RanBPM Contributes To Trkb Signaling And Regulates Brain-Derived Neurotrophic Factor Induced-Neuronal Morphogenesis And Survival

BackgroudBrain-derived neurotrophic factor (BDNF) is an important member of neurotrophin superfamily which has been proved to be essential in vertebrate nervous system. The actions of NTs depend on two entirely distinct classes of transmembrane receptors, the low-affinity pan-neurotrophin receptor 75kD (p75NTR) and the high-affinity TrkB tyrosine kinase receptor. Activated TrkB stimulates several intracellular signal transduction pathways, such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and phospholipase C-y (PLC-y) pathways.RanBPM (Ran-binding protein in the microtubule-organizing center, also known as RanBP9) is ubiquitously expressed in almost all tissues with relatively high expression in brain, heart and kidney. Recent studies demonstrate RanBPM interacts with a broad spectrum of molecules, acting as a scaffolding protein. Particularly, RanBPM interacts with several cell surface receptors, including MET, LFA-1, L1, Axl/Sky and Plexin-A, contributing to different cell signaling pathways.ObjectiveThough RanBPM has been reported to interact with p75NTR and NGF receptor TrkA, little is known about the role of RanBPM in NTs-induced biological activities. Therefore, in the present study, three questions are raised. First, does RanBPM interact with TrkB, the most abundantly expressed neurotrophin receptor in the mammalian CNS? If so, is RanBPM involved in regulating BDNF-triggered TrkB downstream signaling? Third, does RanBPM influence BDNF/TrkB biological functions such as neuronal morphogenesis and survival?Methods 1. Plasmid constructions2. Rat RanBPM cDNA sequence, siRanBPM construction and knocking-down efficiency measurement3. Rat hippocampal neuron culture and Immunocytochemical staining4. Co-immunoprecipitation assay and Western blot5. Establishment of PC1210 cell line-PC12 cells stably overexpressing TrkB6. TrkB and TrkBY515phosphorylation assays, MAPK and PI3K activation assays7. PC1210 cell differentiation assay and dendritic morphogenesis measurement8. PC1210 cell apoptosis assayResults1. Identification of RanBPM as a novel TrkB-interacting proteinFirst, we constructed eukaryotic expression plasmids pcDNA3.1-HA-hRanBPM with a HA tag to human RanBPM cDNA and pcDNA3.1-Flag-rTrkB with a Flag tag to rat TrkB cDNA. Then we confirmed that RanBPM can bind and colocalize with TrkB through co-immunoprecipitation assay and immunocytochemical staining.2. Tyrosine kinase region in TrkB is responsible for its interaction with RanBPMFirst, we constructed TrkB deletion mutants:ACT (C terminal was deleted) and ATK (tyrosine kinase and C terminal were both deleted). We found TrkB ACT but not ATK could be co-immunoprecipitated by RanBPM, which suggests that TrkB TK region is necessary for binding with RanBPM. Then we constructed TrkB mutants TrkB.T1 (a truncated TrkB isoform which only contains a short cytoplasmic region lacking a TK domain) and T1TK (a chimeric receptor with TrkB kinase region transplanted into TrkB.T1), confirming that T1TK but not T1 could bind with RanBPM.3. Kinase activity is important for TrkB/RanBPM interaction; both NT4 and BDNF enhance TrkB/RanBPM interactionWhen K252a, a well-established Trk kinase inhibitor was added to the culture medium, we observed a noticeable decrease of TrkB/RanBPM interaction. Another set of experiment was performed by co-immunoprecipitation of RanBPM and TrkB kinase dead construct (KD), in which ATP binding site was point-mutated to abolish TrkB kinase activity, and we found a much weaker interaction between RanBPM and TrkB KD compared with TrkB WT. Besides, NT4 (30 ng/ml) and BDNF (30 ng/ml) had the equal potency to induce TrkB phosphorylation and enhance TrkB/RanBPM interaction.4. BDNF-induced TrkB and TrkBY515 phosphorylation are not affected by RanBPM, but RanBPM contributes to BDNF-induced MAPK and PI3K signalingThe effect of RanBPM on TrkB or TrkBY515 phosphorylation following BDNF administration was measured at the indicated time by immunoprecipitation of TrkB and immunoblotting with pY99 or anti-phospho-TrkBY515 antibodies. We found neither overexpression nor knocking down of RanBPM had effect on BDNF-induced TrkB or TrkBY515 phosphorylation. Overexpression of RanBPM caused a significant increase in BDNF-induced MAPK and Akt activation in a dose-dependent manner compaired with pcDNA3.1; Knocking-down RanBPM resulted in a remarkably diminished BDNF-induced MAPK and Akt activation compared with control siRNA; RanBPM modulated both the transient and the sustained MAPK activation.5. RanBPM promotes BDNF-induced neuronal morphogenesisCompaired with the control group transfected with pcDNA3.1, overexpression of RanBPM can not only increase BDNF-induced PC1210 differentiation including augment of differentiation index and the average longest neurite length, but also hippocampal neurons'total dendritic branch tip number and dendritic length. Opposite results were obtained when endogenous RanBPM was knocked down.6. RanBPM enhances BDNF-mediated protective effect on cell apoptosisWith the addition of BDNF, there was a marked reduction of apoptotic cells in RanBPM overexpressed PC1210 cells compared with the control group. Concomitantly, significant increase of TUNEL-positive cells was observed in siRanBPM group compared with the control siRNA group. These observations suggest a role of RanBPM in BDNF-mediated protection from serum deprivation induced apoptosis.ConclusionThe scaffolding protein RanBPM could interact with TrkB tyrosine kinase region and play roles in BDNF-triggered TrkB downstream MAPK and PI3K signaling pathways in addition to BDNF/TrkB-induced neuronal morphogenesis and survival.