Spred-2 Interaction With Rab11 Affects Rat Pheochromocytoma PC12 Cell Differentiation

Spred (Sprouty related with EVH1 domain) proteins (Spreds) are tyrosine kinase binding proteins. Spred family members contain an N-terminal Enabled/VASP Homology-1 domain (EVH-1), a central c-Kit binding domain (KBD) and a C-terminal Sprouty-related domain (SPR). Currently, the Spred protein family in mammals mainly consists of Spred-1, Spred-2, Spred-3 and Eve-3. Spred-3 lacks KBD domain while Eve-3 consists of only EVH-1 domain. Spreds inhibit the activation of Ras/Raf/Erk pathway by a wide range of different growth factors and block cell differentiation, but the underlying mechanism is complex and remains to be elucidated. Spred-2 is strongly expressed in different glandular and secretory tissues, mainly localizes in vesicle-like structure and co-localizes with recycling endosomal marker protein Rabll. Rab11 is intracellular vesicle transport-related and promotes neurite outgrowth. Our previous research and studies by other researchers showed that Spred-2 inhibits rat pheochromocytoma cells PC 12 differentiation, but the mechanism is largely unknown. We hypothesized that Rab11 may be involved in Spred-2 regulation of PC 12 cell differentiation. This study found that Spred-2 interacted with Rab11, and co-localized with autophagy marker protein LC3. The main results are summarized as follows:1. Immunofluorescence assays detect the localization of endogenous Spred-2 and endogenous Rab11 in HeLa cells, the results showed that they colocalized in cytoplasmic punctae.293T cells were transfected with Myc-Spred-2 and HA-Rab11, and the whole cell lysates were co-immunoprecipitated with anti-Myc. Immunoblot results showed that Spred-2 interacted with Rab11.2.293T cells were transfected with Myc-Spred-2 wild type or domain deletion mutants and HA-Rab11, and the whole cell lysates were co-immunoprecipitated with anti-Myc. Immunoblot results showed that Spred-2 interacted with Rab11 via its C-terminal domain.3. HeLa cells were transfected with Myc-Spred-2, DsRed-Rabll and green fluorescent protein tag LC3 (GFP-LC3). Immunofluorescence results showed a co-localization between Spred-2, Rab11 and LC3 in cytoplasm.4. HeLa cells were transfected with Myc-Vector or Myc-Spred-2 wild type or SPR domain deletion mutant, with DsRed-Rab11 and GFP-LC3. Immunofluorescence results showed that the SPR domain of Spred-2 was essential for the co-localization between Rab11 and LC3.5. HeLa cells were transfected with DsRed-Rab11 and GFP-LC3, and treated with rapamycin (Rapa) or chloroquine (CQ). Immunofluorescence results showed that Rapa enhanced the overlap of co-localization between Rab11 and LC3, while CQ had a contrary effect.6. PC12 cells were infected with adenoviruses expressing Spred-2 wild type or mutants. Cell differentiation results showed that the LC3 binding sites within the SPR domain of Spred-2 were essential for the inhibition of neurite outgrowth in PC12 cells.In summary, Spred-2 interaction with Rab11 via its C-terminal domain may suppress Rab11-promoted neurite outgrowth of Rab11. Moreover, Spred-2 involve in autophagy flux, and Spred-2-Rab11-LC3 complex which localizes in autolysosome inhibits the PC12 cell differentiation.