PATHWAYS OF RECEPTOR SIGNALING AND CHEMOTAXIS

趋化性是由细胞内的积极和消极作用（适应）途径协调的，其他系统中的受体磷酸化需要关闭下游信号。盘基ostelium使用7-TMR CAR1来感知分泌的cAMP，以协调运动形成对饥饿的反应。聚集形成的关键是大多数car1介导的途径能够快速适应cAMP刺激，以及细胞通过分泌磷酸二酯酶快速破坏cAMP配体的能力。CAR1激活多个网络，包括camp合成途径腺苷酸环化酶A (ACA)，并启动其自身的磷酸化。我们现在证明，受体磷酸化是ACA途径适应所必需的：在持续信号存在的情况下，ACA在表达非磷酸化的CAR1突变体的细胞中不适应。与这些结果一致，我们发现当突变细胞聚集在固体底物上时，cAMP信号的传播明显受到损害，但在聚集过程中仍观察到cAMP振荡的证据，这表明无法抑制cAMP合成在原位得到部分补偿。

Chemotaxis is coordinated by both positively and negatively acting (adapting) intracellular pathways, and receptor phosphorylation in other systems is required to turn off downstream signaling. Dictyostelium uses the 7-TMR CAR1 to sense secreted cAMP to coordinate movement into aggregates in response to starvation. Essential to aggregate formation is the ability for most CAR1-mediated pathways to quickly adapt to a cAMP stimulus and for cells to rapidly destroy the cAMP ligand by secreted phosphodiesterases. CAR1 activates multiple networks, including the cAMP-synthesizing-pathway adenylyl cyclase A (ACA), and also initiates its own phosphorylation. We now demonstrate that receptor phosphorylation is required for adaptation of the ACA pathway: ACA does not adapt in cells expressing non-phosphorylatable CAR1 mutants in the presence of persistent signal. Consistent with these results, we find that propagation of the cAMP signal is significantly compromised when mutant cells are allowed to aggregate on a solid substrate, but evidence of cAMP oscillations during aggregation is still observed, suggesting that the inability to suppress cAMP synthesis is partially compensated in situ. AKT and PKBR1 are two related protein kinases that play pivotal roles in Dictyostelium chemotaxis. PKBR1 is anchored constitutively at the plasma membrane via a myristoylation tag, whereas AKT possesses a PH-domain and is transiently recruited to the membrane upon the PI3K-dependent accumulation of the lipid moiety PIP3. We have found that PKBR1 and AKT are phosphorylated upon stimulation by both the growth-specific chemoattractant folate and the developmental-specific chemoattractant cAMP. We systematically investigated components involved in cAMP regulation of AKT/PKBR1 and show a connection of CAR1 to Ga2bg; downstream, the GefA-RasC pathway is activated. The RasC effector PI3K is required for AKT function, but not for PKBR1, and in pten-null cells, only AKT activity is enhanced. 2 protein kinase activities are required to regulate AKT/ PKBR1. Phosphorylation within the activation loop is mediated by PDK1, while phosphorylation within the C-terminal HM region requires TORC2. HM phosphorylations on AKT and PKBR1 are dependent on Rictor/Pia, SIN1/RIP3, and LST8 and full TORC2 integrity, strongly suggesting TORC2 functions in chemotaxis and development through phosphorylation of AKT and PKBR1.