TRIM22 Activates MAPK Signaling by Regulating the Transcription of SPHK2 and Accelerating the Degradation of Raf-1 in Glioblastoma

Abstract Background Tripartite motif (TRIM) 22 and mitogen-activated protein kinase (MAPK) signaling pathways play a critical role in tumor growth and therapeutic resistance of glioblastoma (GBM) respectively. However, the molecular mechanism between TRIM22 and MAPK signaling remains to be clarified. Methods We constructed TRIM22 knockout cell lines for molecular biology experiments, detected potential DNA fragments binding to TRIM22 by ChIP-Seq technology, and verified the sequencing results by ChIP-qPCR and CUT&Tag technology. In addition, we constructed different TRIM22 mutants to detect the binding of proteins in MAPK signaling pathway. Finally, the therapeutic effect was verified in NOD/SCID mice. The difference between the two groups of data conforming to the normal distribution was tested by Student t-test. Results Here, we found for the first time that TRIM22 acts as a transcription factor in the nucleus and binds to exon 2 of the transcript (NM_001204160) of SPHK2 gene to regulate its expression by ChIP-Seq technology, thus indirectly affecting the downstream MAPK signaling pathway. Knockout of TRIM22 using Cas9-sgRNAs resulted in decreased mRNA level of SPHK2 in GBM cells, while overexpression of TRIM22 enhanced it. The ERK1/2 driven luciferase reporter construct identified TRIM22 as a potential activator of MAPK signaling. Knockout and overexpression of TRIM22 regulate the inhibition and activation of MAPK signaling through its RING-finger domain. Co-immunoprecipitation demonstrated that TRIM22 bound to the negative regulator Raf-1 of MAPK signaling and accelerated its degradation by inducing K48-linked ubiquitination. The combination of the two is related to the CC domain and SPRY domain of TRIM22 and the C1D domain of Raf-1. TRIM22 also forms a complex with the downstream regulator ERK1/2 of MAPK and promotes K63-linked ubiquitination, resulting in the phosphorylation of ERK1/2. In addition, in vitro and in situ xenotransplantation models, SPHK2 inhibitor (K145), ERK1/2 inhibitor (Selumetinib) and non-phosphorylated mutant Raf-1S338A inhibited the growth promoting properties of TRIM22 in GBM cell line. Conclusions In conclusion, our study shows that TRIM22 regulates SPHK2 transcription as a transcription factor, indirectly affects MAPK signaling, and activates MAPK signaling through post-translational modification of two critical regulators of MAPK signaling in GBM cells.