PURIFICATION AND MOLECULAR-CLONING OF SH2-CONTAINING AND SH3-CONTAINING INOSITOL POLYPHOSPHATE-5-PHOSPHATASE, WHICH IS INVOLVED IN THE SIGNALING PATHWAY OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR, ERYTHROPOIETIN, AND BCR-ABL

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase re ceptors to Ras and make tyrosine-kinase functionally associated with r eceptors and pas in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domai ns bind to proteins containing proline-rich regions or tyrosine-phosph orylated proteins and contribute to the association of Grb2/Ash and Sh e with other signaling molecules. However, there could remain unidenti fied signaling molecules that physically and functionally interact wit h these adapter proteins and have biologically important roles in the signaling pathways, By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135- kD protein (pp135) are associated with Grb2/Ash. We have also found th at they become tyrosine-phosphorylated by treatment of a human leukemi a cell line, UT-7, with granulocyte-macrophage colony-stimulating fact or (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) enc oding the pp135 using a cDNA probe, which was obtained by the degenera te polymerase chain reaction based on a peptide sequence of the purifi ed pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino a cid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which co ntains two motifs conserved among the inositol polyphosphate-5-phospha tase proteins, was shown to have the inositol polyphosphate-5-phosphat ase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CS F. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with S hc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important rol e in the signaling pathway triggered by GM-CSF and in the transformati on of hematopoietic cells caused by Bcr-Abl. (C) 1997 by The American Society of Hematology.