Pergolizzi Barbara, Bracco Enrico, Bozzaro Salvatore
Department of Clinical and Biological Sciences, University of Torino, AOU S. Luigi, Orbassano (TO) 10043, Italy.
Department of Oncology, University of Torino, AOU S. Luigi, Orbassano (TO) 10043, Italy.
J Cell Sci. 2017 Feb 1;130(3):551-562. doi: 10.1242/jcs.194225. Epub 2017 Jan 3.
Cyclic AMP (cAMP) binding to G-protein-coupled receptors (GPCRs) orchestrates chemotaxis and development in Dictyostelium. By activating the RasC-TORC2-PKB (PKB is also known as AKT in mammals) module, cAMP regulates cell polarization during chemotaxis. TORC2 also mediates GPCR-dependent stimulation of adenylyl cyclase A (ACA), enhancing cAMP relay and developmental gene expression. Thus, mutants defective in the TORC2 Pia subunit (also known as Rictor in mammals) are impaired in chemotaxis and development. Near-saturation mutagenesis of a Pia mutant by random gene disruption led to selection of two suppressor mutants in which spontaneous chemotaxis and development were restored. PKB phosphorylation and chemotactic cell polarization were rescued, whereas Pia-dependent ACA stimulation was not restored but bypassed, leading to cAMP-dependent developmental gene expression. Knocking out the gene encoding the adenylylcyclase B (ACB) in the parental strain showed ACB to be essential for this process. The gene tagged in the suppressor mutants encodes a newly unidentified HECT ubiquitin ligase that is homologous to mammalian HERC1, but harbours a pleckstrin homology domain. Expression of the isolated wild-type HECT domain, but not a mutant HECT C5185S form, from this protein was sufficient to reconstitute the parental phenotype. The new ubiquitin ligase appears to regulate cell sensitivity to cAMP signalling and TORC2-dependent PKB phosphorylation.
环磷酸腺苷（cAMP）与G蛋白偶联受体（GPCRs）结合，协调盘基网柄菌的趋化性和发育过程。通过激活RasC-TORC2-PKB模块（PKB在哺乳动物中也称为AKT），cAMP在趋化过程中调节细胞极化。TORC2还介导GPCR依赖性的腺苷酸环化酶A（ACA）刺激，增强cAMP信号传递和发育基因表达。因此，TORC2 Pia亚基（在哺乳动物中也称为Rictor）有缺陷的突变体在趋化性和发育方面受损。通过随机基因破坏对Pia突变体进行近饱和诱变，筛选出两个抑制突变体，其中自发趋化性和发育得以恢复。PKB磷酸化和趋化细胞极化得到挽救，而Pia依赖性的ACA刺激未恢复但被绕过，导致cAMP依赖性发育基因表达。在亲本菌株中敲除编码腺苷酸环化酶B（ACB）的基因表明，ACB对这一过程至关重要。抑制突变体中标记的基因编码一种新发现的HECT泛素连接酶，它与哺乳动物HERC1同源，但含有一个普列克底物蛋白同源结构域。从该蛋白中分离出的野生型HECT结构域而非突变型HECT C5185S形式的表达足以恢复亲本表型。这种新的泛素连接酶似乎调节细胞对cAMP信号的敏感性和TORC2依赖性的PKB磷酸化。
