Zhou K, Takegawa K, Emr S D, Firtel R A
Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, La Jolla 92093-0634, USA.
Mol Cell Biol. 1995 Oct;15(10):5645-56. doi: 10.1128/MCB.15.10.5645.
Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5 encodes a functional homolog of yeast Vps34p. These observations indicate that in D. discoideum, different PI kinases regulate distinct cellular processes, including cell growth, development, and protein trafficking.
三类磷脂酰肌醇（PI）激酶可将PI转化为PI(3)磷酸、PI(4)磷酸、PI(4,5)二磷酸和PI(3,4,5)三磷酸。这些磷酸肌醇已被证明在囊泡介导的蛋白质分选过程中发挥作用，并且它们作为第二信使信号分子来调节细胞生长。为了进一步阐明磷酸肌醇的调节机制和功能，我们从盘基网柄菌中克隆了编码五种假定PI激酶的基因。数据库分析表明，盘基网柄菌PIK1（DdPIK1）、-2和-3与哺乳动物p110 PI 3激酶关系最为密切，DdPIK5与酵母Vps34p PI 3激酶最为接近，而DdPIK4与PI 4激酶同源性最高。连同其他已知的PI激酶，已经定义了一个PI激酶基因超家族，所有编码的蛋白质都共享一个共同的高度保守的催化核心结构域。DdPIK1、-2和-3可能具有冗余功能，因为破坏任何单个基因对盘基网柄菌的生长或发育都没有影响。然而，破坏两个相关性最高的基因DdPIK1和DdPIK2的菌株表现出生长和发育缺陷，而DdPIK1和DdPIK3以及DdPIK2和DdPIK3的双敲除似乎是致死的。缺失Ddpik1和Ddpik2的细胞比野生型细胞小，当附着在培养皿上时，在与细菌共生以及在无细胞培养基中生长都很缓慢，但在无细胞培养基中悬浮时无法生长。当将缺失Ddpik1和Ddpik2的细胞接种用于多细胞发育时，它们形成了具有多个尖端的聚集体，并产生异常的子实体。DdPIK5（酿酒酵母VPS34的假定同源物）的反义表达导致盘基网柄菌细胞在细菌草坪上生长缺陷和发育异常。DdPIK5补充了粟酒裂殖酵母缺失Svps34突变菌株的温度敏感型生长缺陷，表明DdPIK5编码酵母Vps34p的功能同源物。这些观察结果表明，在盘基网柄菌中，不同的PI激酶调节不同的细胞过程，包括细胞生长、发育和蛋白质运输。
