Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1199-1205
Multiple Forms of the SH2-Containing Inositol Phosphatase,
SHIP, Are Generated by C-Terminal Truncation
Jacqueline E. Damen,
Ling Liu,
Mark D. Ware,
Marina Ermolaeva,
Philip W. Majerus, and
Gerald Krystal
From the Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, British
Columbia, Canada; and Washington University School of Medicine, St
Louis, MO.
The SH2-containing inositol phosphatase, SHIP, often
appears as multiple bands in anti-SHIP immunoblots. To characterize
these bands, antisera were generated against the N-terminal (anti-N), mid-region (anti-M), and C-terminal (anti-C) portions of SHIP. Immunoprecipitation and immunoblotting studies showed that 145-, 135-, 125-, and 110-kD bands were detected in lysates from the murine
hematopoietic cell line, DA-ER, with either anti-N or anti-M antisera,
whereas only the 145- and 135-kD bands were recognized by the anti-C
antiserum. This finding suggested that the smaller proteins might be
C-terminal truncations of the full-length SHIP. To confirm this and
determine if these proteins arose through alternate splicing or
posttranslational cleavage, a 5
-hemagglutin (HA)-tagged
full-length SHIP cDNA was expressed in these cells. We observed, via
Western analysis with anti-HA antibodies, the same 4 bands with either
anti-N or anti-M and only the 145- and 135-kD bands with anti-C
immunoprecipitation. After interleukin-3 stimulation of
HA-SHIP-expressing DA-ER cells, only the 145-kD form coprecipitated
with Shc, raising the possibility that different forms of SHIP may have
distinct intracellular sites. This was confirmed by subcellular
fractionation, which showed that only the 110-kD form is present in the
cytoskeleton of DA-ER cells. This 110-kD form possesses the same
PIP3 5-ptase activity as the 145-kD form and can be
generated from the latter in vitro by digestion with calpain. It is
therefore possible that the different forms of SHIP are generated in
vivo by calpain-mediated C-terminal truncations and perform distinct
functions within hematopoietic cells.
© 1998 by The American Society of Hematology.
