Functions and Mechanisms of SAC Phosphoinositide Phosphatases in Plants

Phosphatidylinositol (PtdIns) is one type of phospholipid comprising an inositol head group and two fatty acid chains covalently linked to the diacylglycerol group. In addition to their roles as compositions of cell membranes, phosphorylated PtdIns derivatives, termed phosphoinositides, execute a wide range of regulatory functions. PtdIns can be phosphorylated by various lipid kinases at 3-, 4- and/or 5- hydroxyls of the inositol ring, and the phosphorylated forms, including PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P2, PtdIns(4,5)P2, can be reversibly dephosphorylated by distinct lipid phosphatases. Amongst many other types, the SUPPRESSOR OF ACTIN (SAC) family of phosphoinositide phosphatases recently emerged as important regulators in multiple growth and developmental processes in plants. Here, we review recent advances on the biological functions, cellular activities, and molecular mechanisms of SAC domain-containing phosphoinositide phosphatases in plants. With a focus on those studies in the model plant Arabidopsis thaliana together with progresses in other plants, we highlight the important roles of subcellular localizations and substrate preferences of various SAC isoforms in their functions.

Downregulation of Lipid Phosphate Phosphatase 3 correlates with Tumor-Infiltrating Immune Cells in Oral Cancer

Purpose Sphingosine-1-phosphate (S1P), a potent oncogenic lipid. Intracellular levels of S1P are tightly regulated by eight S1P metabolizing enzymes. S1P is synthesized by phosphorylation of sphingosine which is catalyzed by two sphingosine kinases (SphK1 and SphK2). Five lipid phosphatases (two S1P phosphatases and three lipid phosphate phosphatases) reversibly convert S1P back to sphingosine. S1P is ultimately irreversibly degraded by S1P lyase. The role of sphingosine-1-phosphate (S1P) metabolizing enzymes in oral squamous cell carcinoma (OSCC) has not been fully studied. Methods In the current study, we have determined the protein expression of four S1P metabolizing enzymes, namely sphingosine Kinase (SphK) -1, SphK2, S1P phosphatase 1 (SGPP1), and lipid phosphate phosphatase 3 (LPP3) by immunohistochemistry (IHC) and western botting in tumor tissues of 46 OSCC patients and normal oral mucosa (N = 6). Further, we determined the associations of expression of S1P metabolizing enzymes with clinicopathological features of OSCC patients. Results SphK2 and LPP3 exhibit low IRS in OSCC tumors. Importantly, expression of SphK2 and LPP3 was downregulated in malignant cells compared to non-malignant mucosa. Further, LPP3 expression negatively correlated with TNM staging of patients (ρ = -0.307, p = 0.043). Importantly, TCGA analysis revealed that LPP3 expression was positively correlated with infiltration of B cells, neutrophils, macrophages, and dendritic cells in the HNSCC tumors. Conclusion In conclusion, our data show that expression of SphK2 and LPP3 is decreased in OSCC tumors compared to normal mucosa. Thus, LPP3 could represent a potential prognostic marker and therapeutic target for OSCC.

The Leishmania donovani LDBPK_220120.1 Gene Encodes for an Atypical Dual Specificity Lipid-Like Phosphatase Expressed in Promastigotes and Amastigotes; Substrate Specificity, Intracellular Localizations, and Putative Role(s)

