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

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. Retinoic acid (RA), 2 a form of vitamin A, can cause activation of MAPK signaling leading to induced cell differentiation and G 0 cell cycle arrest (1-2). Because of this, RA has been used therapeutically for the chemoprevention and treatment of cancer (3), notably acute promyelocytic leukemia, making its mechanism of action of significant interest. The HL-60 human myeloblastic leukemia cell line serves as a model for studying differentiation induction therapy and the mechanism of RA action (4 -6). The HL-60 cell line was established from peripheral blood leukocytes of a patient originally diagnosed with acute promyelocytic leukemia (4), which was retrospectively re-evaluated to be acute myeloblastic leukemia (7). HL-60 cells undergo G 0 cell cycle arrest and myeloid differentiation in response to RA or monocytic differentiation in response to 1,25-dihydroxyvitamin D 3 . Induced differentiation depends on hyperactive MAPK signaling (1, 8). c-Cbl contributes propulsion to this process (9, 10). It is part of a signaling complex connected to the CD38 receptor that activates the RAF/MEK/ ERK MAPK signaling axis to drive differentiation and G 0 cell cycle arrest (9).The 120-kDa product of the c-Cbl protooncogene is a prominent component of tyrosine kinase signaling cascades downstream of activated cell surface receptors, including the epidermal growth factor receptor, the B cell receptor, Fc receptor, and cytokine receptors (11,12). 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). The Ring finger domain of c-Cbl presents the most conserved region among Cbl family proteins, and it is implicated as an important element in the function of Cbl proteins (17)(18)(19). The TKB domain contains a four-helix bundle, EF-hand calcium-binding domain, and a variant SH2 domain that binds to phosphotyrosine residues (13,20). Several features of the Cbl/Zap-70 complex (13) suggest that the four-helix bundle, EF-hand, and SH2 domains together form an interactive structure that is crucial for phosphoprotein recognition. Studies by Thien et al. (21) indicated that the TKB mutation, G306E, fails to bind phosphotyrosine residues and is a loss of function mutation in the c-Cbl TKB domain.Cbl interacts with a number of intracellular signaling molecules including various receptors, adaptors, cytoskeletal proteins, ubiquitin, and structural proteins via its various domains (22,23). One of the receptors is CD38. The human c...

Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity

“…This suggests that the c-Cbl/CD38 interaction may cooperatively drive MAPK signaling and other aspects of ATRA therapy. This is consistent with a report that a c-Cbl tyrosine kinase binding domain mutant (G306E) that does not bind CD38 also fails to drive MAPK signaling and differentiation [16]. …”

“…Cells were harvested, fixed, and permeabilized as previously described [16]. Cells were resuspended in 200 –l of PBS containing 5 –l of primary rabbit anti-SLP-76 or rabbit anti-Vav1 and mouse anti-CD38 antibodies and then stained with Alexa-350 and 430-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies, respectively, from Invitrogen.…”

“…CD38 forms a complex with c-Cbl [15,16] and CD38 agonist ligand interaction results in c-Cbl phosphorylation [3]. c-Cbl is an E3 ubiquitin ligase and adaptor molecule that, like CD38, promotes mitogen-activated protein kinase (MAPK) signaling and ATRA-induced differentiation when overexpressed [3,15,16].…”

“…c-Cbl is an E3 ubiquitin ligase and adaptor molecule that, like CD38, promotes mitogen-activated protein kinase (MAPK) signaling and ATRA-induced differentiation when overexpressed [3,15,16]. This suggests that the c-Cbl/CD38 interaction may cooperatively drive MAPK signaling and other aspects of ATRA therapy.…”

“…After ATRA treatment Vav1/c-Cbl complexes are detectable in the cytosol, while Vav1/SLP-76 interactions are predominant in nuclei [24]. SLP-76 is also upregulated after ATRA and forms a complex with c-Cbl [16]. Co-expression of SLP-76 with CSF-1/c-FMS enhances ATRA-induced ERK activation, G0 cell cycle arrest, and a number of additional differentiation markers [21].…”