Myotubularin phosphoinositide phosphatases: cellular functions and disease pathophysiology

The myotubularin family of phosphoinositide phosphatases includes several members mutated in neuromuscular diseases or associated with metabolic syndrome, obesity, and cancer. Catalytically dead phosphatases regulate their active homologs by heterodimerization and potentially represent key players in the phosphatase-kinase balance. Although the enzymatic specificity for phosphoinositides indicates a role for myotubularins in endocytosis and membrane trafficking, recent findings in cellular and animal models suggest that myotubularins regulate additional processes including cell proliferation and differentiation, autophagy, cytokinesis, and cytoskeletal and cell junction dynamics. In this review, we discuss how myotubularins regulate such diverse processes, emphasizing newly identified functions in a physiological and pathological context. A better understanding of myotubularin pathophysiology will pave the way towards therapeutic strategies.     

A phylogenetic survey of myotubularin genes of eukaryotes: distribution,protein structure,evolution, and gene expression

Background  

Phosphorylated phosphatidylinositol (PtdIns) lipids, produced and modified by PtdIns kinases and phosphatases, are critical to the regulation of diverse cellular functions. The myotubularin PtdIns-phosphate phosphatases have been well characterized in yeast and especially animals, where multiple isoforms, both catalytically active and inactive, occur. Myotubularin mutations bring about disruption of cellular membrane trafficking, and in humans, disease. Previous studies have suggested that myotubularins are widely distributed amongst eukaryotes, but key evolutionary questions concerning the origin of different myotubularin isoforms remain unanswered, and little is known about the function of these proteins in most organisms.     

Myotubularin phosphatases: policing 3-phosphoinositides

In eukaryotic cells, phosphatidylinositol is subject to differential phosphorylation, resulting in the production of seven distinct phosphatidylinositol phosphates, often referred to as phosphoinositides (PIs). PIs have numerous distinct roles in cellular regulation and membrane trafficking. Recently, myotubularin family PI 3-phosphatases have emerged as key regulators of phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, two PIs that regulate traffic within the endosomal-lysosomal pathway. Mutations in several myotubularin genes lead to myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy. Strikingly, nearly half of the members of the human myotubularin family appear to be catalytically inactive. Several inactive myotubularins have essential functions in mammals. Recent work in mammalian cells and model organisms is shedding light on the roles of myotubularins in membrane traffic.     

The structure and regulation of myotubularin phosphatases

The human neuromuscular diseases X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B are caused by mutations in myotubularin family proteins. The myotubularins are a unique subfamily of protein tyrosine phosphatases that utilize inositol phospholipids, rather than phosphoproteins, as substrates. Recent structural studies, including the first crystal structure of a myotubularin family protein, have defined the structural features that are characteristic of the family and revealed the molecular basis of their unique substrate specificity. Interestingly, the myotubularin family contains a subgroup of proteins that are catalytically inactive. Recent biochemical studies have established that the inactive myotubularins function as adaptors for the active members and play an important regulatory role within the family.     

Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway.     

AtMTM1, a novel mitochondrial protein,may be involved in activation of the manganese-containing superoxide dismutase in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mtm1p is essential for the posttranslational activation of manganese-containing superoxide dismutase (SOD2) in Saccharomyces cerevisiae; however, whether the same holds true for Arabidopsis thaliana is unknown. In this study, by using the yeast mtm1 mutant complementation method, we identified a putative MTM gene (AtMTM1, At4g27940) that is necessary for SOD2 activation. Further, analysis of SOD activity revealed that an SOD2 defect is rescued in the yeast mutant Y07288 harboring the AtMTM1 gene. Related mRNA-level analysis showed the AtMTM1 gene is induced by paraquat but not by hydrogen peroxide, which indicates that this gene is related to the superoxide scavenger SOD. In addition, an AtMTM1::GFP fusion construct was transiently expressed in the protoplasts, and it was localized to the mitochondria. Furthermore, sequence deletion analysis of AtMTM1 revealed that the code region (amino acid (aa) 60–198) of Mtm1p plays an important role in localization of the protein to the mitochondria. Regulation of AtMTM1 gene expression was analyzed using a fusion construct of the 1,766 bp AtMTM1 promoter and the GUS (β-glucuronidase) reporter gene. The screen identified GUS reporter gene expression in the developing cotyledons, leaves, roots, stems, and flowers but not in the siliques. Our results suggest that AtMTM1 encodes a mitochondrial protein that may be playing an important role in activation of MnSOD1 in Arabidopsis.     

Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis

The WNK (With No Lysine K) serine-threonine kinases have been shown to be osmosensitive regulators and are critical for cell volume homeostasis in humans. We previously identified a soybean root-specific WNK homolog, GmWNK1, which is important for normal late root development by fine-tuning regulation of ABA levels. However, the functions of WNKs in plant osmotic stress response remains uncertain. In this study, we generated transgenic Arabidopsis plants with constitutive expression of GmWNK1. We found that these transgenic plants had increased endogenous ABA levels and altered expression of ABA-responsive genes, and exhibited a significantly enhanced tolerance to NaCl and osmotic stresses during seed germination and seedling development. These findings suggest that, in addition to regulating root development, GmWNK1 also regulates ABA-responsive gene expression and/or interacts with other stress related signals, thereby modulating osmotic stress responses. Thus, these results suggest that WNKs are members of an evolutionarily conserved kinase family that modulates cellular response to osmotic stresses in both animal and plants.     

