C-Cbl Tyrosine Kinase-binding Domain Mutant G306E Abolishes the Interaction of c-Cbl with CD38 and Fails to Promote Retinoic Acid-induced Cell Differentiation and G0 Arrest

Miaoqing Shen,Andrew Yen

doi:10.1074/jbc.m109.014241

Abstract

Retinoic acid (RA) causes HL-60 human myeloblastic leukemia cell myeloid differentiation that is dependent on MAPK signaling. The process is propelled by c-Cbl, which binds the CD38 receptor as part of a signaling complex generating MAPK signaling. Here we report that the capability of c-Cbl to do this is lost in the G306E tyrosine kinase-binding domain mutant. Unlike wild-type (WT) c-Cbl, the G306E mutant c-Cbl fails to propel RA-induced differentiation, and disrupts the normal association with CD38. The G306E mutant does, like WT c-Cbl, co-immunoprecipitate with Vav, Slp-76, and p38. But unlike WT c-Cbl, does not cause MAPK signaling. In contrast, the C381A Ring finger domain mutant functions like WT c-Cbl. It binds CD38 and is part of the same apparent c-Cbl/Slp-76/Vav/p38 signaling complex. The C381A mutant causes MAPK signaling and propels RA-induced differentiation. In addition to HL-60 cells and their WT or mutant c-Cbl stable transfectants, the c-Cbl/Vav/Slp-76 complex is also found in NB4 cells where c-Cbl was previously also found to bind CD38. The data are consistent with a model in which the G306E mutant c-Cbl forms a signaling complex that includes Slp-76, Vav, and p38; but does not drive MAPK signaling because it fails to bind the CD38 receptor. Without the G306E mutation the c-Cbl unites CD38 with the signaling complex and delivers a MAPK signal that drives RA-induced differentiation. The results demonstrate the importance of the Gly306 residue in the ability of c-Cbl to propel RA-induced differentiation.

Highlights

Institutes of Health of the USPHS and New York State Stem Cell Science. 1 To whom correspondence should be addressed: T4-008, VRT, Ithaca, NY differentiation induction therapy and the mechanism of Retinoic acid (RA) action (4 – 6)
Cbl proteins have a highly conserved N-terminal domain termed the tyrosine kinase-binding (TKB) domain, which binds to phosphotyrosines on activated receptor-tyrosine kinases and other signaling proteins [13, 14], a short linker region, and a Ring finger domain that binds to ubiquitin-conjugating enzymes [15, 16]
Cell Culture—Human myeloblastic leukemia cells (HL-60) and their derivatives (WT c-Cbl, c-Cbl mutants G306E and C381A stable transfectants) were grown in a humidified atmosphere of 5% CO2 at 37 °C and maintained in RPMI 1640 supplemented with 5% fetal bovine serum (Invitrogen)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Human myeloblastic leukemia cells (HL-60) and their derivatives (WT c-Cbl, c-Cbl mutants G306E and C381A stable transfectants) were grown in a humidified atmosphere of 5% CO2 at 37 °C and maintained in RPMI 1640 supplemented with 5% fetal bovine serum (Invitrogen). Following incubation and two washes, cells were stained with Alexa 647-conjugated antiphospho-p44/42 MAPK antibody (Cell Signaling, Beverly, MA) for 1 h and analyzed by flow cytometry using 633-nm red laser excitation with emitted fluorescence reflected from a dichroic 735 long-pass through a 660/20 band-pass. Cells were incubated at room temperature for 1 h and analyzed by flow cytometry (BD LSRII) using 488-nm excitation and collected through 550 long-pass dichroic and a 576/26 band-pass filters. Cells were resuspended in 200 ␮l of PBS containing 5 ␮l of primary antibodies and stained with Alexa 350- and 430-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Invitrogen). The antibodies of mouse anti-c-Cbl (BD Biosciences) and rabbit anti-Vav (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-SLP76 or anti-p38 (Cell Signaling, Beverly, MA) were used in c-Cbl/Vav or c-Cbl/Slp or c-Cbl/p38 FRET measurements.

RESULTS

Findings

DISCUSSION