Interaction of TRAF6 with MAST205 regulates NF-kappaB activation and MAST205 stability

The binding of immune complexes to macrophage Fcgamma receptor results in a subsequent inhibition of lipopolysaccharide-stimulated interleukin-12 synthesis without affecting the induction of tumor necrosis factor-alpha. RNA interference targeting MAST205, a 205-kDa serine/threonine kinase, and transfection of dominant negative MAST205 mutants also mimic this type II macrophage phenotype. Our previous epistasis experiments suggested that the position of MAST205 in the TLR4 signal pathway was proximal to the IkappaB kinase complex. We now report that MAST205 forms a complex with TRAF6, resulting in the inhibition of TRAF6 NF-kappaB activation. We have identified a peptide (residues 218-233) from the N terminus of MAST205 that, when coupled to a protein transduction domain, inhibits the lipopolysaccharide-stimulated activation of NF-kappaB, modulates the size of the MAST205.TRAF6 complex, and inhibits ubiquitination of TRAF6. A dominant negative N-terminal MAST205 deletion mutant also inhibits TRAF6 ubiquitination. The domain required for degradation of MAST205 after Fcgamma receptor activation resides within the N-terminal 261 residues, and degradation is triggered by protein kinase C isoform phosphorylation of Ser/Thr residues. These results suggest that MAST205 functions as a scaffolding protein controlling TRAF6 activity and, therefore, plays an important role in regulating inflammatory responses.     

TLRR (lrrc67) interacts with PP1 and is associated with a cytoskeletal complex in the testis

Background information. Spermatozoa are formed via a complex series of cellular transformations, including acrosome and flagellum formation, nuclear condensation and elongation and removal of residual cytoplasm. Nuclear elongation is accompanied by the formation of a unique cytoskeletal structure, the manchette. We have previously identified a leucine‐rich repeat protein that we have named TLRR (testis leucine‐rich repeat), associated with the manchette that contains a PP1 (protein phosphatase‐1)‐binding site. Leucine‐rich repeat proteins often mediate protein–protein interactions; therefore, we hypothesize that TLRR acts as a scaffold to link signalling molecules, including PP1, to the manchette near potential substrate proteins important for spermatogenesis. Results. TLRR and PP1 interact with one another as demonstrated by co‐immunoprecipitation and the yeast two‐hybrid assay. TLRR binds more strongly to PP1γ2 than it does to PP1α. Anti‐phosphoserine antibodies immunoprecipitate TLRR from testis lysate, indicating that TLRR is a phosphoprotein. TLRR is part of a complex in testis that includes cytoskeletal proteins and constituents of the ubiquitin–proteasome pathway. The TLRR complex purified from 3T3 cells contains similar proteins, co‐localizes with microtubules and is enriched at the microtubule‐organizing centre. TLRR is also detected near the centrosome of elongated, but not mid‐stage, spermatids. Conclusion. We demonstrate here that TLRR interacts with PP1, particularly the testis‐specific isoform, PP1γ2. Immunoaffinity purification confirms that TLRR is associated with the spermatid cytoskeleton. In addition, proteins involved in protein stability are part of the TLRR complex. These results support our hypothesis that TLRR links signalling molecules to the spermatid cytoskeleton in order to regulate important substrates involved in spermatid transformation. The translocation of TLRR from the manchette to the centrosome region suggests a possible role for this protein in tail formation. Our finding that TLRR is associated with microtubules in cultured cells suggests that TLRR may play a common role in modulating the cytoskeleton in other cell types besides male germ cells.     

