The Role Of Npm1 Mutation Protein In TRAF6-mediated Akt Ubiquitination And Activation In Acute Myeloid Leukemia

PART I The interaction between NPM1 mutation protein and Akt in leukemia cellsNucleophosmin 1(NPM1) is the most commonly mutated gene in acute myeloid leukemia(AML). Mutations leading to aberrant cytoplasmic localization of nucleophosmin is the main characteristics in all cases of AML with mutated NPM1. PKB/AKT(Protein Kinase B) play an important role in tumorigenesis by regulating cell proliferation, differentiation, apoptosis, migrating, metastasis and tumor angiogenesis, and constitutive Akt activation is detectable in 50% of acute myeloid leukemia samples. Results from our previous study demonstrated that knowdown of NPM1 mutation protein expression by si RNA in leukemia cells desceased the levels of phosphorylated Akt, Recently studies have demonstrated that wild NPM1 protein could interact with Akt To explore the interaction between NPM1 mutation protein and Akt, we first detected whether endogenous NPM1 mutation protein interact with Akt in OCI-AML3 leukemia cells carrying the NPM1 mutation by co-immunoprecipitation experiments. In addition, Flag-NPM1 m A and HA-Akt or HA–Akt-△PH were cotransfected into 293 T cells and immunoprecipitated with anti-HA to detect HA-Akt, HA–Akt-△PH or Flag-NPM1 m A in the immunoprecipitation complex. The results showed that endogenous Akt protein interacted with NPM1 mutation protein in OCI-AML3 cells;Exogenous HA-Akt interacted with NPM1 mutation protein, but HA–Akt-△PH lost the ability to interact with NPM1 mutation protein. The results indicate that NPM1 mutation protein interact with Akt, and the PH domain of Akt is responsible for their interaction.PART II TRAF6 regulates Akt ubiquitination and activation in Leukemia cells carrying the NPM1 mutationAkt signaling pathway play an important role in leukemogenesis, and constitutive Akt activation is the result of autocrine IGF-1 signaling in 70% of acute myeloid leukemia samples. Recently studies demonstrated that E3 ligases TRAF6 triggered K63-linked ubiquitination of Akt in response to IGF-1 growth factor stimuli,, which facilitates Akt membrane recruitment and subsequent phosphorylation. To explore whether TRAF6 regulates Akt ubiquitination and activation in leukemia cells, firstly, the levels of TRAF6, p-Akt(S473) and p-Akt(T308) expression in leukemia cell lines were detected by Western blot,;Secondly, co-immunoprecipitation assay was used to explore the interaction between TRAF6 and Akt; Next, Western blot assay was used to explore whether knockdown of TRAF6 affected Akt phosphorylation and ubiquitination level. Recruitment of Akt to the cell membrane is critical for Akt activation, we also investigated whether knockdown of TRAF6 affected Akt membrane translocation. The results showed that TRAF6, p-Akt(S473), p-Akt(T308) expressed in six leukemia cell line; The endogenous Akt and TRAF6 interaction was observed in OCI-AML3 cells and was at least partially dependent on IGF-1 treatment.; Knockdown of TRAF6 expression impaired Akt phosphorylation and ubiquitination; Moreover, knockdown of TRAF6 expression blocked IGF-1 inducing Akt membrane localization. The results indicate that TRAF6 regulates Akt ubiquitination and activation in leukemia cells carrying the NPM1 mutation.PART III NPM1 mutation protein facilitate Akt membrane translocation and phosphorylation activation as a potential "adaptor"Recently studies demonstrated E3 ligases TRAF6 triggered K63-linked ubiquitination of Akt, which facilitated Akt membrane recruitment and subsequent phosphorylation. However, how the Akt ubiquitination contributes to the Akt membrane localization remains elusive? As the K63-linked ubiquitination plays an important role in protein/protein interaction, the K63-linked ubiquitination of Akt on its PH domain may serve as a molecular platform to recruit the essential adaptors for Akt, in turn facilitating Akt membrane recruitment and activation. Considering the the interaction between NPM1 mutation protein and Akt, we aimed to identify whether NPM1 mutation protein regulated Akt membrane translocation and phosphorylation activation as a potential "adaptor". To examine whether NPM1 mutation protein affected Akt ubiquitination, HA-Akt and His-Ub, along with Flag-TRAF6 or/and Flag-NPM1 m A were cotransfected into 293 T cells, and Akt ubiquitination was detected by Western blot; We next investigated whether knockdown of NPM1 mutation protein affected Akt phosphorylation and membrane translocation, Akt phosphorylation and membrane localization were analyzed by Western blot; We also investigated the effect of overexpression NPM1 mutation protein on Akt membrane localization.The results indicate that NPM1 mutation protein does not appear to affect Akt ubiquitination. Instead, NPM1 mutation protein may be a potential adaptor,which facilitates Akt membrane recruitment and subsequent phosphorylation.