A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.
Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2-and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAK Pro-2 ) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.The x-ray crystal structures of Src and Hck (a Src family member) in their inactive form have revealed intramolecular interactions that function to regulate these protein-tyrosine kinases (PTKs) 1 (1, 2). As expected, the tyrosine phosphorylated, negative regulatory element binds to the SH2 domain (1, 2). In addition, the SH3 domain binds to a polypeptide linking the SH2 and catalytic domains which assumes a polyproline type II helix that is structurally similar to SH3-binding sites (3)(4)(5). In this conformation, ␣-helix C in the small lobe of the catalytic domain is displaced altering the conformation of the ATP-binding site. It is noteworthy that the sequences that bind the SH2 and SH3 domains do not conform to high affinity binding sites (3, 6 -8).Dephosphorylation of the negative regulatory element is one mechanism by which these PTKs can be activated. Consequently, this element fails to bind the SH2 domain and the inactive conformation cannot be maintained. A second mechanism by which the Src-like kinases could be activated is by the disruption of the weaker intramolecular SH2-SH3 interactions by the formation of stronger intermolecular SH2-SH3 domain interactions. It has been shown that disruption of the SH2-or SH3-mediated intramolecular interactions in vitro enhances the activity of the enzyme (9 -12). It is likely that similar mechanisms operate in vivo and that complex formation between Src and its binding partners, like FAK, results in its activation. FAK is a 125-kDa PTK that localizes to focal adhesions and functions in integrin signaling (13,14). Integrin-dependent cell adhesion or cross-linking of cell surface integrins induces the tyrosine phosphorylation of FAK and stimulates its activity (14 -18). The major site of FAK autophosphorylation is Tyr-397, whereas other sites of FAK phosphorylation, e.g. Tyr-576, -577, and -925, are phosphorylated by Src family PTKs (19 -21). The sequence flanking Tyr-397 conforms to a high affinity binding site for the Src SH2 domain and serves as a bind...
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.
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