Two members of the human testis-specific serine/threonine (Ser/Thr) kinase family, TSSK 1 and TSSK 2, were cloned and sequenced from a human testis adaptor-ligated cDNA library using a PCR strategy. Within the cDNA, open reading frames (ORF) were defined encoding proteins of 367 and 358 amino acids respectively, as well as conserved kinase domains typical of the superfamily of Ser/Thr kinases. Both genes were intronless and mapped to chromosomes 5 and 22 respectively. The human and mouse homologues of TSSK 1 and TSSK 2, together with TSSK 3 and SSTK/FKSG82, constitute a kinase subfamily closely related to the calmodulin kinases and SNF/nim 1 kinase subfamilies. Similar to the mouse, tissue expression by northern and dot blot analysis revealed that human TSSK 1 and 2 messages are expressed exclusively in the testis. However, mRNA for these kinases can be detected in other tissues using real-time PCR. In addition, TSKS, the human homologue of a putative substrate of TSSK 1 and 2, was cloned. TSKS had an ORF of 592 amino acids and was also expressed exclusively in the testis as demonstrated by northern and dot blot analyses; however, lower levels of expression in other tissues were detected using real-time PCR. Human TSSK 2 and TSKS interacted in a yeast two-hybrid system and also co-immunoprecipitated after in vitro translation. TSSK 2 expressed in yeast and bacteria was able to autophosphorylate and also phosphorylated recombinant TSKS in vitro. Antibodies against recombinant TSSK 2 demonstrated that a member of the TSSK family was present in human testis and localized to the equatorial segment of ejaculated human sperm. In contrast, TSKS was only found in the testis. The finding of a TSSK family member in mature sperm suggests that this family of kinases might play a role in sperm function.
Enzymes in the glycolytic pathway of mammalian sperm are modified extensively and are localized in the flagellum, where several are tightly bound to the fibrous sheath. This study provides the first evidence for three novel aldolase isozymes in mouse sperm, two encoded by Aldoart1 and Aldoart2 retrogenes on different chromosomes and another by Aldoa_v2, a splice variant of Aldoa. Phylogenetic analyses and comparative genomics indicate that the retrogenes and splice variant have remained functional and have been under positive selection for millions of years. Their expression is restricted to the male germline and is tightly regulated at both transcriptional and translational levels. All three isozymes are present only in spermatids and sperm and have distinctive features that may be important for localization in the flagellum and/or altered metabolic regulation. Both ALDOART1 and ALDOA_V2 have unusual N-terminal extensions not found in other aldolases. The N-terminal extension of ALDOA_V2 is highly conserved in mammals, suggesting a conserved function in sperm. We hypothesize that the N-terminal extensions are responsible for localizing components of the glycolytic pathway to the fibrous sheath and that this localization is required to provide sufficient ATP along the length of the flagellum to support sperm motility.
Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers.
We report the cloning and characterization of MOEP19, a novel 19 kDa RNA binding protein that marks a defined cortical cytoplasmic domain in oocytes and provides evidence of mammalian oocyte polarity and a form of pre-patterning that persists in zygotes and early embryos through the morula stage. MOEP19 contains a eukaryotic type KH-domain, typical of the KH-domain type I superfamily of RNA binding proteins, and both recombinant and native MOEP19 bind polynucleotides. By immunofluorescence, MOEP19 protein was first detected in primary follicles throughout the ooplasm. As oocytes expanded in size during oogenesis, MOEP19 increased in concentration. MOEP19 localized in the ovulated egg and early zygote as a symmetrical spherical cortical domain underlying the oolemma, deep to the zone of cortical granules. MOEP19 remained restricted to a cortical cytoplasmic crescent in blastomeres of 2-, 4- and 8-cell embryos. The MOEP19 domain was absent in regions underlying cell contacts. In morulae, the MOEP19 domain was found at the apex of outer, polarized blastomeres but was undetectable in blastomeres of the inner cell mass. In early blastocysts, MOEP19 localized in both mural and polar trophectoderm and a subset of embryos showed inner cell mass localization. MOEP19 concentration dramatically declined in late blastocysts. When blastomeres of 4- to 8-cell stages were dissociated, the polarized MOEP19 domain assumed a symmetrically spherical localization, while overnight culture of dissociated blastomeres resulted in formation of re-aggregated embryos in which polarity of the MOEP19 domain was re-established at the blastomere apices. MOEP19 showed no evidence of translation in ovulated eggs, indicating that MOEP19 is a maternal effect gene. The persistence during early development of the MOEP19 cortical oocyte domain as a cortical crescent in blastomers suggests an intrinsic pre-patterning in the egg that is related to the apical-basolateral polarity of the embryo. Although the RNAs bound to MOEP19 are presently unknown, we predict that the MOEP19 domain directs RNAs essential for normal embryonic development to specific locations in the oocyte and early embryo.
Residues 1-434 of the ubiquitin ligase Cbl control epidermal growth factor receptor (EGF-R) signaling by enhancing receptor ubiquitination, downregulation, and lysosomal degradation. Cbl 1-434 comprises a tyrosine kinase-binding domain, linker region, RING finger (RF), and a subset of the RF tail amino acids 420-436. Using full-length alanine substitution mutants, we demonstrate that the Cbl RF tail regulates biochemically distinct EGF-R endocytosis checkpoints: 1) Cbl- and ubiquitin-dependent degradation of hSprouty2 upstream of EGF-R ubiquitination (compromised by Cbl V431A); and 2) Cbl- and EGF-R-dependent dephosphorylation or degradation of the endosomal trafficking regulator Hrs (compromised by Cbl F434A). Deregulated Hrs phosphorylation correlates with the inhibition of both early endosome fusion and EGF-R degradation. This is the first evidence that Cbl can regulate receptor fate by controlling the fusion of sorting endosomes. We postulate that it does so by modulating the generation and loss of tyrosine phosphorylated Hrs.
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