Correlation of the phosphorylation states of pp60c-src with tyrosine kinase activity: the intramolecular pY530-SH2 complex retains significant activity if Y419 is phosphorylated.
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.
Boerner
R J
RJ
Department of Biochemistry, Glaxo Wellcome Inc., Research Triangle Park, North Carolina 27709, USA.
Kassel
D B
DB
Barker
S C
SC
Ellis
B
B
DeLacy
P
P
Knight
W B
WB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D010748:Phosphopeptides; D011994:Recombinant Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D014357:Trypsin