Ran,a GTP-binding protein involved in nucleocytoplasmic transport and microtubule nucleation,relocates from the manchette to the centrosome region during rat spermiogenesis

Ran, a Ras-related GTPase, is required for transporting proteins in and out of the nucleus during interphase and for regulating the assembly of microtubules. cDNA cloning shows that rat testis, like mouse testis, expresses both somatic and testis-specific forms of Ran-GTPase. The presence of a homologous testis-specific form of Ran-GTPase in rodents implies that the Ran-GTPase pathway plays a significant role during sperm development. This suggestions is supported by distinct Ran-GTPase immunolocalization sites identified in developing spermatids. Confocal microscopy demonstrates that Ran-GTPase localizes in the nucleus of round spermatids and along the microtubules of the manchette in elongating spermatids. When the manchette disassembles, Ran-GTPase immunoreactivity is visualized in the centrosome region of maturing spermatids. The circumstantial observation that fractionated manchettes, containing copurified centrin-immunoreactive centrosomes, can organize a three-dimensional lattice in the presence of taxol and GTP, points to the role of Ran-GTPase and associated factors in microtubule nucleation as well as the potential nucleating function of spermatid centrosomes undergoing a reduction process. Electron microscopy demonstrates the presence in manchette preparations of spermatid centrosomes, recognized as such by their association with remnants of the implantation fossa, a dense plate observed only at the basal surface of developing spermatid and sperm nuclei. In addition, we have found importin beta1 immunoreactivity in the nucleus of elongating spermatids, a finding that, together with the presence of Ran-GTPase in the nucleus of round spermatids and the manchette, suggest a potential role of Ran-GTPase machinery in nucleocytoplasmic transport. Our expression and localization analysis, correlated with functional observations in other cell systems, suggest that Ran-GTPase may be involved in both nucleocytoplasmic transport and microtubules assembly, two critical events during the development of functional sperm. In addition, the manchette-to-centrosome Ran-GTPase relocation, together with the similar redistribution of various proteins associated to the manchette, suggest the existence of an intramanchette molecular transport mechanism, which may share molecular analogies with intraflagellar transport.     

Spnr, a murine RNA-binding protein that is localized to cytoplasmic microtubules

Previous studies in transgenic mice have established the importance of the 3' untranslated region (UTR) of the spermatid-specific protamine-1 (Prm-1) mRNA in its translational control during male germ cell development. To clone genes that mediate the translational repression or activation of the Prm-1 mRNA, we screened cDNA expression libraries made with RNA from pachytene spermatocytes and round spermatids, with an RNA probe corresponding to the 3' UTR of Prm-1. We obtained six independent clones that encode Spnr, a spermatid perinuclear RNA- binding protein. Spnr is a 71-kD protein that contains two previously described RNA binding domains. The Spnr mRNA is expressed at high levels in the testis, ovary, and brain, and is present in multiple forms in those tissues. Immunolocalization of the Spnr protein within the testis shows that it is expressed exclusively in postmeiotic germ cells and that it is localized to the manchette, a spermatid-specific microtubular array. Although the Spnr protein is expressed too late to be directly involved in the translational repression of Prm-1 specifically, we suggest that the Spnr protein may be involved in other aspects of spermatid RNA metabolism, such as RNA transport or translational activation.     

Identification of a novel human MAST4 gene, a new member of the microtubule associated serine-threonine kinase family

Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64%, 63%, 59% and 39% identical aminoacid residues with MAST1, MAST2, MAST3 and MASTL respectively. RT-PCR analysis revealed relatively high expression level of MAST4 in most normal human tissues, with an exception of in testis, small intestine, colon and peripheral blood leukocyte.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Testis fascin (FSCN3): a novel paralog of the actin-bundling protein fascin expressed specifically in the elongate spermatid head

