Tyr394 and Tyr505 are autophosphorylated in recombinant Lck protein-tyrosine kinase expressed in Escherichia coli.
The activity of the Src family protein-tyrosine kinase p56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p50csk which negatively modulates p56lck activity. To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S-transferase expression system to express wild-type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase-deficient p56lck with a mutation of the ATP-binding site [K273E]Lck and a double mutant [Y394F, Y505F]Lck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild-type Lck both in vivo and in vitro. Wild-type Lck, [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinase-deficient mutant[K273E]Lck and the double mutant [Y394F, Y505F]Lck did not react with monoclonal anti-phosphotyrosine antibody [anti-Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein-tyrosine kinase activities, and therefore tyrosine phosphorylations of wild-type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that p56lck can undergo autophosphorylation on these two tyrosine residues. We propose that autophosphorylation at Tyr505 of p56lck may represent an accessory mechanism for the down-regulation of the tyrosine kinase activity of p56lck.
Jullien
P
P
Institut National de la Santé et de la Recherche Médicale Unité 332, Université Paris René Descartes, France.
Bougeret
C
C
Camoin
L
L
Bodeus
M
M
Durand
H
H
Disanto
J P
JP
Fischer
S
S
Benarous
R
R
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000201:enzymology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)