Biochemical analysis of interleukin-2 receptor beta chain phosphorylation by p56(lck).
Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor. In this study, we have used subdomains of the IL-2 receptor beta chain (IL-2Rbeta) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56(lck), one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56(lck) phosphorylates in vitro tyrosine residues within the IL-2Rbeta chain but not those within the IL-2Rgamma chain. p56(lck) phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2Rbeta chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either Tyr-355 or Tyr-361. p56(lck) also phosphorylates very efficiently the two tyrosines present in the IL-2Rbeta chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56(lck) with the IL-2Rbeta chain which was found to depend on a short stretch of the IL-2Rbeta chain acidic subdomain, and to be independent of the presence of its tyrosine residues.
Delespine-Carmagnat
M
M
INSERM Unit 461, Faculté de Pharmacie Paris-XI, Châtenay-Malabry, France.
Bouvier
G
G
Allée
G
G
Fagard
R
R
Bertoglio
J
J
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D015375:Receptors, Interleukin-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)