During spermiogenesis, significant morphological changes occur as round spermatids are remodeled into the fusiform shape of mature spermatozoa. These changes are correlated with a reorganization of microfilaments and microtubules in the head and tail regions of elongating spermatids. There is also altered expression of specialized actin- and tubulin-associated proteins. We report the characterization of a novel, spermatid-specific murine paralog of the actin-bundling protein fascin (FSCN1); this paralog is designated testis fascin or FSCN3. Testis fascin is distantly related to fascins but retains its primary sequence organization. cDNA clones of mouse testis fascin predict a 498 amino acid protein of molecular mass 56 kD that shares 29% identity with mouse fascin. Mapping of murine and human FSCN3 genes shows localization to the 7q31.3 chromosome. Northern analysis indicates that FSCN3 expression is highly specific to testis and that in situ hybridization further restricts expression to elongating spermatids. Antibodies raised against recombinant FSCN3 protein identify a band at 56 kD in testis, epididymis, and epididymal spermatozoa, suggesting that testis fascin persists in mature spermatozoa. In accord with the in situ hybridization results, immunofluorescent microscopy localizes testis fascin protein to areas of the anterior spermatid head that match known distributions of F-actin in the dorsal and ventral subacrosomal spaces. It is possible that testis fascin may function in the terminal elongation of the spermatid head and in microfilament rearrangements that accompany fertilization.     

Linkage of manchette microtubules to the nuclear envelope and observations of the role of the manchette in nuclear shaping during spermiogenesis in rodents.

Structural features of the mouse and rat manchette and the role of the manchette in shaping the spermatid nucleus were investigated. Rod-like elements about 10 nm in diameter and 40-70 nm in length were seen linking the innermost microtubules of the manchette and the outer leaflet of the nuclear envelope in step 8 through step 11 rat and mouse spermatids that either had been routinely fixed for electron microscopy or had been isolated and detergent extracted. Rod-like linkers were also seen joining the nuclear ring to the plasma membrane and nuclear envelope. These linkers may ensure that under normal conditions the manchette remains in a defined position relative to these membranous components. A variety of compounds (taxol, cytoxan, and 5-fluorouracil) were found to perturb the manchette and to affect nuclear shaping. In addition, sys and azh mutant mice were used to determine the consequences of defective manchette formation. These genetic conditions and chemical treatments either produced manchettes that were not in their normal position (azh, sys, and taxol) and/or caused the manchette to appear abnormal (azh, sys, cytoxan, 5-fluorouracil, and taxol), and all resulted in a deformation of the step 9-11 spermatid nucleus. In all instances where the manchette was present, either in normal or ectopic locations, the sectioned nuclear envelope was parallel to the long axis of the microtubules of the manchette. In general, areas of the nuclear envelope where the manchette was not present, or where it was expected to be present but was not, were rounded (normal animals, sys, cytoxan). In addition, there are indications using certain compounds (cytoxan and 5-fluorouracil) as well as in the azh and sys mouse that the manchette may exert pressure to deform the nucleus. It is suggested that the rod-like linkages of the manchette ensure that the nuclear envelope remains at a constant distance from the manchette microtubules and that this is a major factor acting to impart nuclear shape changes on a region of the head caudal to the acrosome during the early elongation phase of spermiogenesis. The manchette microtubules, which are also known to be linked together, may act as a scaffold to deform this part of the nucleus from its spherical shape, perhaps in concert with forces initiated by other structural elements. Evidence from sys animals indicates that structural elements, such as the acrosomal complex over the anterior head (acrosome-actin-nuclear envelope), may affect nuclear shaping over the acrosome-covered portion of the spermatid head.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.     

Identification of a novel human MAST4 gene, a new member of human microtubule associated serine/threonine kinase family

Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here, we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64, 63, 59, and 39% identical amino acid residues with MAST1, MAST2, MAST3, and MASTL, respectively. RT-PCR analysis revealed a relatively high expression level of MAST4 in most normal human tissues, with the exception of in testis, small intestine, colon, and peripheral blood leukocyte. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 808–815. The text was submitted by the authors in English. The nucleotide sequences reported in this paper have been submitted to GenBank under accession number: AY830839. These two authors contributed equally to this paper.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.     

