Tyrosine phosphorylation of SHIP promotes its proteasomal degradation
Received 2 March 2010; received in revised form 2 March 2010; accepted 8 March 2010. published online 22 March 2010.
Objective
The activity of the SH2-containing-phosphatidylinositol-5′-phosphatase (SHIP, also known as SHIP1), a critical hematopoietic-restricted negative regulator of the PI3 kinase (PI3K) pathway, is regulated in large part via its protein levels. We sought to determine the mechanism(s) involved in its downregulation by BCR-ABL and by interleukin (IL)-4.
Materials and Methods
We used Ba/F3p210-tetOFF cells to study the downregulation of SHIP by BCR-ABL and bone marrow−derived macrophages to study SHIP's downregulation by IL-4.
Results
We show herein that BCR-ABL downregulates SHIP, but not SHIP2 or PTEN, and this can be blocked with the Src kinase inhibitor PP2, which inhibits the tyrosine phosphorylation of SHIP, or with the proteasomal inhibitor MG-132. We also show, using anti-SHIP immunoprecipitates, that c-Cbl and Cbl-b are associated with SHIP and that BCR-ABL induces SHIP's polyubiquitination. This ubiquitination can be blocked with PP2, consistent with the tyrosine phosphorylation of SHIP acting as a signal for its ubiquitination. In bone marrow−derived macrophages, IL-4 also leads to the proteasomal degradation of SHIP but, unlike in Ba/F3p210-tetOFF cells, SHIP2 is also proteasomally degraded and the degradation of both inositol phosphatases can be prevented with PP2 or MG-132.
Conclusion
Our results suggest that SHIP protein levels can be reduced via BCR-ABL and/or Src family member-induced tyrosine phosphorylation of SHIP because this triggers its polyubiquitination and degradation within the proteasome.
aThe Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada
cDepartment of Immunology and Parasitology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan
dDepartment of Immunobiology, Yale University, School of Medicine and the Howard Hughes Medical Institute, New Haven, Conn., USA
eDivision of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, Calif., USA
fDepartment of Pediatrics, Division of Gastroenterology, BC Children's Hospital and University of British Columbia, Vancouver, BC, Canada
gNSERM U935, Université de Poitiers and Service d'Hématologie et d'Oncologie Biologique, CHU de Poitiers, Poitiers, France
Offprint requests to: Gerald Krystal, Ph.D., The Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada
ABSTRACT