Calcium-dependent isolation of the 36-kilodalton substrate of pp60src-kinase. Fractionation of the phosphorylated and unphosphorylated species.
In this paper, we present a new and simple purification of the 36-kDa protein, a major substrate of both viral and growth factor-receptor associated tyrosine protein kinases, and its complex from normal and Schmidt-Ruppin strain Rous sarcoma virus-transformed chicken embryo fibroblasts that employs a DEAE-Sephacel column and introduces the calcium-dependent adsorption of 36-kDa protein. The use of EGTA step gradients differentially elutes the 36-kDa molecules from the DEAE-Sephacel column. An average total yield of the 36-kDa protein in all fractions approached 80% and represented 0.78% of the [35S] methionine-labeled cellular protein. A purity of 95-99% was obtained with a yield of 60% in the central elution fractions from normal or Rous sarcoma virus-transformed chicken embryo fibroblasts. Furthermore, 2 mM EGTA elutes poorly phosphorylated molecules while heavily phosphorylated 36-kDa protein requires 4 or 6 M EGTA; a small residual fraction is released at 8-10 mM EGTA. If the EGTA step gradients were neutralized with Ca2+ ion, elution of the 36-kDa protein is inhibited. The complex of the 36-kDa protein and the 6-10-kDa protein may not be dependent on the phosphorylation as the associated 6-10-kDa peptide is seen in all fractions containing the 36-kDa protein. Tyrosine phosphorylation of the 36-kDa protein is increased 2-3-fold following a short term incubation of whole cells with micromolar vanadate. The elution pattern (but not intensity) of the 36-kDa protein obtained from lysates of vanadate-treated cells was identical to untreated cell lysates. The additional phosphorylation appears to result from a recruitment of unphosphorylated 36-kDa protein as the position (but not intensity) of the phosphorylated tryptic peptides is unchanged. We conclude that the function of the 36-kDa protein may be calcium ion-dependent and may be influenced by the phosphorylation state of the protein.
D000818:Animals; D017302:Annexins; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002118:Calcium; D002471:Cell Transformation, Neoplastic; D002642:Chick Embryo; D005347:Fibroblasts / Q000032:analysis; D007106:Immune Sera; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D015688:Oncogene Protein pp60(v-src); D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000378:metabolism
D017302:Annexins; D007106:Immune Sera; D008565:Membrane Proteins; D012191:Retroviridae Proteins; D011494:Protein Kinases; D015688:Oncogene Protein pp60(v-src); D002118:Calcium