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. Myotubularin (MTM1)1 is a dual specificity protein-tyrosine phosphatase (PTP)-like enzyme that is mutated in X-linked myotubular myopathy, a severe congenital disorder in which muscle cell development is compromised (1-3). Myogenesis in affected individuals is arrested at a late stage of differentiation/ maturation following myotube formation, and the muscle cells have characteristic large centrally located nuclei (1). The MTM1 protein is the first characterized member of one of the largest families of dual specificity PTPs yet identified (reviewed in Refs. 4 and 5). The MTM family includes at least eight putative catalytically active proteins as well as four forms that are predicted to be enzymatically inactive (4 -7). The inactive MTM proteins contain substitutions at specific residues that are required for catalysis by PTP superfamily enzymes and may function as interaction modules (4 -8). Phylogenetic analysis of MTM family proteins indicates that they can be further divided into at least four distinct subgroups, which include the catalytically active MTM1/MTMR1/MTMR2, MTMR3/MTMR4, and MTMR6/MTMR7/MTMR8 enzymes, as well as the SBF1/LIP-STYX/MTMR10/3-PAP inactive forms (4 -7). Our laboratory and others have previously shown that myotubularin specifically dephosphorylates the D3 position ...

The myotubularin (MTM) family constitutes one of the most highly conserved protein-tyrosine phosphatase subfamilies in eukaryotes. MTM1, the archetypal member of this family, is mutated in X-linked myotubular myopathy, whereas mutations in the MTMrelated (MTMR)2 gene cause the type 4B1 Charcot-Marie-Tooth disease, a severe hereditary motor and sensory neuropathy. In this study, we identified a protein that specifically interacts with MTMR2 but not MTM1. The interacting protein was shown by mass spectrometry to be MTMR5, a catalytically inactive member of the MTM family. We also demonstrate that MTMR2 interacts with MTMR5 via its coiled-coil domain and that mutations in the coiledcoil domain of either MTMR2 or MTMR5 abrogate this interaction. Through this interaction, MTMR5 increases the enzymatic activity of MTMR2 and dictates its subcellular localization. This article demonstrates an active MTM member being regulated by an inactive family member.T he myotubularin (MTM) family constitutes one of the largest and most highly conserved protein-tyrosine phosphatase (PTP) subfamilies in eukaryotes (1-3). The human MTM family of phosphatases includes MTM1͞MTM-related (MTMR)1͞ MTMR2, MTMR3͞MTMR4, and MTMR6͞MTMR7͞MTMR8 subgroups (1, 3). The consensus CX 5 R active site motif is found in the MTM family and the sequence "CSDGWDR" is invariant within all of the enzymatically active members of this family. Most PTPs use phosphoproteins as substrates and specifically dephosphorylate substrates containing only phosphotyrosine sites. Other phosphatases, collectively known as dual-specificity phosphatases, are capable of removing phosphoserines͞ threonines and phosphotyrosines from protein substrates.Initially, MTM1 was reported to be a dual-specificity phosphatase (4-6). However, we and others have demonstrated that MTM1 utilizes the lipid second messenger, phosphatidylinositol 3-phosphate [PI(3)P], as a physiological substrate (7,8). Recent findings demonstrate that other MTMR phosphatases MTMR1, MTMR2, MTMR3, MTMR4, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all active MTM family members (9-12). MTMR2 and MTMR3 have also been shown to dephosphorylate phosphatidylinositol 3,5-bisphosphate (9, 13). PI(3)P plays a key role in membrane trafficking͞vesicular transport processes and serves as a targeting mechanism for proteins containing specific PI(3)P-binding modules such as Fab1͞YOTB͞Vac1p͞EEA1 (FYVE), pleckstrin homology (PH), and Phox homology domains (14-17).To date, two MTMR proteins have been associated with human diseases. The MTM1 gene on chromosome Xq28 is mutated in X-linked myotubular myopathy, a severe congenital muscular disorder characterized by hypotonia and generalized muscle weakness in newborn males (18,19 MTM1 and MTMR2 are highly similar proteins (64% identity, 76% similarity), use the same physiologic substrate, and have a ubiquitous expression pattern (6, 9-12). However, mutations in MTM1 and MTMR2 cause different diseases with different target tissues...