A zinc finger-containing glycine-rich RNA-binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress conditions

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to changing environmental conditions, the reports demonstrating their biological roles are severely limited. Here, we examined the functional roles of a zinc finger-containing GRP, designated atRZ-1a, in Arabidopsis thaliana under drought or salt stress conditions. Transgenic Arabidopsis plants overexpressing atRZ-1a displayed retarded germination and seedling growth compared with the wild-type plants under salt or dehydration stress conditions. In contrast, the loss-of-function mutants of atRZ-1a germinated earlier and grew faster than the wild-type plants under the same stress conditions. Germination of the transgenic plants and mutant lines was influenced by the addition of ABA or glucose, implying that atRZ-1a affects germination in an ABA-dependent way. H(2)O(2) was accumulated at higher levels in the transgenic plants compared with the wild-type plants under stress conditions. The expression of several germination-responsive genes was modulated by atRZ-1a, and proteome analysis revealed that the expression of different classes of genes, including those involved in reactive oxygen species homeostasis and functions, was affected by atRZ-1a under dehydration or salt stress conditions. Taken together, these results suggest that atRZ-1a has a negative impact on seed germination and seedling growth of Arabidopsis under salt or dehydration stress conditions, and imply that atRZ-1a exerts its function by modulating the expression of several genes under stress conditions.     

Gamma-glutamyl transpeptidase in transgenic tobacco plants. Cellular localization, processing, and biochemical properties

gamma-Glutamyl transpeptidase (gamma-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the gamma-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for gamma-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound gamma-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis gamma-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.     

Myotubularin-related Proteins 3 and 4 Interact with Polo-like Kinase 1 and Centrosomal Protein of 55 kDa to Ensure Proper Abscission

The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein–protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.The myotubularins are a subfamily of protein tyrosine phosphatases (PTPs)¹, consisting of sixteen conserved proteins. Despite containing the conserved C(X)₅R catalytic motif found in all protein tyrosine phosphatases, myotubularins harbor active sites that do not dephosphorylate tyrosine, but instead catalyze the conversion of the phosphatidylinositol-type lipids phosphatylinositol 3 phosphate (PI3P) and phosphatylinositol 3,5 phosphate (PI3,5P) to phosphatidylinositol (PI) and phosphatylinositol 5 phosphate (PI5P), respectively (1). Phosphatidylinositols are important molecules in a variety of processes, and as enzymatic regulators, myotubularins may function in cell proliferation, differentiation, survival, and cytoskeletal and junctional dynamics (1, 2). Of the sixteen myotubularins, only nine are active enzymes (supplemental Fig. S1A), as several lack catalytic cysteine residues (3). Myotubularins interact extensively with each other, and interactions between active and inactive pairs are frequent (4). It is thought that inactive myotubularins regulate the activity, substrate binding, and/or localization of their active binding partners (2).Several myotubularins are linked to human disease. Myotubularin (MTM1), the first reported family member, is mutated in X-linked centronuclear myopathy (5), and Myotubularin related protein 14 (MTMR14) is mutated in autosomal centronuclear myopathy (6). Mutations in the active MTMR2 or its inactive binding partner, SET binding factor (SBF)2 (MTMR13), cause Charcot-Marie-Tooth diseases CMT4A and CMT4B, respectively (7–9). MTMR7 and MTMR9 have been associated with metabolic syndrome and obesity (MTMR9) (10, 11), epilepsy (MTMR7/9) (12), and Creutzfeldt-Jakob disease (MTMR7) (13). In addition, misregulation of the active phosphatase MTMR3 contributes to susceptibility to gastric and colon carcinomas (14), oral cancer (15), and lung cancer (16), and contributes to metastasis (15, 17). Aberrant expression of the inactive MTMR11 has been observed in acute myeloid leukemia (18), acute lymphocytic leukemia (19), and Her2-positive breast cancer (20). Generally, myotubularins are thought to integrate different cellular pathways, through both phosphatidylinositol regulation and protein–protein interactions (2). Despite their proposed involvement in a variety of cellular processes as well as disease states, many myotubularins remain poorly characterized, with their precise cellular functions not yet elucidated, and the pathological significance of those functions still unknown.To gain insight on the biological functions of myotubularin family phosphatases, we have used affinity purification coupled to mass spectrometry (AP-MS) to identify protein–protein interactions for each myotubularin. The results expand upon the known repertoire of intra-myotubularin interactions, and, critically, identify specific novel interactions for individual myotubularins, providing valuable clues toward their respective functions. Further investigation revealed an unexpected role for MTMR3 and MTMR4 in abscission (21), the fission event at the end of cytokinesis that severs the final membrane link between divided daughter cells. Future studies of additional identified protein–protein interactions will undoubtedly illuminate the cellular roles of myotubularin family phosphatases.     

Topology, subcellular localization, and sequence diversity of the Mlo family in plants

Barley Mlo defines the founder of a novel class of plant integral membrane proteins. Lack of the wild type protein leads to broad spectrum disease resistance against the pathogenic powdery mildew fungus and deregulated leaf cell death. Scanning N-glycosylation mutagenesis and Mlo-Lep fusion proteins demonstrated that Mlo is membrane-anchored by 7 transmembrane (TM) helices such that the N terminus is located extracellularly and the C terminus intracellularly. Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. A genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana revealed approximately 35 family members, the only abundant gene family encoding 7 TM proteins in higher plants. The sequence variability of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of metazoan 7 TM receptors, the G-protein-coupled receptors.