SRC tail phosphorylation is limited by structural changes in the regulatory tyrosine kinase Csk.
Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.
Lieser
Scot A
SA
Department of Chemistry and Biochemistry and Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.
Shaffer
Jennifer
J
Adams
Joseph A
JA
D000081247:CSK Tyrosine-Protein Kinase; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D015394:Molecular Structure; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human