Yang Jing, Cron Peter, Good Valerie M, Thompson Vivienne, Hemmings Brian A, Barford David
Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, UK.
Nat Struct Biol. 2002 Dec;9(12):940-4. doi: 10.1038/nsb870.
The protein kinase Akt/PKB is stimulated by the phosphorylation of two regulatory residues, Thr 309 of the activation segment and Ser 474 of the hydrophobic motif (HM), that are structurally and functionally conserved within the AGC kinase family. To understand the mechanism of PKB regulation, we determined the crystal structures of activated kinase domains of PKB in complex with a GSK3beta-peptide substrate and an ATP analog. The activated state of the kinase was generated by phosphorylating Thr 309 using PDK1 and mimicking Ser 474 phosphorylation either with the S474D substitution or by replacing the HM of PKB with that of PIFtide, a potent mimic of a phosphorylated HM. Comparison with the inactive PKB structure indicates that the role of Ser 474 phosphorylation is to promote the engagement of the HM with the N-lobe of the kinase domain, promoting a disorder-to-order transition of the alphaC helix. The alphaC helix, by interacting with pThr 309, restructures and orders the activation segment, generating an active kinase conformation. Analysis of the interactions between PKB and the GSK3beta-peptide explains how PKB selects for protein substrates distinct from those of PKA.
蛋白激酶Akt/PKB可通过两个调节性残基（激活环的苏氨酸309和疏水基序（HM）的丝氨酸474）的磷酸化而被激活，这两个残基在AGC激酶家族中在结构和功能上都是保守的。为了理解PKB的调节机制，我们确定了与GSK3β-肽底物和ATP类似物结合的PKB激活激酶结构域的晶体结构。激酶的激活状态是通过使用PDK1磷酸化苏氨酸309，并通过S474D取代或用PIFtide（一种磷酸化HM的有效模拟物）的HM取代PKB的HM来模拟丝氨酸474的磷酸化而产生的。与无活性的PKB结构比较表明，丝氨酸474磷酸化的作用是促进HM与激酶结构域的N叶结合，促进αC螺旋从无序到有序的转变。αC螺旋通过与pThr 309相互作用，重组并使激活环有序化，产生有活性的激酶构象。对PKB与GSK3β-肽之间相互作用的分析解释了PKB如何选择与PKA不同的蛋白质底物。