Dynamic membrane remodeling during intracellular trafficking is controlled by the intricate interplay between lipids and proteins. BAR domains are modules that participate in endocytic processes by binding and deforming the lipid bilayer. Sorting nexin 9 (SNX9), which functions in clathrin-mediated endocytosis, contains a BAR domain, however, the properties of this domain are not well understood. Here we show that SNX9 shares many properties with other BAR domain-containing proteins, such as amphiphysin and endophilin. SNX9 is able to deform the plasma membrane, as well as liposomes, into narrow tubules and recruit N-WASP and dynamin 2 to these tubules via its SH3 domain. SNX9-induced tubulation is antagonized by N-WASP and dynamin 2 while it is enhanced by perturbation of actin dynamics. However, SNX9 also has several unique properties. The tubulating activity requires the BAR and PX domains, as well as the low-complexity (LC) domain, which binds the Arp2/3 complex. SNX9 also binds to PtdIns(4)P-5-kinases via its PX domain and its tubulating activity is regulated by phosphoinositides. In addition, the kinase activity of PtdIns(4)P-5-kinases is stimulated by interaction with SNX9, suggesting a positive feedback interaction between SNX9 and PtdIns(4)P-5-kinases. These results suggest that SNX9 functions in the coordination of membrane remodeling and fission via interactions with actin-regulating proteins, endocytic proteins and PtdIns(4,5)P 2 -metabolizing enzymes.

The family with sequence similarity 20 (Fam20) kinases phosphorylate extracellular substrates and play important roles in biomineralization. Fam20C is the Golgi casein kinase that phosphorylates secretory pathway proteins within Ser-x-Glu/pSer motifs. Mutations in Fam20C cause Raine syndrome, an osteosclerotic bone dysplasia. Here we report the crystal structure of the Fam20C ortholog from Caenorhabditis elegans. The nucleotide-free and Mn/ ADP-bound structures unveil an atypical protein kinase-like fold and highlight residues critical for activity. The position of the regulatory αC helix and the lack of an activation loop indicate an architecture primed for efficient catalysis. Furthermore, several distinct elements, including the presence of disulfide bonds, suggest that the Fam20 family diverged early in the evolution of the protein kinase superfamily. Our results reinforce the structural diversity of protein kinases and have important implications for patients with disorders of biomineralization.P rotein phosphorylation is a fundamental mechanism that regulates numerous physiological processes (1, 2). Protein kinases have evolved to function as dynamic switches relaying signals in response to various stimuli by transferring a phosphate from ATP to target proteins (3-5). Most phosphoproteins are found within the cell, residing in the nuclei and cytosol; however, secreted proteins, including casein and other members of the secretory calciumbinding phosphoprotein family, are phosphorylated as well (6). We recently identified a family of kinases that reside in the secretory pathway and function to phosphorylate extracellular substrates (7,8). One member of this family, Fam20C (family with sequence similarity 20, member C), is the physiological casein kinase that phosphorylates multiple secreted proteins within a Ser-x-Glu/pSer motif (7, 9, 10). The importance of this discovery is underscored by the fact that some 75% of the phosphoproteins identified in human serum and cerebrospinal fluid contain phosphate within this motif (11-13). Furthermore, mutations in FAM20C cause Raine syndrome, a deadly osteosclerotic bone dysplasia characterized by generalized osteosclerosis, ectopic calcifications, and characteristic facial features (14-16). Most individuals with Raine syndrome die within a few weeks after birth; however, nonlethal cases with dental abnormalities and clinical features of hypophosphatemia have been reported (17,18). Loss of Fam20C in mice also results in severe bone and tooth anomalies, as well as hypophosphatemia (19)(20)(21).Two other closely related Fam20C paralogs, Fam20A and Fam20B, are present in humans (22). Fam20B is ubiquitously expressed and phosphorylates xylose within the tetrasaccharide linkage region of proteoglycans (23). This phosphorylation event may influence glycosaminoglycan biosynthesis (24). Genetic deletion of Fam20B in mice results in embryonic lethality at E13.5, and mutations in Danio rerio result in reduced cartilage matrix production and skeletal defects (19,25). The substra...

