Phosphatidylinositol phosphate kinases in the moss Physcomitrella patens

Abstract: Phosphatidylinositol(4,5)bisphosphate, [PtdIns(4,5)P2], is a signalling lipid which is involved in many important processes in animal cells such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels, and nuclear signalling pathways. In contrast to the animal field, much less is known about the roles of PtdIns(4,5)P2 and its synthesizing enzyme, phosphatidylinositol phosphate kinase (PtdInsP kinase), in plants. The two PtdInsP kinases, PpPIPK1 and PpPIPK2, from the moss Physcomitrella patens were studied at genetic, biochemical, and physiological levels. In accordance with PtdInsP kinases class I/II B of flowering plants, sequence analysis of the moss enzymes identified a N-terminal MORN domain, in which 8 MORN motifs are clustered, a linker sequence followed by a ninth MORN motif, and a C-terminal conserved kinase catalytic domain. Biochemical characterization showed that the two moss enzymes exhibit different substrate specificities. PpPIPK1 is regulated in a similar way to other animal and plant PtdInsP kinases: activation by phosphatidic acid and phosphorylation by protein kinase A. The function of the glutamic acid located in the activation loop is conserved as in animal type I PtdInsP kinases: this amino acid determines the substrate specificity in PpPIPK1 and AtPIP5K1. Both PpPIPKs are localized to the plasma membrane in moss protoplasts. A dibasic amino acid pair, KR, located within the activation loop of PpPIPK1 was found to be essential for the lipid kinase activity and plasma membrane localization of the enzyme. Knock out mutants for both enzymes were obtained. A strong phenotype for pipk1, but not for pipk2, single knock out was obtained. In comparison to the wild type, the colony morphology of pipk1 lines was altered due to a dramatic reduction of extension growth of protonema as well as of rhizoids. A more severe phenotype was observed for the double knock out mutant, pipk1-2, characterized by a dramatic reduction in rhizoid length, lack of the caulonemal cell type, and loss of sporophyte production. Complementation of the pipk1 phenotype with PpPIPK1 resulted in full restoration of wild type phenotype. PtdIns(4,5)P2 is known to be an important regulator of several F-actin-binding proteins. Treatment of either regenerating protoplasts or young protonema with F-actin drugs, such as Cytochalasin B and Latruculin B, showed that treated wild type mimicked pipk1 single knock out phenotype. The pipk1-2 knock out lines, which already showed a strong defect in the establishment of polar growth, were not affected by the drugs. Taken together our results show that the moss PtdInsP kinase enzymes play a crucial role in the physiology of plant cells suggesting a role in the regulation of tip growth.