SUMOylation Of 3-Phosphoinositide Dependent Protein Kinase1 Is Required For Glycolysis Dependent CD4~+T Cell Homeostatic Maintenance

Background and Aims:The immune system needs to be finely tuned to fight against infections and to eradicate neoplasms,but requires to be devoid from aberrant immune activation coupled with autoimmunity.Protein post-translational modification(PTM)constitutes a molecular layer of regulation to guarantee the proper intensity of immune response.UBC9 mediated SUMOylation process is dynamicly involved in T cell biology,and various substrates have been identified to be engaged ineither downstream TCR signaling(PKC-θ,IκB,NFAT1,Jun B)or T cell differentiation program(c-MAF,RORγ-T).A previous study revealed the critical function of IRF4 SUMOylation in Treg specialization and effector activity and the deficiency of UBC9 impairs normal thymus developmental process.However,whether SUMOylation affects the maintenance of peripheral Tcon and Treg cellsand the exact underlying mechanisms remain elusive.The current project is designed to address the above scientific questions.Methods and Results:We first examined the phenotype of CD4~+T cell specific UBC9 deficient mice,and found that those mice displayed signs of growth retardation and lymphatic organ atrophy.Subsequent preliminary mechanistic studies suggested that cell proliferation,not apoptosis,is the main potential driver.In vitro proliferation and differentiation studies confirmed the defect in proliferation rather than the differentiation efficacy of various CD4~+ T cell subsets.Chimeric model of bone marrow transplantation further demonstrated that UBC9 deficiency caused above changes via endogenous pathways,while mice with FOXP3+Treg specific UBC9 deletion confirmed that UBC9 deficiency could lead to progressive reduction of Treg cells.Given the defects in both Tcon and Treg cell homeostasis,we proposed that a common pathway is responsible for the observed abnormalities.Seahorse metabolic analysis revealed that the levels of glycolysis and oxidative phosphorylation of UBC9 decifient CD4~+T cells significantly decreased.Real time quantitative PCR confirmed that the expression of glycolysis related genes was downregulated,and the glucose uptake assays further confirmed this notion from the functional perspective.PDPK1 is a key kinase involved in the regulation of cell metabolism.Western Blot results showed that the activation of PDPK1 pathway was decreased in UBC9 knockout cells.At the same time,bioinformatic analysis combined with co-immunoprecipitations confirmed that PDPK1 is an important SUMOylation target in CD4~+T cells.We elucidated that K299 is the main SUMOylation site of PDPK1 by site directed mutagenesis.Phosphoprotein mass spectrometry analysis further proved that disruption of PDPK1 SUMOylation leads to the abnormal activation of its downstream signaling.Finally,we demonstrated that disruption of PDPK1 SUMOylation did not affect CD4~+T cell differentiation,but significantly reduced its proliferative capability by transduction of lentiviral particles carrying wild-type PDPK1 and PDPK1-K299 R mutant.Conclusions:Our data support that UBC9 mediated SUMOylation of PDPK1 plays an essential role in peripheral CD4~+T cell proliferation,which involves both conventional T cell(Tcon)and regulatory T cell(Treg),but its impact on Treg cells is much more potent.As a result,CD4~+T cells deficieny in Ubc9 are characterized by the progressive reduction of Treg cells along with increased proportion of effector CD4~+T cells.Mechanistically,PDPK1(3-phosphoinositide dependent protein kinase1)was identified as a novel SUMOylated substrate,which occurs mainly on Lysine299(K299)located within the protein kinase domain.Loss of PDPK1 SUMOylation impedes its auto-phosphorylation on Ser241,which results in hypo-activation of its downstream m TORC1 signaling and incompetence of cell proliferation.Altogether,these results support that SUMOylation of PDPK1 impacts CD4~+T cell proliferation through regulation of PDPK1-m TORC1 mediated glycolytic process.