A PHOSPHATIDYLINOSITOL (PI) KINASE GENE FAMILY IN DICTYOSTELIUM-DISCOIDEUM - BIOLOGICAL ROLES OF PUTATIVE MAMMALIAN P110 AND YEAST VPS34P PI 3-KINASE HOMOLOGS DURING GROWTH AND DEVELOPMENT

Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3 )phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trispho sphate. These phosphoinositides have been shown to function in vesicle -mediated protein sorting, and they serve as second-messenger signalin g molecules for regulating cell growth. To further elucidate the mecha nism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Datab ase analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 ar e most closely related to the mammalian p110 PI 4-kinase, DdPIK5 is cl osest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous t o PI 4- kinases. Together with other known PI kinases, a superfamily o f PI kinase genes has been defined, with all of the encoded proteins s haring a common highly conserved catalytic core domain. DdPIK1, -2, an d -3 may have redundant functions because disruption of any single gen e had no effect on D. discoideum growth or development. However, strai ns in which both of the two most highly related genes, DdPIK1 and DdPI K2, 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 gro w in suspension in axenic medium. When Delta Ddpik1 Delta Ddpik2 null cells were plated for multicellular development, they formed aggregate s having multiple tips and produced abnormal fruiting bodies. Antisens e expression of DdPIK5 (a putative homolog of the Saccharomyces cerevi siae VPS34) led to a defect in the grow th of D. discoideum cells on b acterial lawns and abnormal development. DdPIK5 complemented the tempe rature-sensitive growth defect of a Schizosaccharomyces pombe Delta Sv ps34 mutant strain, suggesting DdPIK5 encodes a functional homolog of yeast Vps34p. These observations indicate that in D. discoideum, diffe rent PI kinases regulate distinct cellular processes, including cell g rowth, development, and protein trafficking.