REGULATION OF DEVELOPMENTAL GENE EXPRESSION

组织胚胎体计划是控制早期后生动物发育的主要必要条件，形态因子信号是建立和指定细胞命运的中心。其中一种途径涉及Wnt/无翅信号，包括7-跨膜（7-TM）靶受体和下游效应蛋白激酶GSK3b。我们已经提出，在决定细胞命运的背景下，盘基骨菌中的拮抗7-TM cAMP受体（CAR）通路在GSK3聚集。我们在Dictyostelium中发现了一种新的酪氨酸激酶ZAK1，它是cAMP在发育过程中激活GSK3所必需的。我们还发现重组ZAK1可以磷酸化，从而在体外激活哺乳动物GSK3b。这项研究可能揭示涉及蛋白酪氨酸激酶的新机制，该机制可能对其他系统中的细胞命运规范或肿瘤抑制至关重要。在盘基骨柱发育的早期，cAMP会脉动性地释放到细胞外介质中，从而指导趋化迁移和诱导基因表达。细胞外cAMP信号通过调节腺苷酸环化酶（AC）活性的特定受体/ g蛋白途径通过细胞膜传导。在cAMP信号的存在下，交流电是短暂激活的，但在持续cAMP信号下，交流电会迅速适应。虽然Gbg与AC激活有关，但对cAMP反应的适应机制尚不清楚。我们在Dictyostelium中发现了一种新的Ga (Ga9)，它与抑制性Ga亚类的蛋白高度相似，我们的数据表明Ga9可能参与了适应途径。ga9 -null也对一种新发现的因子敏感，这种因子是由发育中的盘基骨柱细胞分泌的，该细胞增强了对cAMP的化学反应。ga9 -null比野生型反应更快，细胞密度更低。我们已将因子纯化到均匀性，两个蛋白被单独分离并正在测序。这些观察结果表明，Ga9是指导细胞运动和感知细胞密度的复杂信号网络的一部分。盘状骨的发育需要cAMP受体1 （CAR1）来传递cAMP信号。CAR1的表达受cAMP调控。新的转录因子CRTF与CAR1早期启动子内的一个基本元件结合，并介导cAMP对CAR1的诱导。我们将CRTF纯化至均匀性，并分离其cDNA。CRTF估计分子量为100kDa，具有新颖的锌指dna结合基序。crtf缺失的细胞在CAR1表达和聚集方面存在缺陷。与car1-nulls一样，高密度电镀时CRTF-nulls可以诱导聚集，但与car1-nulls不同，晚期CRTF-nulls的发展非常延迟，效率极低，并且在终末分化中受阻。后一种缺陷不能通过直接激活camp依赖性蛋白激酶PKA来绕过，PKA是早期发育缺陷的有效抑制因子。这些数据表明，CRTF在发育后期具有PKA下游功能的新作用。-盘基骨柱，cAMP，受体，激酶，G蛋白，模式形成，转录

Organizing the embryonic body plan is a major requisite governing early metazoan development, with morphogen signaling central to establish and specify cell fates. One of these pathways involves Wnt/wingless signaling and includes 7-transmembrane (7-TM) target receptors and a downstream effector, the protein kinase GSK3b. We have suggested that antagonistic 7-TM cAMP receptor (CAR) pathways in Dictyostelium converge at GSK3 in the context of cell fate determination. We have identified a novel tyrosine kinase, ZAK1, in Dictyostelium that is required for activation of GSK3 during development by cAMP. We also show that recombinant ZAK1 can phosphorylate and consequently activate mammalian GSK3b in vitro. This study may reveal new mechanisms involving protein tyrosine kinases that may be critical for cell fate specification or tumor suppression in other systems. During early Dictyostelium development there is a pulsatile release of cAMP into the extracellular media that directs chemotatic migration and induces gene expression. The extracellular cAMP signal is transduced through the membrane by specific receptor/G-protein pathways that regulate the activity of adenylyl cyclase (AC). AC is transiently activated in the presence of a cAMP signal, but rapidly adapts to a persistent cAMP signal. While Gbg is implicated in AC activation, the mechanism for adaptation of the response to cAMP has been unknown. We have identified a novel Ga (Ga9) in Dictyostelium that is highly similar to proteins of the inhibitory Ga subclass, and our data suggest that Ga9 may participate in the adaptation pathway. Ga9-nulls are also hypersensitive to a newly identified factor secreted by developing Dictyostelium cells that potentiates the chemotatic response to cAMP. Ga9-nulls respond more quickly and at lower cell densities than wild- type. We have purified the factor to homogeneity and two proteins were individually isolated and are being sequenced. These observations suggest that Ga9 is part of a complex signaling network that directs cell movement and senses cell density. Dictyostelium development requires cAMP receptor 1 (CAR1) to relay cAMP signals. Expression of the CAR1 is regulated by cAMP. The novel transcription factor CRTF binds to an essential element within the CAR1 early promoter and mediates CAR1 induction by cAMP. We have purified CRTF to homogeneity and isolated its cDNA. CRTF has an estimated molecular weight of 100kDa with a novel Zn-finger DNA-binding motif. CRTF-null cells are defective in CAR1 expression and in aggregation. Like car1-nulls, CRTF-nulls can be induced to aggregate when plated at high density, but unlike car1- nulls, late CRTF-null development is very delayed, extremely inefficient, and blocked in terminal differentiation. This latter defect cannot be bypassed by direct activation of cAMP-dependent protein kinase PKA, a potent suppressor of early developmental defects. These data suggest a novel role for CRTF late in development that functions downstream of PKA. - Dictyostelium, cAMP, receptors, kinases, G proteins, pattern formation, transcription