Functional Analysis Of ClipR-59in Mouse Muscle Cell Differentiation

ClipR-59(Cytoplasmic linker protein related59kD protein) is a membrane associated protein characterized by four distinct structural features including a Glu-Pro rich domain, three ankryin repeats, two putative CAP-Gly(cytoskeleton-associated protein glycine-rich) domains and one Membrane Binding Domain (MBD). In adipocyptes, ClipR-59interacts with Akt and modulates Akt plasma membrane compartmentalization, enhances AS160phosphorylation, thereby promote Glut4membrane translocation and glucose transport. ClipR-59also interacts with TNF receptor to regulate TNF signaling and cell apoptosis. Inactivation of ClipR-59in mice resulted in perinatal lethality in part due to muscle malfunction, implying that this gene plays a crucial role in muscle development. In this project, the aim is to screen the new interaction protein of ClipR-59by yeast two-hybrid system, reveal the complex’s function and identify their molecular mechanism.We carried out a yeast two-hybrid screen using a murine embryonic fibroblast F422A-3T3cDNA library using ClipR-59as the bait. Several potential ClipR-59-interaction proteins were identified in this screen. One of them, Elmo2, was particularly interesting because it was recently reported to play an essential role in myoblast fusion through Elmo2-Dock180-Racl signal pathway. We found that ClipR-59interacts with Elmo2, enhance Racl activity and modulates myoblast fusion.Firstly, the ClipR-59and Elmo2were identified to interact with each other in vivo and vitro through Co-immunoprecipitation assay, and the two proteins had the same subcellular localization by cell staining.Secondly, we examined ClipR-59expression during differentiation of C2C12cells into myotuble. Expression of ClipR-59was increased following C2C12differentiation. This change in expression of ClipR-59is correlated to muscle cell differentiation as the expression of myogenin and myosin heavy chain (MHC), markers of muscle cell differentiation, were also increased. We next introduced a validated ClipR-59shRNA into C2C12cells via lentiviral gene transfer; Compare with control cells, interfering with ClipR-59expression in C2C12cells reduced the number of myotuble(>3nuclei in each fiber) by more than80%. There was a marginal difference in the expression of myogenin between control and ClipR-59shRNA-expressing C2C12cells, this demonstrates that ClipR-59plays a role in muscle differentiation at the step of myoblast fusion.Then, we generated a series of Elmo2mutants in which different portions of Elmo2were deleted, and confirmed that the PH domain of Elmo2is required for the interaction between Elmo2and ClipR-59. In the same way, we identified that the E/P domain in ClipR-59that mediates the interaction between Ehno2and ClipR-59. What’s more, we found that ClipR-59, Elmo2and Dock180are present in the same complex through Co-immunoprecipitation assay; and interaction of ClipR-59with Elmo2enhances the cellular levels of active of Racl in GST(Glutathione S-transferase)-CRIB(Cdc24/Racl interactive binding domain of p21activated kinase) pull down assay, and knock down ClipR-59in C2C12cell, decrease the cellular levels of active of Racl. In the following, we introduced wildtype and Elmo2interaction defective2E/P ClipR-59into C2C12cells and examined how forcing expression of ClipR-59affected C2C12differentiation. Forcing expression of ClipR-59modestly enhanced C2C12differentiation (50%vs60%) presumably because there is a sufficient amount of endogenous ClipR-59in the cells. However, in sharp contrast, forcing expression of AE/P ClipR-59reduced C2C12cell myoblast fusion by about50%.Next, we co-transduced C2C12cells with lentiviral vector expressing ClipR-59shRNA and pMigrl expression vector that expresses ClipR-59or ΔE/P ClipR-59and examined that whether C2C12differentiation suppressed by ClipR-59shRNA could be rescued, respectively. Then, viral transduced cells were subjected to differentiation. The control cells were that co-transduced with lentiviral vector expressing ClipR-59shRNA and pMigrl empty vector, or lentiviral vector expressing luciferase shRNA and pMigrl empty vector. As a result, we observed that ClipR-59expression in ClipR-59shRNA expressing cells clearly increased the level of differentiation which was at a comparable level with luciferase shRNA expressing cells. On the other hand, expression of ClipR-59ΔE/P has no such effect.At last, we found that ClipR-59and Elmo2interaction is regulated by Insulin through GST-Pull down assay; what’s interesting, the Elmo2is a tyrosine phosphorylation protein regulated by Insulin with P-Tyr IP assay, the713Y is the major phosphorylation site through point mutant assay.In all, our studies suggest that, by interacting with Elmo2, ClipR-59facilitates Racl activation and thereby myoblast fusion. The founding of this research will supply a theory basis for further research of muscle development in vivo.