Miralem Tihomir, Lerner-Marmarosh Nicole, Gibbs Peter E M, Jenkins Jermaine L, Heimiller Chelsea, Maines Mahin D
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA
FASEB J. 2016 Aug;30(8):2926-44. doi: 10.1096/fj.201600330RR. Epub 2016 May 10.
Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.
胆绿素还原酶A（BVR）和Akt同工酶在胰岛素/PI3K/MAPK途径中具有重叠的多效性功能。人BVR（hBVR）还可将血红素加氧酶活性产物胆绿素还原为胆红素，并被胰岛素受体激酶（IRK）直接激活。Akt同工酶（Akt1 - 3）位于IRK下游，在S（473）自磷酸化之前，先由磷脂酰肌醇依赖性激酶1（PDK1）磷酸化T（308）而被激活。Akt（RxRxxSF）和PDK1（RFxFPxFS）结合基序存在于hBVR中。Akt对糖原合酶激酶3（GSK3）α/β亚型的磷酸化会抑制其活性；未磷酸化的GSK3β会抑制各种基因的激活。我们研究了hBVR在PDK1/Akt1/GSK3信号传导中的作用以及Akt1在hBVR磷酸化中的作用。hBVR可独立于其激酶活性激活Akt1在S（473）位点的磷酸化。hBVR和Akt1可共免疫沉淀，细胞内荧光共振能量转移（FRET）和谷胱甘肽S - 转移酶下拉分析确定Akt1的pleckstrin同源结构域为相互作用结构域。hBVR可独立于其激酶活性激活Akt1在S（473）位点的磷酸化。定点突变、质谱分析和动力学分析确定hBVR（225）RNRYLSF序列中的S（230）为Akt1的作用靶点。下划线氨基酸是信号基序的必需残基。在细胞中，hBVR激活的Akt1增加了GSK3α/β和O类转录因子3（FoxO3）的磷酸化，并抑制了总GSK3活性；hBVR的缺失解除了抑制并刺激了葡萄糖摄取。免疫沉淀分析表明，PDK1和hBVR通过hBVR的PDK1结合（161）RFGFPAFS基序相互作用，并形成PDK1/hBVR/Akt1复合物。siRNA介导的hBVR敲低阻断了复合物的形成。研究结果确定hBVR是Akt1此前未知的共激活因子，是Akt1/GSK3途径的关键介质，并确定了hBVR在PDK1激活Akt1中的关键作用。 - 米拉莱姆，T.，勒纳 - 马尔马罗什，N.，吉布斯，P.E.M.，詹金斯，J.L.，海米勒，C.，梅因斯，M.D. 人胆绿素还原酶与Akt/蛋白激酶B和磷脂酰肌醇依赖性激酶1的相互作用调节糖原合酶激酶3活性：Akt激活的新机制
