Autophosphorylation of Akt at threonine 72 and serine 246. A potential mechanism of regulation of Akt kinase activity.
Activation of the serine/threonine protein kinase Akt is a multistep process. We here propose that the kinase activity of Akt is regulated via autophosphorylation in trans at two putative sites (threonine 72 and serine 246) that lie in the characteristic Akt substrate motif (RXRXX(S/T)). Incubation of Akt immunoprecipitated from transfected cells with a pre-activated Akt recombinant protein and gamma-32P-labeled ATP led to marked incorporation of radioactivity in wild-type Akt but not Akt/T72A/S246A mutant. Western blot analysis using a phosphorylated Akt substrate-specific antibody of Akt immunoprecipitated from transfected cells confirmed the autophosphorylation of wild-type Akt but not Akt/T72A/S246A mutant in insulin-like growth factor-1 (IGF-1)-stimulated cells. Autophosphorylation of Akt on Thr-72 and Ser-246 appeared to require prior phosphorylation of Akt on Thr-308 and Ser-473. Compared with wild-type Akt, Akt/T72A/S246A mutant exhibited markedly reduced basal Akt kinase activity and response to cellular stimulation by insulin-like growth factor-1, and also conferred less cellular resistance to doxorubicin-induced apoptosis. The findings from these pilot studies suggest that Akt regulates its kinase activity through autophosphorylation. Further investigation of this potential novel regulatory mechanism by which Akt performs its cellular functions is warranted.
Li
Xinqun
X
Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Lu
Yang
Y
Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Jin
Weidong
W
Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Liang
Ke
K
Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Mills
Gordon B
GB
Department of Molecular Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030.
Fan
Zhen
Z
Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030. Electronic address: zfan@mdanderson.org.
D000595:Amino Acid Sequence; D017209:Apoptosis; D001665:Binding Sites; D045744:Cell Line, Tumor; D004317:Doxorubicin; D006801:Humans; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000378:metabolism*
D010769:Phosphothreonine; D010768:Phosphoserine; D004317:Doxorubicin; D051057:Proto-Oncogene Proteins c-akt