Selective effect of poly(lysine) on the enhancement of the lyn tyrosine protein kinase activity

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Abstract
A tyrosine protein kinase (TPK-I), isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and recently identified as the product of the lyn oncogene [Brunati, A. M., Donella-Deana, A., Ralph, S., Marchiori, F., Borin, G., Fischer, S. & Pinna, L. A. (1991) Biochim. Biophys. Acta 1901, 123-126], is stimulated by a variety of effectors, including poly(lysine), heparin and very high NaCl concentrations. The efficacy of these compounds is variably dependent on the nature of the phosphoacceptor peptide substrates. Here we show that poly(lysine), but neither NaCl nor heparin, specifically enhances the phosphorylation efficiency of lyn TPK for the peptide EDNEYTA (src peptide). It reproduces the main autophosphorylation site of pp60c-src (Tyr416), which is entirely conserved in lyn TPK. The favourable effect of poly(lysine) is accounted for by both a dramatic drop of the Km value (70 microM versus 670 microM) and more than a threefold increase in Vmax. The effect is not so evident with a variety of different peptides, either unrelated to the src peptide (e.g. angiotensin II, AAYAA) or derived from the src peptide by single or multiple substitutions of the residues located on the N-terminal side of tyrosine. In particular, the responsiveness to poly(lysine) is dramatically reduced whenever alanine is replaced for either asparagine at position -2 or glutamic acid at position -1, either in the src heptapeptide or in its shorter derivative, the pentapeptide NEYTA. In sharp contrast, the favourable effect of 2 M NaCl, which is invariably accounted for only by an increased Vmax, is especially evident with peptides like angiotensin II and AAYAA, whose phosphorylation is less sensitive to poly(lysine) stimulation. The phosphorylation of the src peptides are actually inhibited rather than stimulated by 2 M NaCl. Consistent with this, lyn TPK autophosphorylation is also dramatically stimulated by poly(lysine) while being inhibited by 2 M NaCl. These data show that poly(lysine) deeply alters the selectivity of lyn TPK by conferring to it an enhanced activity and an especially high affinity toward peptides that reproduce the conserved autophosphorylation site of the TPK of the src family. It is suggested that endogenous compound, whose effect is mimicked by poly(lysine) in vitro, may play a relevant role in determining the specificity of lyn TPK in vivo and possibly of other TPK of the src family.