Hierarchical phosphorylation of a 50-kDa protein by protein tyrosine kinases TPK-IIB and C-Fgr, and its identification as HS1 hematopoietic-lineage cell-specific protein.
TPK-IIB is an acidophilic protein tyrosine kinase devoid of autophosphorylation activity and unrelated to the Src family kinases [Marin, O., Donella-Deana, A., Brunati, A.M., Fischer, S. & Pinna, L. A. (1991) J. Biol. Chem. 266, 17798-17803]. Here, we describe the purification to homogeneity of a 50-kDa prominent substrate (p50) of TPK-IIB from rat spleen homogenates. Microsequence analysis of fragments from purified p50 discloses its homology to HS1, a hematopoietic-lineage cell-specific protein implicated in B-cell-antigen receptor-mediated signalling [Yamanashi, Y., Okada, M., Semba, T., Yamori, T., Umemori, H., Tsunasawa, S., Toyoshima, K., Kitamura, D., Watanabe, T. & Yamamoto, T. (1993) Proc. Natl Acad. Sci. USA 90, 3631-3635]. p50 is an excellent substrate for TPK-IIB, exhibiting very favourable kinetic constants (Km = 0.07 microM, kcat = 1.5) and incorporating up to 4 mol P/mol protein. p50 is, however, a weak substrate for the Src-related protein kinases Lyn and c-Fgr. Once phosphorylated by TPK-IIB, however, p50 is converted into a good substrate for c-Fgr and, to a lesser extent, for Lyn. p50 phosphorylated by TPK-IIB associates with c-Fgr and Lyn, as judged by co-immunoprecipitation with anti-Fgr IgG and anti-Lyn IgG, respectively. These data suggest the involvement of TPK-IIB in B-cell-antigen receptor-mediated signalling, and support the idea that phosphorylation by TPK-IIB might be a prerequisite for the recruitment of certain protein substrates by Src-related protein tyrosine kinases.
Brunati
A M
AM
Dipartimento di Chimica Biologica, Università di Padova, Italy.
Ruzzene
M
M
James
P
P
Guerra
B
B
Pinna
L A
LA
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000081247:CSK Tyrosine-Protein Kinase; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D013154:Spleen / Q000378:metabolism; D019061:src-Family Kinases
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; C061909:HCLS1 protein, human; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human