Gregory S. Taylor

Phoxy Lipids: Revealing PX Domains as Phosphoinositide Binding Modules

and functional insights into the PX domain as a novel The coordination of cellular signaling events requires phosphoinositide binding module. both spatial and temporal regulation. A recurring theme PX Domains as Regulators of Membrane among eukaryotic organisms is the use of phosphoinosiand Protein Trafficking tide-specific binding domains to direct proteins to disThe mechanisms underlying membrane trafficking and crete sites within cells where their function(s) are revesicular fusion have been intensively studied using the quired. To date, a number of distinct, highly conserved vacuolar sorting pathway of the budding yeast, Saccharphosphoinositide binding motifs have been identified, omyces cerevisiae, as a model system. Vam7p, a the most well-characterized of which include the C2 PX-containing protein that is an essential component of (PKC conserved region 2), ENTH (epsin N-terminal hovacuolar sorting, is related to the SNAP-23/25 mammamology) (Ford et al., 2001; Itoh et al., 2001), FYVE (Fab1, lian t-SNAREs (Schultz et al., 2000). SNARE proteins, YOTB, Vac1, and EEA1), and PH (pleckstrin homology) which are found on both cytoplasmic donor vesicles domains (Schultz et al., 2000; see references in reviews (v-SNAREs) and the acceptor organelles to which they by Gillooly et al. 2001; Hurley and Meyer, 2001; and are targeted (t-SNAREs), serve to modulate the specificOdorizzi et al., 2000). These domains recruit proteins to ity of docking and fusion between donor and acceptor specific regions in cells via their interactions with inositol membranes. Vam7p has an N-terminal PX domain, a lipids and may also serve as allosteric regulators of C-terminal coiled-coil t-SNARE motif (Schultz et al., enzyme activity or protein-protein interactions. 2000), and localizes to the vacuolar membrane as part The PX domain was initially identified as a conserved of a vacuolar SNARE complex (Sato et al., 1998; Ungermotif of 130 residues within the p40 and p47 mann and Wickner, 1998). Vam7p has previously been subunits of the neutrophil NADPH oxidase superoxide shown to physically interact with Vam3p, which also generating complex (Ponting, 1996). The PX motif can localizes to vacuolar membranes (Sato et al., 1998; Unalso be found in a wide variety of proteins involved in germann and Wickner, 1998). Mutation of conserved cell signaling pathways (phospholipases D1 and D2, PI residues within the Vam7p PX domain create synthetic 3-kinase, and Spo14p), vesicular trafficking and yeast vacuolar sorting defects when combined with a tempervacuolar morphology (human sorting nexins; yeast ature-sensitive vam3 allele, suggesting that the PX doVps5p, Vps17p, Vam7p, and Mvp1p), and control of main is necessary for Vam7p function (Sato et al., 1998). yeast bud emergence and cell polarity (Bem1p and Further, Vam7p does not contain known membrane tarBem3p) (Ponting, 1996). Currently, at least 57 human geting motifs, prompting the question of how it associand 15 yeast proteins that contain PX domains have ates with vacuolar membranes. In an effort to identify been identified (Schultz et al., 2000). the role of the PX domain of Vam7p in vacuolar sorting, Although the function of PX domains has remained Cheever and colleagues have investigated whether it unclear, previous studies have implied a possible role may serve as a targeting module to direct the localization in regulating the subcellular localization of their correof Vam7p (Cheever et al., 2001). sponding proteins. For example, a significant number In this report, several lines of evidence corroborate of PX proteins are localized to membranes or vesicular that the PX domain functions to localize Vam7p to vacustructures within cells (Schultz et al., 2000). In the case olar membranes (Cheever et al., 2001). First, either a PX of the NADPH oxidase complex, translocation from the domain point mutation or deletion results in mislocalizacytosol to membranes occurs upon activation of the tion of Vam7p to the cytoplasm. Because a Vam7p muneutrophil respiratory burst response (see references tant lacking the C-terminal coiled-coil motif required for in Babior, 1999). In addition, many of the PX proteins its interaction with Vam3p still localizes to the vacuolar involved in membrane trafficking processes do not conmembrane, the authors conclude that the PX domain tain recognizable membrane targeting motifs (Schultz alone is both necessary and sufficient to direct memet al., 2000). Taken together, these observations have brane targeting of Vam7p. It is of note that a Vam7p provided a strong rationale for investigating whether PX mutant localizes to endosomes in a yeast strain in which domains might function as membrane targeting modendosome-to-vacuole sorting is blocked (vps4 ). Here, ules. In a series of papers in Nature Cell Biology this they further observe that this localization is similar to month, the PX domains of the NADPH oxidase compothat of FYVE-domain-containing proteins, which are nents, p40 and p47, the yeast vacuolar SNARE, known to target the membrane phosphoinositide, PI(3)P

Crystal Structure of a Phosphoinositide Phosphatase, MTMR2: Insights into Myotubular Myopathy and Charcot-Marie-Tooth Syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.

