Identification of csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure.
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 vivo 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.
Joukov
V
V
Molecular/Cancer Biology Laboratory, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), FIN-00014, Finland.
Vihinen
M
M
Vainikka
S
S
Sowadski
J M
JM
Alitalo
K
K
Bergman
M
M
D000081247:CSK Tyrosine-Protein Kinase; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases
D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human