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.

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of fulllength SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.Sorting nexin 9 (SNX9), 2 also known as SH3PX1, is a member of the sorting nexin superfamily characterized by the presence of a phospholipid-binding motif, the PX domain. Sorting nexin family proteins contribute to protein sorting in cells by their ability to bind specific lipids and to form protein-protein complexes. SNX9, initially identified as a protein interacting with the metalloproteases MDC9 and MDC15 (1), is composed of an N-terminal Src homology 3 domain, a low complexity region, a PX domain, and a C-terminal Bin/Amphiphysin/Rvs (BAR) domain (2-4). It forms a complex with dynamin-2 and regulates the recruitment of dynamin-2 to the membrane (5). It also enhances the assembly of dynamin and increases its GTPase activity (6). Other endocytic molecules, namely AP-2 (adaptor protein complex 2) and clathrin, also bind to the low complexity region of SNX9 in a cooperative manner (2). Through these interactions, SNX9 plays an important role in clathrin-mediated endocytosis in non-neuronal cells (2, 6).Dynamin is centrally involved in clathrin-mediated endocytosis (7,8). It self-assembles around the necks of invaginated clathrin-coated pits and releases vesicles from the membrane via GTP hydrolysis (9). It is composed of several domains. The N-terminal nucleotide-binding domain is responsible for GTP hydrolysis, and the C-terminal proline-rich domain (PRD) links it to several SH3 domain-containing proteins such as Grb2, amphiphysin, and endophilin (10 -12). The central pleckstrin homology domain controls its binding to membrane phospholipids (13), and a coiled-coil domain (also called the GTPase effector domain) is involved in its self-assembly and in regulating its GTPase activity.The affinity between the pleckstrin homology domain of dynamin and lipids is not high enough to translocate dynamin from the cytosol to the p...

BackgroundEpigallocatechin-3-gallate (EGCG), one of the major catechins in green tea, is a potential chemopreventive agent for various cancers. The aim of this study was to examine the effect of EGCG on the expression of heat shock proteins (HSPs) and tumor suppression.MethodsCell colony formation was evaluated by a soft agar assay. Transcriptional activity of HSP70 and HSP90 was determined by luciferase reporter assay. An EGCG-HSPs complex was prepared using EGCG attached to the cyanogen bromide (CNBr)-activated Sepharose 4B. In vivo effect of EGCG on tumor growth was examined in a xenograft model.ResultsTreatment with EGCG decreased cell proliferation and colony formation of MCF-7 human breast cancer cells. EGCG specifically inhibited the expression of HSP70 and HSP90 by inhibiting the promoter activity of HSP70 and HSP90. Pretreatment with EGCG increased the stress sensitivity of MCF-7 cells upon heat shock (44°C for 1 h) or oxidative stress (H2O2, 500 μM for 24 h). Moreover, treatment with EGCG (10 mg/kg) in a xenograft model resulted in delayed tumor incidence and reduced tumor size, as well as the inhibition of HSP70 and HSP90 expression.ConclusionsOverall, these findings demonstrate that HSP70 and HSP90 are potent molecular targets of EGCG and suggest EGCG as a drug candidate for the treatment of human cancer.

Heat shock transcription factor 1 (HSF1) is activated by pathophysiologic stresses and activation leads to an increased cellular level of heat shock proteins (Hsp(s)). Although the activation of HSF1 occurs via multiple stress-induced processes such as hyperphosphorylation, the exact cellular mechanism of HSF1 activation is still unclear. Here we show polo-like kinase 1 (PLK1) and HSF1 interact in vivo using the tandem affinity purification system. Although the interaction between HSF1 and PLK1 is increased by thermal stress, overexpression of PLK1 did not affect HSF1 trimerization or DNA binding activity. This interaction results in the phosphorylation of HSF1 on serine 419 by PLK1. Interestingly, mutation of serine 419 to alanine inhibited heat-stress induced HSF1 nuclear translocation. Our results suggest that the phosphorylation of HSF1 by PLK1 is an essential step for HSF1 nuclear translocation by heat stress.