Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.

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 (Siaα2-3Galß1-4Xylß1) 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.

Receptor mediated endocytosis 8 is a novel PI(3)P binding protein regulated by myotubularin-related 2.

Myotubularin related protein 2 (MTMR2) is a member of the myotubularin family of phosphoinositide lipid phosphatases. Although MTMR2 dephosphorylates the phosphoinositides PI(3)P and PI(3,5)P2, the phosphoinositide binding proteins that are regulated by MTMR2 are poorly characterized. In this study, phosphoinositide affinity chromatography coupled to mass spectrometry identified receptor mediated endocytosis 8 (RME-8) as a novel PI(3)P binding protein. RME-8 co-localized with the PI(3)P marker DsRed-FYVE, while the N-terminal region of RME-8 is required for PI(3)P and PI(3,5)P(2) binding in vitro. Depletion of PI(3)P by MTMR2 S58A or wortmannin treatment attenuated RME-8 endosomal localization and co-localization with EGFR on early endosomes. Our results suggest a model in which the localization of RME-8 to endosomal compartments is spatially mediated by PI(3)P binding and temporally regulated by MTMR2 activity.

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 Ser(58), 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.

Phosphatase PRL-3 is a direct regulatory target of TGFbeta in colon cancer metastasis.

Metastasis causes most deaths from cancer yet mechanistic understanding and therapeutic options remain limited. Overexpression of the phosphatase PRL-3 (phosphatase of regenerating liver) is associated with metastasis of colon cancer. Here, we show that PRL-3 is a direct target of signaling by TGFß, which is broadly implicated in progression and metastasis. We found that suppression of PRL-3 expression by TGFß was mediated by Smad-dependent inhibition of PRL-3 transcription at the level of promoter activity. PRL-3 activation stimulated PI3K/AKT signaling that caused resistance to stress-induced apoptosis. PRL-3 overexpression promoted metastatic colonization in an orthotopic mouse model of colon cancer, whereas PRL-3 knockdown reduced metastatic potential. Altered metastatic phenotypes were not derivative of primary tumor development or local invasion but could be attributed to PRL-3-mediated cell survival. Our findings suggest that inhibiting PRL-3 expression might be an important mechanism through which TGFß suppresses metastasis in colon cancer. In addition, our findings suggest that loss of TGFß signaling, which occurs commonly during colon cancer progression, is sufficient to activate a PRL-3-mediated cell survival pathway that can selectively promote metastasis. Therefore, a major implication of our findings is that PRL-3 antagonists may offer significant value for antimetastatic therapy in patients with colon cancer.

Distinguishing mitochondrial inner membrane orientation of dual specific phosphatase 18 and 21.

Dual specificity phosphatase (DSP) 18 and 21 are members of a poorly understood subfamily of protein tyrosine phosphatases (PTP) that are unique in their ability to dephosphorylate both phosphotyrosine and phosphoserine/threonine residues in vitro. Because of the difficulty in identifying substrate specificity, determining subcellular localization can help to resolve biological function of these phosphatases. DSP18 and DSP21 are targeted to mitochondria by internal localization signals. Surprisingly, DSP18 and DSP21 are both peripherally associated with the mitochondrial inner membrane, however, DSP18 is oriented toward the intermembrane space while DSP21 is facing the matrix compartment. This chapter describes methodology for purification of recombinant protein and demonstration of phosphatase activity, for mitochondrial purification and subfractionation of mitochondria to determine submitochondrial localization and for determining membrane orientation and strength of membrane association.

Incidence of postoperative infections in non-immunocompromised pediatric patients with low absolute neutrophil counts preoperatively.

PURPOSE: To discover the incidence of postoperative surgical site infections in non-immunocompromised pediatric patients with an absolute neutrophil count (ANC) < or = 1,000 preoperatively. METHODS: Office and hospital charts of consecutive patients with preoperative ANC < or = 1,000 undergoing elective surgery over a three-year period were reviewed for evidence of postoperative surgical site infection. RESULTS: Six of 200 patients with preoperative ANCs < or = 1,000 developed a postoperative surgical site infection, an infection rate of 3.0%. One of 35 patients with preoperative ANCs < or = 500 developed a surgical site infection. The mean ANC of the study population was 800 (range 200 to 1,000); the mean ANC of the group with postoperative infection likewise was 800 (range 500 to 1,000). CONCLUSIONS: There was a postoperative surgical site infection rate of 3.0% in non-immunocompromised children who had a preoperative ANC < or = 1,000, similar to the overall surgical site infection rate in children. Cancellation of elective procedures in non-immunocompromised children with ANC < or = 1,000 is not warranted on the grounds of concern about postoperative infection.

