The Study Of The Mechanism That Lysine Methyltrasferase SMYD2 Regulates BMP Signaling Pathway

In recent years,protein lysine methylation has been detected in non-histone proteins as well as histones.Increasing evidence indicates that analogous to phosphorylation and acetylation,lysine methylation plays broad roles in regulating protein function.Among the known methylated proteins,most are chromosomal or nuclear proteins,and few are cytoplasm-or membrane-associated signaling proteins.Therefore,in this study we aimed to investigate the function of non-histone methylation in intracellular signaling.TGF-? superfamily signaling pathway not only regulates cellular proliferation,differentiation and homeostasis,but also plays important roles in embryonic development,bone formation and regeneration,and organogenesis.Abnormalities in TGF-? or BMP signal pathways often cause various disorders such as pulmonary hypertension and neoplasm.SMYD family proteins are lysine methyltransferases that contain a split SET catalytic domain,some of them mainly reside in cytoplasm,and catalyze different non-histone substrates.Through a RNA interference-based functional screen targeting the SMYD family methyltransferases,we found that SMYD2 knockdown specifically impaired BMP-induced activation of ID1,ID2,ID3 and SMAD6,but had no effect onTGF-?-induced activation of PAI-1,p21 and SMAD7.Furthermore,BMP-induced SMAD1/5 phosphorylation was also impaired upon SMYD2 knockdown.In support,we found that SMYD2 knockdown by shRNA or knockout by CRISPR-Cas9 editing also resulted in reduced SMAD1/5 phosphorylation and impaired induction of BMP target genes.Further analysis revealed that loss of SMYD2 impairs BMP-induced SMAD1 nuclear localization and interaction with Co-SMAD.Treatment with an SMYD2-specific enzymatic inhibitor inhibited BMP-induced SMAD1/5 phosphorylation suggesting that SMYD2 regulates BMP pathway in an enzymatic activity-dependent manner.Protein-protein interaction assay showed that SMYD2 interacts with multiple proteins in TGF/BMP pathways.However,in vitro and in vivo methylation assay showed that SMYD2 specifically methylates the BMP pathway-specific receptor BMPR2.The methylation on BMPR2 was mapped to the kinase domain of BMPR2.Together,our study suggests SMYD2 may positively regulate BMP signaling by directly methylating BMPR2,which in turn influences SMAD1/5 phosphorylation and activation of BMP target gene expression.