The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species

Albert Weijland, Gitte Neubauer, Sara A. Courtneidge, Matthias Mann, Rik K. Wierenga, Giulio Superti-Furga

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unsepa rated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (k(cat)) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The k(cat) values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min-1), but the apparent K(cat values differ significantly (approximately 3 μM and 10 μM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K(m) values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.

Original languageEnglish (US)
Pages (from-to)756-764
Number of pages9
JournalEuropean Journal of Biochemistry
Volume240
Issue number3
DOIs
StatePublished - Jan 1 1996

Keywords

  • Auto-phosphorylation
  • Electrospray mass spectrometry
  • Phosphorylation mapping
  • Schizosaccharomyces pombe
  • Src protein tyrosine kinase

ASJC Scopus subject areas

  • Biochemistry

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