Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85.
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.
Koegl
M
M
European Molecular Biology Laboratory, Heidelberg, Germany.
Kypta
R M
RM
Bergman
M
M
Alitalo
K
K
Courtneidge
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002846:Chromatography, Affinity; D015536:Down-Regulation; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000302:isolation & purification* / Q000378:metabolism; D018411:Spodoptera; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D019061:src-Family Kinases
D000911:Antibodies, Monoclonal; D000081247:CSK Tyrosine-Protein Kinase; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides; D017853:Phosphotransferases (Alcohol Group Acceptor); D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src); D014443:Tyrosine; D019061:src-Family Kinases