The regulation of cell growth has been intensively studied for a decade and PI3K/Akt/mTOR/p70S6k cascade has been assigned to a major signaling pathway modulating the cell growth. Much work has focused on unveiling the signaling components of the pathway and has revealed many regulators including AMP kinase, Tuberous sclerosis complex 1/2 (TSC1/2), and Rheb GTPase. In many cells, an increase in levels of intracellular cyclic AMP (cAMP) diminishes cell growth and promotes differentiation, and in certain conditions cAMP is even antagonistic to the effect of growth factors. Here, I showed that cAMP has inhibitory effects on the phosphatidylinositol 3-kinase (PI3K)/PDK1/Akt/p70S6k signaling pathway. cAMP potently inhibits the activities of p70S6k and Akt by inhibiting PDK1 translocation to the plasma membrane. Finally, cAMP was found to inhibit the lipid kinase activity of PI3K and to decrease the levels of phosphatidylinositol 3, 4, 5-triphosphate ($PIP_3$) in vivo, which are required for the membrane localization of PDK1. Collectively, these data strongly support that the cAMP-dependent signaling pathway inhibits p70S6k and Akt activity by blocking the coupling between Akt and its upstream regulators, PDK1, in the plasma membrane.
Next, to further investigate the regulators of p70S6k, I identified UNC-51-like kinase (ULK), a novel signaling component regulating p70S6k through Drosophila genetic screening. The reduction of dULK gene dosage enhanced cell growth caused by overexpression of dS6k and partially restored the decreased cell size, the lethality, and developmental delay in dTor-deficient mutant fly. The lethality of dULK-deficient fly was also rescued by the reduction of dS6k or dPDK1 expression. In addition, the phosphorylation and activation of p70S6k were potently inhibited by ULK in a ULK activity-dependent manner in HEK293T cells. Furthermore, my data indicated that ULK could phosphorylate Ser 389 residue of PDK1 linker region and modulate the function of PDK1 to induce the Thr 389 phosphorylation of p70S6k. In contrast, Akt and p90RSK, other physiological targets of PDK1, were independent from ULK. Therefore, it can be proposed that the phosphorylation of PDK1 at Ser 389 by ULK is specifically involved in the regulation of p70S6k. In short, p70S6k can be regulated by various mechanisms, adjusting the cell growth in response to challengeable environment.
세포 성장을 조절하는 기작에 대한 연구는 지난 10여 년간 상당히 진행되어, PI3K/Akt/mTOR/p70S6k 신호전달계가 그 중추를 담당한다는 사실이 잘 알려져 있다. 최근에는 신호전달을 매개하는 TSC1/2, Rheb, AMP kinase 등의 단백질들의 기능이 알려지면서 세포 성장 조절에 대한 이해가 심화되었다. 본 연구에서는, 세포 성장을 저해하고 분화를 촉진한다고 알려진 cAMP가 PI3K/Akt/p70S6k 신호 전달계에 미치는 영향과, 새로운 p70S6k의 조절자에 대해 설명하였다. cAMP는 PDK1이 세포막으로 이동하는 것을 방해하여, Akt와 p70S6k의 활성화를 저해하였다. 이 결과는 cAMP가 PI3K의 활성을 낮추어서 생긴 것임을 보였다. 따라서, 지금까지 알려진 cAMP의 생체 내 효과는 PI3K 신호 전달계를 저해함으로써 나타나는 현상으로 생각된다. 한편, p70S6k의 새로운 조절자를 찾기 위해 초파리를 이용하여 게놈 전체에 대한 탐색을 수행하였다. 그 결과, ULK가 TOR의 신호 전달을 방해하여, p70S6k의 활성을 저해하는 것으로 나타났다. 이러한 현상은, ULK가 PDK1의 세린 389번을 직접 인산화하여, PDK1이 p70S6k의 쓰레오닌 389번을 인산화하는 능력을 저해함으로써 일어난다. 이러한 현상은, ULK가 PDK1의 세린 389번을 직접 인산화하여, PDK1이 p70S6k의 쓰레오닌 389번을 인산화하는 능력을 저해함으로써 일어난다. 놀라운 것은, ULK의 이러한 효과가 PDK1의 다른 기질인 Akt, p90RSK에는 미치지 않고, 오직 p70S6k에 특수하다는 것이다. 요컨대, 생체는 세포 성장을 조절하기 위해, 다양한 방법으로 p70S6k를 조절한다.
