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
H. Odai et al., 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, Blood, 89(8), 1997, pp. 2745-2756
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.