Cloning and characterization of SH2-containing inositol 5-phosphatase, SHIP.

摘要

A 145-kDa protein originally cloned from murine cells and named SH2-containing inositol 5-phosphatase (SHIP), becomes both tyrosine phosphorylated and associated with the adapter protein Shc following stimulation of hematopoietic cells with a wide variety of extracellular stimuli. Here we describe the cloning of the human homologue of SHIP from a human megakaryocytic cell line (MO7e) λgt11 cDNA library. Northern blot analysis indicates human SHIP gene is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of the cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with murine SHIP. Contained within the defined open reading frame is an N-terminal, Group I Src homology 2 (SH2) domain, two NPxY-containing phosphotyrosine binding (PTB) domain ligand motifs, a C-terminal proline-rich region, and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization mapped human SHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37. Examination of proteins capable of binding the six major PxxP motifs of SHIP identified Grb2 and kinases of the Src family as potential binding partners. Both Grb2 and Lyn bound SHIP in unstimulated WEHI 231 B cells but only Lyn binding increased with activation of the B cell antigen receptor (BCR). Experiments with the Src family kinase inhibitor, PP2, revealed that SHIP's tyrosine phosphorylation is regulated by Src-like kinases following activation of BCR, interleukin-3 (IL-3) receptor, and stem cell factor (SCF) receptor signalling. Functional analysis of various SHIP domains was undertaken by reintroduction of wild type and or mutanted forms of SHIP into bone marrow-derived mast cells obtained form SHIP−/− mice. Results revealed that the phosphatase domain and proline-rich C-terminus were both vital to SHIP's ability to hydrolyze SCF-induced phosphatidylinositol 3,4,5-P ₃ (PtdIns3,4,5P₃), inhibit extracellular calcium entry, and prevent degranulation in these cells, while the inability of SHIP to interact with Shc only partially affected SHIP's regulation of these processes.