The intracellular protozoan parasites of the Leishmania genus are responsible for Leishmaniases, vector borne diseases with a wide range of clinical manifestations. Leishmania (L.) donovani causes visceral leishmaniasis (kala azar), the most severe of these diseases. Along their biological cycle, Leishmania parasites undergo distinct developmental transitions including metacyclogenesis and differentiation of metacyclic promastigotes (MPs) to amastigotes. Metacyclogenesis inside the phlebotomine sandfly host’s midgut converts the procyclic dividing promastigotes to non-dividing infective MPs eventually injected into the skin of mammalian hosts and phagocytosed by macrophages where the MPs are converted inside modified phagolysosomes to the intracellular amastigotes. These developmental transitions involve dramatic changes in cell size and shape and reformatting of the flagellum requiring thus membrane and cytoskeleton remodeling in which phosphoinositide (PI) signaling and metabolism must play central roles. This study reports on the LDBPK_220120.1 gene, the L. donovani ortholog of LmjF.22.0250 from L. major that encodes a phosphatase from the “Atypical Lipid Phosphatases” (ALPs) enzyme family. We confirmed the expression of the LDBPK_220120.1 gene product in both L. donovani promastigotes and axenic amastigotes and showed that it behaves in vitro as a Dual Specificity P-Tyr and monophosphorylated [PI(3)P and PI(4)P] PI phosphatase and therefore named it LdTyrPIP_22 (Leishmaniad onovani Tyrosine PI Phosphatase, gene locus at chromosome 22). By immunofluorescence confocal microscopy we localized the LdTyrPIP_22 in several intracellular sites in the cell body of L. donovani promastigotes and amastigotes and in the flagellum. A temperature and pH shift from 25°C to 37°C and from pH 7 to 5.5, induced a pronounced recruitment of LdTyrPIP_22 epitopes to the flagellar pocket and a redistribution around the nucleus. These results suggest possible role(s) for this P-Tyr/PI phosphatase in the regulation of processes initiated or upregulated by this temperature/pH shift that contribute to the developmental transition from MPs to amastigotes inside the mammalian host macrophages.

PTEN and Other PtdIns(3,4,5)P3 Lipid Phosphatases in Breast Cancer

The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. PtdIns(3,4,5)P3 can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P2. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced PIPP expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although PIPP depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression.

Phosphoinositide lipids in primary cilia biology

Primary cilia are solitary signalling organelles projecting from the surface of most cell types. Although the ciliary membrane is continuous with the plasma membrane it exhibits a unique phospholipid composition, a feature essential for normal cilia formation and function. Recent studies have illustrated that distinct phosphoinositide lipid species localise to specific cilia subdomains, and have begun to build a ‘phosphoinositide map’ of the cilium. The abundance and localisation of phosphoinositides are tightly regulated by the opposing actions of lipid kinases and lipid phosphatases that have also been recently discovered at cilia. The critical role of phosphoinositides in cilia biology is highlighted by the devastating consequences of genetic defects in cilia-associated phosphoinositide regulatory enzymes leading to ciliopathy phenotypes in humans and experimental mouse and zebrafish models. Here we provide a general introduction to primary cilia and the roles phosphoinositides play in cilia biology. In addition to increasing our understanding of fundamental cilia biology, this rapidly expanding field may inform novel approaches to treat ciliopathy syndromes caused by deregulated phosphoinositide metabolism.

Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting βHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting βHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

Characteristics of the First Protein Tyrosine Phosphatase with Phytase Activity from a Soil Metagenome

Protein tyrosine phosphatases (PTPs) fulfil multiple key regulatory functions. Within the group of PTPs, the atypical lipid phosphatases (ALPs) are known for their role as virulence factors associated with human pathogens. Another group of PTPs, which is capable of using inositol-hexakisphosphate (InsP6) as substrate, are known as phytases. Phytases play major roles in the environmental phosphorus cycle, biotechnology, and pathogenesis. So far, all functionally characterized PTPs, including ALPs and PTP-phytases, have been derived exclusively from isolated microorganisms. In this study, screening of a soil-derived metagenomic library resulted in identification of a gene (pho16B), encoding a PTP, which shares structural characteristics with the ALPs. In addition, the characterization of the gene product (Pho16B) revealed the capability of the protein to use InsP6 as substrate, and the potential of soil as a source of phytases with so far unknown characteristics. Thus, Pho16B represents the first functional environmentally derived PTP-phytase. The enzyme has a molecular mass of 38 kDa. The enzyme is promiscuous, showing highest activity and affinity toward naphthyl phosphate (Km 0.966 mM). Pho16B contains the HCXXGKDR[TA]G submotif of PTP-ALPs, and it is structurally related to PtpB of Mycobacterium tuberculosis. This study demonstrates the presence and functionality of an environmental gene codifying a PTP-phytase homologous to enzymes closely associated to bacterial pathogenicity.