Detection of a physical and functional interaction between Csk and Lck which involves the SH2 domain of Csk and is mediated by autophosphorylation of Lck on tyrosine 394.
The COOH-terminal Src kinase (Csk) is responsible for the phosphorylation of the conserved, negative regulatory, carboxyl-terminal tyrosine of most of the Src family protein tyrosine kinases. Up to now, no stable binding of Csk to Src kinases has been detected. We therefore decided to analyze this interaction using two systems which allow detection of transient interaction. We produced and purified recombinant proteins in the glutathione S-transferase prokaryotic expression system. First, using real-time biospecific interaction analysis (BIAcore(TM)), we detected in vitro a specific interaction between Csk and one of its substrates Lck, a lymphocyte-specific member of the Src family. This interaction requires the autophosphorylation of Lck on tyrosine 394 (the phosphorylation of which is correlated with an increase of the kinase activity) and involves a functional Csk SH2 domain. Second, using the yeast two-hybrid system, we confirmed in vivo the physical interaction between Csk and Lck. Furthermore, in vitro we showed that autophosphorylation of Lck on tyrosine 394 enhances the phosphorylation of Lck by Csk on the negative regulatory site, tyrosine 505, suggesting that activated Lck serves preferentially as substrate for Csk. These findings might explain the mechanism(s) by which Csk interacts with most of Src kinases to down-regulate their kinase activity.
Bougeret
C
C
INSERM, Institut Cochin de Génétique Moleculaire/Université Paris, France.
Delaunay
T
T
Romero
F
F
Jullien
P
P
Sabe
H
H
Hanafusa
H
H
Benarous
R
R
Fischer
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D017124:Conserved Sequence; D005982:Glutathione Transferase / Q000096:biosynthesis; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014443:Tyrosine; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases