SUMOylation is an important post-translational modification, and Akt SUMOylation 
was found to regulate cell proliferation, tumorigenesis and cell cycle, but the 
molecular mechanism of Akt SUMOylation is less well known. Here, we show both 
endogenous and ectopic Akt SUMOylation and Lys276 is the major SUMO acceptor on 
Akt. Further, Akt SUMOylation is Akt phosphorylation dependent and Akt 
SUMOylation increases Akt kinase activity without affecting the phosphorylation 
level of Akt. Moreover, endogenous Akt SUMOylation is enhanced by insulin 
treatment and this is Akt activity dependent. Heat-shock stimulus also increases 
Akt SUMOylation and it is also Akt activity dependent. Endogenous Akt 
SUMOylation is also found in the rat brain and it is enhanced by insulin-like 
growth factor-1 stimulation. In addition, Akt directly phosphorylates Ubc9 at 
Thr35 and phosphorylates SUMO1 at Thr76. Ubc9 phosphorylation at Thr35 promotes 
Ubc9 thioester bond formation and SUMO1 phosphorylation at Thr76 stabilizes the 
SUMO1 protein. Through these distinct mechanisms, Akt SUMOylation regulates 
global SUMOylation, including Akt and Ubc9 SUMOylation, and substrate 
SUMOylation specificity, including STAT1 and CREB SUMOylation, in different 
manners. Akt SUMOylation also enhances phosphatase and tensin homolog (PTEN) 
SUMOylation through Akt phosphorylation of Ubc9 and SUMO1, which serves as an 
endogenous mechanism to stop the positive feedback loop resulted from Akt 
activation. Further, Akt SUMOylation increases cyclin D1 expression and cell 
proliferation, and these effects are also mediated through Ubc9 phosphorylation 
at Thr35 and SUMO1 phosphorylation at Thr76. Here, we have identified a novel 
mechanism for SUMOylation regulation. Because of the important role Akt plays in 
tumorigenesis, this mechanism may also be involved in Akt-regulated 
tumorigenesis.