PTEN and myotubularin phosphoinositide phosphatases: bringing bioinformatics to the lab bench

Phosphoinositides play an integral role in a diverse array of cellular signaling processes. Although considerable effort has been directed toward characterizing the kinases that produce inositol lipid second messengers, the study of phosphatases that oppose these kinases remains limited. Current research is focused on the identification of novel lipid phosphatases such as PTEN and myotubularin, their physiologic substrates, signaling pathways and links to human diseases. The use of bioinformatics in conjunction with genetic analyses in model organisms will be essential in elucidating the roles of these enzymes in regulating phosphoinositide-mediated cellular signaling.

Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis

Significance Proteoglycans are cellular proteins modified with long chains of repeating sugar residues connected to serine residues within the protein core by a short tetrasaccharide linker. Proteoglycans perform critical cellular functions such as formation of the extracellular matrix, binding to a diverse array of molecules, and regulation of cell motility, adhesion, and cell–cell communication. We show here that family with sequence similarity 20, member B (Fam20B) is a xylose kinase that phosphorylates a xylose sugar residue within the proteoglycan tetrasaccharide linkage. Xylose phosphorylation dramatically stimulates the activity of galactosyltransferase II (GalT-II, B3GalT6), an enzyme that adds galactose to the growing linkage. Cells lacking Fam20B cannot extend the tetrasaccharide linkage and thus have immature and nonfunctional proteoglycan, a phenotype remarkably similar to Ehlers-Danlos syndrome caused by inactivating GalT-II mutations. Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2–3Galβ1–4Xylβ1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.

PTEN and myotubularins: families of phosphoinositide phosphatases.

Publisher Summary This chapter discusses PTEN and Mytobularins, families of Phosphoinositides phosphatases, playing crucial roles in a variety of complex cellular processes. The involvement of phosphoinositides in such a striking number of physiologic processes has led to intensive study of the protein effectors, including lipid kinases and phosphatases that control the spatial and temporal levels of these signaling molecules. PTEN was initially identified as a human tumor suppressor gene located on chromosome 10q23.3, a region frequently mutated in human brain, prostrate, kidney, and breast cancers. The myotubularins represent a second family of protein tyrosine phosphatase-like enzymes that utilize inositol lipids as physiologic substrates. Myotubularin (MTM1), the archetypical member of this group of phosphatases, was initially identified in patients with X-linked, recessive myotubular myopathy, a severe congenital disorder in which muscle cell development is compromised. The chapter describes methodologies that are successful in assessing the lipid phosphatase activity of PTEN and myotubularin family enzymes. These approaches are useful as they can readily be adapted to analyze the activity of most any lipid phosphatases by simply altering simple reaction parameters such as pH, ionic strength, and substrate concentration.

Myotubularin Regulates Akt-dependent Survival Signaling via Phosphatidylinositol 3-Phosphate

Myotubularin is a 3-phosphoinositide phosphatase that is mutated in X-linked myotubular myopathy, a severe neonatal disorder in which skeletal muscle development and/or regeneration is impaired. In this report we provide evidence that siRNA-mediated silencing of myotubularin expression markedly inhibits growth factor-stimulated Akt phosphorylation, leading to activation of caspase-dependent pro-apoptotic signaling in HeLa cells and primary human skeletal muscle myotubes. Myotubularin silencing also inhibits Akt-dependent signaling through the mammalian target of rapamycin complex 1 as assessed by p70 S6-kinase and 4E-BP1 phosphorylation. Similarly, phosphorylation of FoxO transcription factors is also significantly reduced in myotubularin-deficient cells. Our data further suggest that inhibition of Akt activation and downstream survival signaling in myotubularin-deficient cells is caused by accumulation of the MTMR substrate lipid phosphatidylinositol 3-phosphate generated from the type II phosphatidylinositol 3-kinase PIK3C2B. Our findings are significant because they suggest that myotubularin regulates Akt activation via a cellular pool of phosphatidylinositol 3-phosphate that is distinct from that generated by the type III phosphatidylinositol 3-kinase hVps34. Because impaired Akt signaling has been tightly linked to skeletal muscle atrophy, we hypothesize that loss of Akt-dependent growth/survival cues due to impaired myotubularin function may be a critical factor underlying the severe skeletal muscle atrophy characteristic of muscle fibers in patients with X-linked myotubular myopathy.

Endosomal Targeting of the Phosphoinositide 3-Phosphatase MTMR2 is Regulated by an N-Terminal Phosphorylation Site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser58, or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.