Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference.
The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography. The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa). Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized. The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II. Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris. Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer. Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively). All three peptides were shown to be substrates for the recombinant tyrosine kinase. Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases.
Radziejewski
C
C
Laboratory of Virology, Rockefeller University, New York, New York 10021.
Miller
W T
WT
Mobashery
S
S
Goldberg
A R
AR
Kaiser
E T
ET
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification*; D016392:Proto-Oncogene Proteins pp60(c-src); D011994:Recombinant Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)