Akin to c-Raf, Lyn bound to RB, specifically to pS608RB. differentiation. Part of this mechanism reflects promoting cell cycle arrest via ATRA-induced upregulation of the p27 Kip1 CDKI. Roscovitine also enhanced the ATRA-induced nuclear enrichment of other signaling molecules traditionally perceived as cytoplasmic promoters of proliferation, but now known to promote differentiation; in particular: SFKs, Lyn, Fgr; adaptor proteins, c-Cbl, SLP-76; a guanine exchange factor, Vav1; and a transcription factor, IRF-1. Akin to c-Raf, Lyn bound to RB, specifically to pS608RB. Lyn-pS608RB association was greatly diminished by ATRA and essentially lost in ATRA plus roscovitine treated cells. Interestingly Lyn-KD enhanced such ATRA-induced nuclear signaling and differentiation and made roscovitine more effective. ATRA thus mobilized traditionally cytoplasmic signaling molecules to the nucleus where they drove Oxaceprol differentiation which were further enhanced by roscovitine. retinoic acid (ATRA), a retinoid metabolite of vitamin A, regulates gene expression [1] in a number of physiological processes, including morphogenesis, vision, growth, metabolism, differentiation and cellular homeostasis [2]. For cancer chemotherapy, ATRA is prominent as a differentiation-inducing therapeutic for acute promyelocytic leukemia (APL) [3, 4], which is a FAB (French American British classification) M3 subtype of acute myeloid leukemia (AML). APL is cytogenetically characterized by a t (15;17) (q22; q12) translocation that results in a PML-RAR fusion protein seminal to the disease [5]. The classical paradigm of ATRA-induced differentiation in leukemia cells focuses on RAR and retinoid X receptors, which are transcription factors activated by binding to their ligands. However, other signaling pathways, particularly mitogen-activated protein kinase (MAPK), have been found to be necessary for RAR and RXR to transcriptionally activate and induce differentiation and G1/G0 cell cycle arrest [6C8]. The Raf/Mek/Erk axis is imbedded in the ATRA-induced signalsome which also includes Src family kinases Fgr and Lyn, PI3K, c-Cbl, SLP-76, Vav1, 14-3-3 and KSR1, Rabbit polyclonal to AnnexinA1 plus transcription factors AhR and IRF1 [9C12]. HL-60 cells have been an archetype model for analyzing effects of ATRA < 0.05 comparing ATRA-treated samples to ATRA/roscovitine-treated samples. (D) TATA binding protein (TBP) was the loading control. (E) Roscovitine augments ATRA-induced reduction of nuclear Lyn interaction with pS608 phosphorylated RB tumor suppressor protein. Co-immunoprecipitation was done using Lyn as bait. (F) Nuclear RB binds Lyn in ATRA and ATRA plus roscovitine treated cells. Co-immunoprecipitation was done in treated cells using RB as bait. Vav also binds RB in these cells. An equal amount of pre-cleared nuclear lysate was collected 72 h post treatment and incubated overnight with 1:100 concentration of the precipitating antibody with magnetic beads and resolved on 12 % polyacrylamide gels. All blots shown are representative of three replicates. Given that the above results show the presence of SFKs in the nucleus, we explored the association between Lyn and RB. Immunoprecipitation showed that Lyn complexed with RB and in particular its S608 phosphorylated form. ATRA reduced the amount of Lyn complexed with pS608 RB. At the same time Lyn expression in the nucleus was enhanced by ATRA in addition to gains from relieving the amount bound to pS608 RB. Roscovitine enhanced these ATRA-induced effects. Roscovitine Oxaceprol thus again potentiated ATRA effects, but it did not cause such effects by itself (Figure 2E). While Lyn binding to pS608 RB was greatly diminished in ATRA treated cells and essentially lost in ATRA plus roscovitine treated Oxaceprol cells, Lyn binding to RB was detectable in both (Figure 2F), consistent with preferential binding to non-pS608 phosphorylated RB in the treated cells. Interestingly, like Lyn, Vav likewise binds RB. ATRA plus roscovitine co-treatment enhances nuclear VAV1 expression Vav1 is a GEF found in both the cytoplasm and nuclear compartments and is the only member of the Vav family expressed in hematopoietic cells [41]. Vav was identified as a component of the cytoplasmic signalsome that drives differentiation [29]. We explored whether Vav was regulated by ATRA and roscovitine as were the related signaling molecules, c-Raf and Lyn, which were also signalsome components. Cells were untreated controls or treated with ATRA, roscovitine or ATRA plus roscovitine. After 72 h of culture, we collected the cell lysate, extracted nuclear protein, and analyzed the expression of Vav. ATRA alone up-regulated nuclear Vav1 expression, and co-treatment with roscovitine caused further nuclear enrichment (Figure 3A). We next searched for Vav partners of regulatory significance in the nucleus. Using immunoprecipitation with RB as bait, we also noted a novel Vav1-RB interaction in the nucleus (Figure 2F). We infer that.