C-Cbl Negative Feedback P-selectin Induce β2 Integrin Activity And SHKBP1 Inhibit EGFR Signal Down-regulation After Activation

c-Cbl is a ring finger type E3 ubiquitin ligase, which promotes ubiquitination and degradation of activated tyrosine kinase and receptor tyrosine kinase. P-selectin glycoprotein ligand-1 (PSGL-1, CD162) was found to directly mediate integrin activation upon P-selectin binding in leukocyte, and our previous study revealed a detailed PSGL-1-Integrin signaling pathway involving Src kinase, Naf1 and Phosphoinositide-3 kinase (PI3K). And Src kinase family is an important type of tyrosine kinase, several members of Src kinase suan as Fgr, Lyn and Yes were found to be downregulated by c-Cbl ubiquitin ligase. Here we find that c-Cbl is involved in this signal pathway with negatively regulates Src kinase activity. Upon P-selectin stimulation, the activity of Src in neutrophil was up-regulated first and then down-regulated soon, c-Cbl is responsible for this down regulation by ubiquitinated the activated Src and then target to proteasome degradation. Nafl and PI3K activity are negatively regulated soon after activated by P-selectin binding as well as Neutrophil adhesion on fibrinogen coated plate. Src, c-Cbl and Nafl were found recruitment to lipid raft under P-selectin incubation. While Src is activated with P-selectin PSGL-1 binding, the activated Src interacts with c-Cbl and phosphorylates c-Cbl tyrosine residues 700, 731 and 774 in Src activation dependent. Further study implies that knockdown c-Cbl can rescue neutrophil adhesion and over-express c-Cbl ubiquitin ligase diffident mutant (CblY371F) will reduce neutrophil adhesion by P-selectin binding. In this study, we reported a negative feedback machanism in PSGL-1-Integrin signaling pathway with c-Cbl down-regulates activated Src kinase.Upon EGF stimulation, it binds with EGFR and let EGFR dimerization, then EGFR self-phosphorylation and binds with c-Cbl through its SH2 conserved domain, and induces c-Cbl phosphoryaltion. The phosphoryated c-Cbl activates its ubiquitin ligase activity and ubiqutinates EGFR, engages CIN85-Endophilin complex in cytoplasm and forms an endosome with Dynamin and Clathrin from cell membrane pitting and shedding. With endosome sorting, some EGFR recycle to cell surface, the others degradated target to lysosome. Recent research finds that the mutation of the phsophorylated or ubiquitinated site in EGFR will increase its expression and cause the tissue canceration. So it is very necessary to find probable mechanism which inhibits down-reguliaton after EGFR activation.CIN85 is an 85 kD protein first find as a Cbl binding protein. It consists with 3 SH3 domains at N terminal, a proline rich repeat and a C terminal coiled-coil fragment asα-helix. CIN85 is an adaptor protein participates in protein interaction and signal transduction, and the C terminal coiled-coil decides it location and self-polymerization. Recently, CIN85 was found to join in down-regulation of EGFR signal.We find a gene named SHKBP1 from yeast two hybrid screening, and find it can interact with CIN85 from its protein sequence analysis and its description. Co-precipitation experiment and yeast two hybrid screen use SHKBP1 as bait confirm their interaction. Further study find the SHKBP1 and CIN85 interaction can inhibit c-Cbl and CIN85 interaction, and disturb down-regulation activity of EGFR signal by c-Cbl-CIN85-Enduphilin complex. We find SHKBP1 can inhibit EGFR degradation, and will increase transcription after EGFR activation. Our study indicate a probable mechanism which can inhibit EGFR signal down-regulation after activation, this may supply further study.PDCD2L(PDCD2 like) is named as its C termainal PCDC2_C conserved domain like PDCD2, so it's considered significantly in apoptosis. But our previous study find it doesn't affect STAT expression in cell apoptosis, and itself level hasn't change much neither. Apoptosis hasn't increase while over-expression of PDCD2L in 293T. We find PDCD2L and PDCD2 have a role in inflammation, under LPS stimulation, TNF-αrelease in PDCD2L and PDCD2 overexpressed cell reduced. We further use Pull-down assay to screen suitable protein which can interact with PDCD2L to study PDCD2L function. We get 12 positive clones and two of them are LDHA and LDHB.LDH composes with 5 isoenzymes, it's a tetramers combined with two subunits LDHA and LDHB. The expression of LDHA and LDHB in mammalian development is tissue specific, so the level of LDH in serum and the distribution of different isoenzymes are an indicator with inflammation and tumor pathology progress. We confirm the interaction of PDCD2L and LDHA/B with co-precipitation, and make sure that it isn't the PDCD2_C domain that interacts with LDHA/B but the N terminal. Further study to determine the role of this interaction in inflammation will deploy recently. Our study may serve as a clue to research PDCD2L function.