REGULATION OF DEVELOPMENTAL GENE EXPRESSION

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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