Molecular basis for substrate recognition by MTMR2, a myotubularin family phosphoinositide phosphatase.

Myotubularins, a large family of catalytically active and inactive proteins, belong to a unique subgroup of protein tyrosine phosphatases that use inositol phospholipids, rather than phosphoproteins, as physiological substrates. Here, by integrating crystallographic and deuterium-exchange mass spectrometry studies of human myotubularin-related protein-2 (MTMR2) in complex with phosphoinositides, we define the molecular basis for this unique substrate specificity. Phosphoinositide substrates bind in a pocket located on a positively charged face of the protein, suggesting an electrostatic mechanism for membrane targeting. A flexible, hydrophobic helix makes extensive interactions with the diacylglycerol moieties of substrates, explaining the specificity for membrane-bound phosphoinositides. An extensive H-bonding network and charge-charge interactions within the active site pocket determine phosphoinositide headgroup specificity. The conservation of these specificity determinants within the active, but not the inactive, myotubularins provides insight into the functional differences between the active and inactive members.

Living unrelated donor renal transplantation: a single center experience.

PURPOSE: Living, genetically unrelated donor renal transplantation (LURT) is being performed with increasing frequency. We evaluated our single center experience with LURT and compared this to a cohort of living related donor renal transplants (LRT) to evaluate the short-term success of LURT at our center. MATERIALS AND METHODS: We identified 99 consecutive patients who underwent LURT at our center and had at least 1 year of followup data. A control cohort of 99 patients who underwent LRT at our center matched for age, number of transplants and date of transplant was also identified. One-year graft and patient survival, and serum creatinine levels at 1, 3, 6 and 12 months were compared between the groups. Our data were compared with national and international data. RESULTS: At our center 1-year graft survival was 95% in the LURT and LRT cohorts. One-year LURT patient survival was 99% compared with 97% in the LRT group and the serum creatinine levels were not significantly different. CONCLUSIONS: Patients undergoing LURT at our center have excellent 1-year graft and patient survival compared with LRT performed at our center, and national and international LURT. Genetically unrelated kidney donors should continue to be used to expand the kidney donor pool.

Assaying phosphoinositide phosphatases.

The roles of phosphoinositide second messengers as signaling molecules in a vast array of cellular processes including cell growth, metabolism, vesicular transport, programmed cell death, and responses to extracellular signals are only beginning to be understood. The recent identification of novel phosphoinositide signaling molecules underscores the need for methodology with which to characterize the enzymes responsible for regulating cellular phosphoinositide levels. One of the ways in which cells control these lipids is through dephosphorylation by phosphoinositide phosphatases, which oppose and regulate the actions of phosphoinositide kinases. We describe herein two rapid and simple assays for characterizing phosphoinositide phosphatases that can be used to provide a basis for understanding the activity and specificity of these enzymes.

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.

Myotubularin and MTMR2, phosphatidylinositol 3-phosphatases mutated in myotubular myopathy and type 4B Charcot-Marie-Tooth disease.

Myotubularin is the archetype of a family of highly conserved protein-tyrosine phosphatase-like enzymes. The myotubularin gene, MTM1, is mutated in the genetic disorder, X-linked myotubular myopathy. We and others have previously shown that myotubularin utilizes the lipid second messenger, phosphatidylinositol 3-phosphate (PI(3)P), as a physiologic substrate. We demonstrate here that the myotubularin-related protein MTMR2, which is mutated in the neurodegenerative disorder, type 4B Charcot-Marie-Tooth disease, is also highly specific for PI(3)P as a substrate. Furthermore, the MTM-related phosphatases MTMR1, MTMR3, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all myotubularin family enzymes. A direct comparison of the lipid phosphatase activities of recombinant myotubularin and MTMR2 demonstrates that their enzymatic properties are indistinguishable, indicating that the lack of functional redundancy between these proteins is likely to be due to factors other than the utilization of different physiologic substrates. To this end, we have analyzed myotubularin and MTMR2 transcripts during induced differentiation of cultured murine C2C12 myoblasts and find that their expression is divergently regulated. In addition, myotubularin and MTMR2 enhanced green fluorescent protein fusion proteins exhibit overlapping but distinct patterns of subcellular localization. Finally, we provide evidence that myotubularin, but not MTMR2, can modulate the levels of endosomal PI(3)P. From these data, we conclude that the developmental expression and subcellular localization of myotubularin and MTMR2 are differentially regulated, resulting in their utilization of specific cellular pools of PI(3)P.