Multiple forms of the SH2-containing inositol phosphatase, SHIP, are generated by C-terminal truncation.
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.
Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, British Columbia, Canada.
J E
JE
Damen
L
L
Liu
M D
MD
Ware
M
M
Ermolaeva
P W
PW
Majerus
G
G
Krystal
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002154:Calpain / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D003599:Cytoskeleton / Q000201:enzymology; D051380:GRB2 Adaptor Protein; D006207:Half-Life; D006412:Hematopoietic Stem Cells / Q000201:enzymology; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007527:Isoenzymes; D011506:Proteins; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D002154:Calpain