Isolation from spleen of a 57-kDa protein substrate of the tyrosine kinase Lyn. Identification as a protein related to protein disulfide-isomerase and localisation of the phosphorylation sites.
A 57-kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src-like tyrosine kinase Lyn purified from the same source with a Km of 0.34 microM. The tyrosine kinases c-Fgr, Fyn, C-terminal Src kinase and p72syk, as well as the Ser/Thr-specific cAMP-dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C-terminal Src kinase, which acts to down-regulate the Src-like protein-tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide-isomerase which belongs to the superfamily of Cys-Gly-His-Cys-containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C-terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti-Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested 'processive phosphorylation' model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s).
Donella-Deana
A
A
Dipartimento di Chimica Biologica, University of Padova, Italy.
James
P
P
Staudenmann
W
W
Cesaro
L
L
Marin
O
O
Brunati
A M
AM
Ruzzene
M
M
Pinna
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D019704:Protein Disulfide-Isomerases; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013154:Spleen / Q000378:metabolism*; D013379:Substrate Specificity; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D011506:Proteins; D019061:src-Family Kinases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases