REGULATION OF DEVELOPMENTAL GENE EXPRESSION

组织胚胎体计划是管理早期后生动物发育的主要必要条件，其中形态发生素信号传导是建立和指定细胞命运的中心。这些途径之一涉及Wnt/无翅信号传导，包括7-跨膜（7-TM）靶受体和下游效应子，蛋白激酶GSK 3b。我们已经表明，拮抗7-TM cAMP受体（CAR）途径在网骨藻收敛在GSK 3的背景下，细胞命运的决定。我们已经确定了一种新的酪氨酸激酶，ZAK 1，在Dictyosteoprotein，这是必需的激活GSK 3在发展过程中的cAMP。我们还表明，重组ZAK 1可以磷酸化，从而激活哺乳动物GSK 3b在体外。这项研究可能揭示涉及蛋白酪氨酸激酶的新机制，这些蛋白酪氨酸激酶可能对其他系统中的细胞命运规范或肿瘤抑制至关重要。在早期网骨藻发育过程中，cAMP脉冲式释放到细胞外介质中，指导趋化性迁移并诱导基因表达。细胞外cAMP信号通过调节腺苷酸环化酶（AC）活性的特异性受体/G蛋白途径通过膜转导。AC在cAMP信号存在下被瞬时激活，但迅速适应持续的cAMP信号。虽然Gbg与AC激活有关，但对cAMP应答的适应机制尚不清楚。我们已经确定了一个新的镓（镓9）在网骨藻是高度相似的抑制性镓亚类的蛋白质，我们的数据表明，镓9可能参与适应途径。Ga 9-nulls也对由发育中的网骨细胞分泌的新鉴定的因子过敏，所述因子增强对cAMP的趋化反应。与野生型相比，Ga 9-空序列响应更快，细胞密度更低。我们已经将该因子纯化至均一，并将两种蛋白质单独分离并进行测序。这些观察结果表明，Ga 9是一个复杂的信号网络的一部分，该网络指导细胞运动并感知细胞密度。网骨藻的发育需要cAMP受体1（CAR 1）来传递cAMP信号。CAR 1的表达受cAMP调节。新的转录因子CRTF与CAR 1早期启动子内的必需元件结合，并通过cAMP介导CAR 1诱导。我们纯化了CRTF，并分离了其cDNA。CRTF的分子量约为100 kDa，具有一个新的锌指DNA结合基序。CRTF缺失细胞在CAR 1表达和聚集方面有缺陷。与car 1-nulls一样，CRTF-nulls在高密度接种时可以被诱导聚集，但与car 1- nulls不同，晚期CRTF-nulls的发育非常延迟，效率极低，并在终末分化中受阻。后一种缺陷不能通过cAMP依赖性蛋白激酶PKA的直接激活而绕过，PKA是早期发育缺陷的有效抑制剂。这些数据表明CRTF在PKA下游发挥作用的发育后期的新作用。- 网骨藻，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