Study On The Function And Mechanism Of Linear Ubiquitination Of PIM1 Kinase

PIM proteins belong to a family of serine/threonine kinases family, including PIM1, PIM2 and PIM3 three members, with greatly overlapping functions. The PIM kinases play pivotal roles in the regulation of tumorigenesis, cell cycle, apoptosis, metabolism, autophagy and stemness maintenance of embryonic stem cells. It has been reported that PIM protein is overexpressed in a variety of malignant tumors and plays an important role in tumorigenesis and metastasis, which has become a novel target of anticancer drugs. In 1984, PIM1 was identified as a common integration site for murine leukemia virus that can encode 2 isoforms with sizes of 34 and 44 k Da through alternative initiation sites. It is a stress--response kinase and its expression can be induced by various cytokines, mitogens and hormones such as GM-CSF, IL-2, IL-3, IL-5, IL-7, IL-9, IL-12, IL-15, erythropoietin, PMA and so on. PIM1 involves in tumorigenesis, cell cycle, apoptosis and other important functions by phosphorylating different substrate proteins. The important signaling pathways Jak-STAT and NF-kB can promote PIM1 gene transcription and increase PIM1 expression level. But high expression of PIM1 will inhibit sustained activation of these two pathways. PIM1 has been shown to stimulate cell cycle progression at various stages. PIM1 could enhance G1/S transition through phosphorylation of Cdc25 A and induce G2/M transition through phosphorylation of Cdc25 C. Also, PIM1 protein accelerates the cell cycle progression phosphorylating p27Kip1, the CDK inhibitor, and promotes the proteasomal degradation of p27. PIM1 can phosphorylate proapoptosis protein Bad at residue Ser112 which leads to proteasomal degradation of BAD and protects cells from the proapoptotic effects of Bad. PIM1 can inhibit mitochondria membrane protein Drp1 mitochondrial translocation, via phosphorylating and protect the cells from death. PIM1 has become a new target for treatment of ischemic myocardial damage.Ubiquitination is involved in a multitude of processes including cell-cycle progression, transcriptional regulation, DNA repair, signal transduction and protein turnover by the proteasome, etc. The ubiquitination modification consists of monoubiquitination and poly-ubiquitination, which have been extensively studied. In recent year, a novel type of head-to-tail linked linear poly-ubiquitination modification which have been generated by the linear ubiquitin chain assembly complex ubiquitin ligase complex was identified which plays critical roles in the regulation of canonical NF-kB pathway. Linear ubiquitination of NEMO acts as a scaffold for the IKK complex and accumulation of IKK induces trans-phosphorylation-mediated activation of IKKb, which phosphorylates IkBa followed by the proteasomal degradation of the inhibitory protein. Finally, canonical NF-kB proteins typically formed. NEMO is the protein with linear ubiquitination modification, which has one linear UBAN motif-binds preferentially to linear di-ubiquitin. Based on the characteristic of linear ubiquitination modification, we hope to find novel linear ubiquitination substrate and explore their biological function.We obtained PIM1 gene fragment by PCR technology in vitro. Hind III and Eco R I restriction endonuclease cut PIM1 fragments and pc DNA3.0 carrier segments with the same enzyme cut sites. T4 DNA ligase was used to insert PIM1 gene into carrier. E3 enzyme and the Flag-PIM1 were overexpressed into HEK293 T cell, obtains PIM1 protein with linear ubiquitin chain. Furthermore, mass spectrometry analysis showed that a special peptide fragments with GGMQIF(G-Glycine, M-Methionine, Q-Glutamine, I-Isoleucine F-Phenylalanine) amino acid sequence from PIM1 protein enzymatic samples has the signature sequence of linear ubiquitin chain, indicating the existence of linear ubiquitination modifications of PIM1.PIM1 protein plays important roles in cells dependent on its kinase activity. We investigated the effect of linear ubiquitination modification on the kinase activity of PIM1 via kinase assays in vitro experiment. The kinase reaction system includes purified histone H3 as PIM1 kinase substrate, ATP as energy supplier, PIM1 as kinase, and phosphorylation of histone H3 by Aurora kinase as the positive control. The results showed that the PIM1 kinase activity is inhibited by linear ubiquitination, which suggests that linear ubiquitination of PIM1 can inhibit the function of PIM1 kinase.In summary, we found a novel type of post-translational modification of PIM1---linear ubiquitination, and identified two linear ubiquitination sites by mass spectrum analysis. Further kinase assays showed that linear ubiquitination of PIM1 can inhibit the kinase activity. Our study will provide new clues for the biological function of PIM1 protein and the studies on anti-tumor therapy based on PIM1 protein.