Koegl, R. M. Kypta, M. Bergman, K. Alitalo, Sara Courtneidge

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

To analyse the regulation of Src family tyrosine kinases in vitro we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation. from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

Original languageEnglish (US)
Pages (from-to)737-744
Number of pages8
JournalBiochemical Journal
Volume302
Issue number3
StatePublished - 1994
Externally publishedYes

Fingerprint

Phosphatidylinositol 3-Kinase
src Homology Domains
Purification
Phosphopeptides
Phosphotyrosine
Proteins
Phosphotransferases
Phosphorylation
src-Family Kinases
Baculoviridae
Distillation columns
Ion Exchange Chromatography
Chromatography
Kinetic parameters
Insects
Ion exchange
Down-Regulation
Resins
Salts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rapid and efficient purification of Src homology 2 domain-containing proteins : Fyn, Csk and phosphatidylinositol 3-kinase p85. / Koegl, M.; Kypta, R. M.; Bergman, M.; Alitalo, K.; Courtneidge, Sara.

In: Biochemical Journal, Vol. 302, No. 3, 1994, p. 737-744.

Research output: Contribution to journalArticle

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AB - To analyse the regulation of Src family tyrosine kinases in vitro we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation. from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

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