Correlation of the Phosphorylation States of pp60^c-src with Tyrosine Kinase Activity:  The Intramolecular pY530−SH2 Complex Retains Significant Activity If Y419 Is Phosphorylated

Abstract

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.

Intramolecular Regulatory Interactions in the Src Family Kinase Hck Probed by Mutagenesis of a Conserved Tryptophan Residue

“…The purpose of these experiments was to determine whether the C-terminal tail becomes disengaged from the SH2 domain after autophosphorylation on Tyr 416 . Previous experiments on Src demonstrated that a form of Src with an intramolecular interaction between the SH2 domain and the phosphorylated tail was able to autophosphorylate at Tyr 416 (9). We wished to address the question of whether autophosphorylation leads to a subsequent disruption of the SH2-tail interaction.…”

“…Phosphorylation of Tyr 527 by c-Src kinase (Csk) produces an intramolecular interaction between the phosphorylated tail and the SH2 domain that inhibits Src kinase activity (4 -7). Src can also autophosphorylate Tyr 527 , but it is not the primary autophosphorylation site (8,9). The major autophosphorylation site, Tyr 416 , lies in the activation segment, a flexible portion of the catalytic domain near the active site.…”

Altered regulation of Src upon cell detachment protects human lung adenocarcinoma cells from anoikis

“…Phosphorylation of Tyr-419/416 can also activate Src activity in the context of simultaneous phosphorylation at Tyr-530/527 (Boerner et al, 1996;Sun et al, 1998), suggesting that active Src phosphorylated at Tyr-419/416 can not be fully inactivated by phosphorylation at Tyr-530/527 alone. This supports the findings that inactive Src may be the target of other kinases that can phosphorylate Tyr-419/416 and cause Src activation (Chiang and Sefton, 2000).…”

Selected glimpses into the activation and function of Src kinase

“…vSrc) has more modest e ects (Snyder and Bishop, 1984;Snyder et al, 1983), suggesting that other alterations can perturb the normal regulatory processes imparted by the activation loop. Phosphorylation of Tyr(416/ 419) can also activate Src activity in the context of simultaneous phosphorylation at Tyr(527/530) (Boerner et al, 1996;Sun et al, 1998), suggesting ®rst, that active Src phosphorylated at Tyr(416/419) cannot be fully inactivated by phosphorylation at Tyr(527/530) alone, but also requires dephosphorylation of Tyr(416/ 419). Secondly, this supports recent ®ndings that inactive Src family members may be the target of other kinases that can phosphorylate Tyr(416/419) and cause their activation (Chiang and Sefton, 2000).…”