Rapid digestion of pp60c-src tyrosine kinase (src TK) in combination with electrospray ionization mass spectrometry enabled the determination of the time course for autophosphorylation of three tyrosine sites (Y338, Y419, and Y530) and a correlation with src TK activity. A form of src TK was purified from baculovirus-infected cells which contains only Y338 partially phosphorylated. Incubation with MgATP increases the phosphorylation of all three sites. The autophosphorylation and dephosphorylation of Y419 are directly correlated with the level of src TK activity. The role of Y338 phosphorylation is unknown. Conditions resulting in complete autophosphorylation of Y530 were identified by electrospray ionization mass spectrometry. Surface plasmon resonance detection and size exclusion chromatography provide direct evidence for an intramolecular pY530-SH2 complex, supporting previous models [Matsuda, M., Mayer, B.J., Fukui, Y., & Hanafusa, H. (1990) Science 248, 1537-1539]. Contrary to these models, when the enzyme is fully phosphorylated on Y530, phosphorylated on Y419, and present only as the intramolecular pY530-SH2 complex, 20% of the kinase activity is retained. In addition, the k(m)'s for substrates are unaffected. Disruption of the pY530-SH2 interaction and activation of kinase activity by a high-affinity SH2 ligand yield a Kactivation which is 200-fold larger than the Kd for ligand binding to the uncomplexed src SH2 domain. These data suggest a Keq of 200 (unitless) for the intramolecular association of pY530 with the SH2 domain. We propose that the pY530-SH2 interaction modulates signal transduction by down-regulating src TK activity 5-fold, and perhaps more importantly by inhibiting protein-protein interactions with the SH2 domain. These results have significant implications relative to the development of SH2 ligands as therapeutics to control aberrant signal transduction. These ligands will be 200-fold less effective at inhibiting protein-protein interactions versus down-regulated src TK than versus activated src TK. This should minimize activation of src TK activity in normal cells and lead to an increased therapeutic index.

The kinetic mechanism of the pp60c-src tyrosine kinase (src TK) reaction was investigated in the forward and reverse directions. In the forward direction, initial velocities obtained by varying ATP and the peptide (FGE)3Y(GEF)2GD indicated a sequential addition of the two substrates. The peptide analog, (FGE)3F(GEF)2GD, was a competitive inhibitor versus the peptide substrate and a noncompetitive inhibitor versus MgATP. Interestingly, the tyrosine hydroxyl group imparts only a 6-fold increase in binding. AMP-PCP was a competitive inhibitor versus MgATP and a noncompetitive inhibitor versus the peptide substrate. These results prove that the addition of substrates is random. Furthermore, there appears to be little binding synergy as the KiMgATP approximately equal to 2.4KmMgATP. The phosphorylated peptide (FGE)3-pY-(GEF)2GD was a competitive inhibitor versus peptide and a noncompetitive inhibitor against MgATP, suggesting that a dead end complex can form between MgATP, the phosphorylated peptide product, and the enzyme. The reverse reaction was investigated by varying ADP and the phosphopeptide. (FGE)3-pY-(GEF)2GD. The initial velocity pattern was indicative of a sequential mechanism. There was even less binding synergy in the reverse direction as the KiMgADP approximately equal to 1.4KmMgADP. AMP-CP was a competitive inhibitor versus MgADP and a noncompetitive inhibitor versus the phosphopeptide. (FGE)3F(GEF)2GD was a competitive inhibitor versus the phosphopeptide and a noncompetitive inhibitor versus MgADP. These data prove that addition of the substrates in the reverse direction is random. (FGE)3Y(GEF)2GD was a competitive inhibitor against peptide substrate and a noncompetitive inhibitor against MgADP; therefore a dead end complex can form between MgADP, (FGE)3Y(GEF)2GD, and the enzyme. These results indicate that the src TK reaction follows a sequential bi-biequilibrium random mechanism in both directions, with dead end complexes forming when either MgATP and (FGE)3-pY-(GEF)2GD or MgADP and (FGE)3Y(GEF)2GD bind to the enzyme. The kinetic constants determined from the forward and reverse reactions were used in the Haldane equation to determine a K(eq) constant for the forward reaction of 10.1, corresponding to a delta G of -1.4 kcal/mol. This further confirms that the O-P bond of phosphotyrosine is similar in energy to that of the gamma-phosphoryl of MgATP.

Summar~Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor o~ (TNFol) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNFo~ signal transduction. Because TNFot is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNFcc-induced PGE2 production. We found that sphingosine enhanced TNFot-induced PGE2 production by as much as 18-fold over TNFot alone. Sphingosine appeared to stimulate TNFot-induced PGE2 production independent of TNFc~-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNFc*-induced PGE2 production or the stimulatory effect of sphingosine. TNFot stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNFot nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNFo~-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 rain) exposure of cells to sphingosine dramatically increased TNFot-induced PGE2 production. A potential mechanism by which sphingosine could increase TNFo~-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNFot and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNFc~ on phospholipase A2 activity. It appears that sphingosine affects TNFolinduced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.

The minimum length required for phosphorylation of a peptide by pp60 c-src tyrosine kinase (srcTK) was delineated in this work. Budde (M. D. Anderson University of Texas, personal communication) suggested that the peptide (FGE) 3 Y(GEF) 2 GD (peptide I) was a "good" srcTK substrate. Peptide I yielded a 251-fold higher k cat /K m than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK. This was due to a 38-fold lower K m and a 6.6-fold increase in k cat . N-terminal truncation of up to 8 residues in a series of peptides yielded only a 3-fold decrease in activity. Removal of the final N-terminal residue resulted in a 10-fold loss in substrate activity, primarily as a result of an increase in the K m . C-terminal truncations ending in the amide yielded no significant loss in activity until the Y؉3 residue was removed, which resulted in a 73-fold decrease in k cat /K m relative to peptide I. The latter was due primarily to an increase in K m . The results from peptides truncated on both termini suggest that subsite recognition N-and C-terminal relative to the site of phosphorylation can be examined independently. In addition, the observation that only 5 residues are required for significant substrate activity suggests that small molecule inhibitors based upon interactions with the phosphoacceptor site may be developed.Protein tyrosine kinases (TKs) 1 were initially discovered as either oncogenes or proto-oncogene products, pointing to their therapeutic potential as targets in cancer (for a review of oncogenes, see Pimentel (1989a and1989b)). The phosphorylation equilibrium of protein tyrosine residues is regulated by cytokines and growth factors, pointing to roles for these equilibria in signal transduction pathways. Therefore, control of these equilibria has the potential for therapeutic intervention in diseases including cancer, inflammatory diseases, and diabetes, to name a few.While initially TKs were thought to be nonspecific, more recent work has demonstrated that they do indeed phosphorylate specific substrates in vivo (for example, see Ogawa et al. (1994)). The study of these enzymes has been hampered by the lack of specific peptide substrates. The specificity requirements for amino acid sequences remain to be elucidated. While several groups proposed that the specificity of TKs was not governed by the amino acid sequence surrounding the tyrosine (Tinker et al., 1988;Radziejewski et al., 1989), more recent studies have suggested that there is specific recognition of proximal residues. For example, Garcia et al. (1993) reported differences in the peptide sequences recognized by pp60 v-src 2 and v-abl TK. These workers found that substitution of N 3 for D in the YϪ1 4 position of KKSRGDYMTMQIG, a peptide based upon a phosphorylation site of insulin receptor substrate-1, resulted in complete loss of substrate activity for both TKs. Substitution of I for M in the Yϩ1 position resulted in a 4-fold loss in catalytic efficiency for pp60 v-src but a 10-fold increase in the catalytic...

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