Binding of PTEN to specific PDZ domains contributes to PTEN protein stability and phosphorylation by microtubule-associated serine/threonine kinases

The tumor suppressor phosphatase PTEN is a key regulator of cell growth and apoptosis that interacts with PDZ domains from regulatory proteins, including MAGI-1/2/3, hDlg, and MAST205. Here we identified novel PTEN-binding PDZ domains within the MAST205-related proteins, syntrophin-associated serine/threonine kinase and MAST3, characterized the regions of PTEN involved in its interaction with distinctive PDZ domains, and analyzed the functional consequences on PTEN of PDZ domain binding. Using a panel of PTEN mutations, as well as PTEN chimeras containing distinct domains of the related protein TPTE, we found that the PTP and C2 domains of PTEN do not affect PDZ domain binding and that the C-terminal tail of PTEN (residues 350-403) provides selectivity to recognize specific PDZ domains from MAGI-2, hDlg, and MAST205. Binding of PTEN to the PDZ-2 domain from MAGI-2 increased PTEN protein stability. Furthermore, binding of PTEN to the PDZ domains from microtubule-associated serine/threonine kinases facilitated PTEN phosphorylation at its C terminus by these kinases. Our results suggest an important role for the C-terminal region of PTEN in the selective association with scaffolding and/or regulatory molecules and provide evidence that PDZ domain binding stabilizes PTEN and targets this tumor suppressor for phosphorylation by microtubule-associated serine/threonine kinases.     

Microtubule configurations and post-translational alpha-tubulin modifications during mammalian spermatogenesis

The mechanisms underlying cell cycle progression and differentiation are tightly entwined with changes associated in the structure and composition of the cytoskeleton. Mammalian spermatogenesis is a highly intricate process that involves differentiation and polarization of the round spermatid. We found that pachytene spermatocytes and round spermatids have most of the microtubules randomly distributed in a cortical network without any apparent centrosome. The Golgi apparatus faces the acrosomal vesicle and some microtubules contact its surface. In round spermatids, at step 7, there is an increase in short microtubules around and over the nucleus. These microtubules are located between the rims of the acrosome and may be the very first sign in the formation of the manchette. This new microtubular configuration is correlated with the beginning of the migration of the Golgi apparatus from the acrosomal region towards the opposite pole of the cell. Next, the cortical microtubules form a bundle running around the nucleus perpendicular to the main axis of the cell. At later stages, the nuclear microtubules increase in size and a fully formed manchette appears at stage 9. On the other hand, acetylated tubulin is present in a few microtubules in pachytene spermatocytes and in the axial filament (precursor of the sperm tail) in round spermatids. Our results suggest that at step 7, the spermatid undergoes a major microtubular reordering that induces or allows organelle movement and prepares the cell for the formation of the manchette and further nuclear shaping. This new microtubular configuration is associated with an increase in short microtubules over the nucleus that may correspond to the initial step of the manchette formation. The new structure of the cytoskeleton may be associated with major migratory events occurring at this step of differentiation.     

Abnormal manchette development in spermatids of azh/azh mutant mice

A study of manchette development during spermiogenesis in azh/azh mutant mice was carried out by thin-section transmission electron microscopy with the goal of determining which of the initial steps in spermatid development are aberrant. In the homozygous mutant, spermatogenesis was quantitatively normal; but 100% of the sperm nuclei produced had abnormal shapes. The first defect, observed in steps 8-9, was the abnormal positioning of many manchette microtubules. These microtubules were directed towards regions of the plasma membrane not normally associated with manchette formation, in addition to being located at the caudal rim of the acrosome in the normal region of manchette formation. At steps 10-12, sheets of manchette microtubules were often in ectopic positions along the plasma membrane, rather than in association with the nuclear membrane as well. The fine structural appearance of the manchette was generally normal; the defect appeared to be in its positioning within the cell. In many step 8-10 spermatids nuclear invaginations and evaginations were observed, always associated with irregularities in the position of some of the manchette microtubules; these illustrate the capacity of manchette microtubules to deform nuclear shape. The nuclear irregularities remained throughout spermiogenesis. These observations are consistent with the hypothesis that the manchette is involved in at least some aspects of sperm nuclear shaping and that the improper positioning of manchette formation is a likely candidate for the primary abnormality resulting from a defective allele at the azh locus.