Zeng C, Anjard C, Primpke G, Wetterauer B, Wille S, Nellen W
Department of Genetics, Kassel University, Heinrich-Plett-Str. 40 D-34132 Kassel, Germany.
Differentiation. 2001 Feb;67(1-2):25-32. doi: 10.1046/j.1432-0436.2001.067001025.x.
The gdt1 gene is a negative regulator of the growth-differentiation-transition (GDT) in Dictyostelium. gdt1- cells express the GDT marker discoidin earlier and at higher levels and prematurely enter the differentiation pathway. Protein kinase A is a positive regulator of the GDT and is required for multicellular development. Disruption of the PKA catalytic subunit or overexpression of a constitutively active mutant of the regulatory subunit results in cells which do not form multicellular aggregates and which show strongly reduced levels of discoidin. We have created PKA-/gdt1- double mutants and show that these display high levels of discoidin expression but no aggregation, suggesting that gdt1 may be a downstream target of PKA in a branched signaling cascade initiating differentiation. Data obtained with the PKA inhibitor H89 support these result: in wild type cells H89 inhibits discoidin expression while in gdt1- mutants there is no obvious effect. However, since PKA-/gdt1- cells display less discoidin expression than the single gdt1 mutant, we propose that PKA and gdt1 are in two parallel interacting pathways. To get insight into the mechanism how PKA may block gdt1, we have tested two putative PKA phosphorylation sites in the protein and found that one of them is efficiently phosphorylated by PKA in vitro. A model for the interplay between PKA and gdt1 during the growth-differentiation-transition is discussed.
gdt1基因是盘基网柄菌生长-分化-转变（GDT）过程中的负调控因子。gdt1基因缺失的细胞更早且更高水平地表达GDT标志物盘状菌素，并过早进入分化途径。蛋白激酶A是GDT的正调控因子，是多细胞发育所必需的。PKA催化亚基的缺失或调节亚基组成型活性突变体的过表达会导致细胞无法形成多细胞聚集体，且盘状菌素水平大幅降低。我们构建了PKA基因缺失/gdt1基因缺失的双突变体，结果显示这些双突变体表现出高水平的盘状菌素表达但没有聚集体形成，这表明在启动分化的分支信号级联反应中，gdt1可能是PKA的下游靶点。用PKA抑制剂H89获得的数据支持了这些结果：在野生型细胞中，H89抑制盘状菌素表达，而在gdt1基因缺失的突变体中则没有明显影响。然而，由于PKA基因缺失/gdt1基因缺失的细胞比单一gdt1突变体表现出更低的盘状菌素表达，我们提出PKA和gdt1处于两条平行的相互作用途径中。为了深入了解PKA可能阻断gdt1的机制，我们测试了该蛋白中两个假定的PKA磷酸化位点，发现其中一个位点在体外能被PKA有效磷酸化。文中还讨论了PKA和gdt1在生长-分化-转变过程中的相互作用模型。
