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In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain-containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud. [ABSTRACT FROM AUTHOR]

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1-/- and Hmgb2-/- mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-κB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders. [ABSTRACT FROM AUTHOR]

Sperm motility is regulated by protein phosphorylation. The 66 kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues associated with the motility initiation. In order to understand the regulatory mechanism of sperm motility, the 66 kDa protein was identified in the present study. The 66 kDa protein was purified by 2-D gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry, liquid chromatography-tandem mass spectrometry and peptide sequencer. The 66 kDa protein was tubulin β chain. The 66 kDa protein is one of the tubulin β chain isoforms and phosphorylated in relation to the motility initiation. (Reprod Med Biol 2004; 3: 133–139) [ABSTRACT FROM AUTHOR]

In our previous studies (Fujinoki et al., 2001, 2003), we reported that two types of 36 kDa proteins, designated 36K-A protein and 36K-B protein, obtained from hamster sperm flagella, are associated with motility activation and phosphorylated in a cAMP-dependent manner at serine residues. In the present experiments, we focused on the hamster (Mesocricetus auratus) 36K-A protein, which was analyzed by peptide mass finger printing and amino acid sequencing. The results suggest that 36K-A protein is a pyruvate dehydrogenase E1 component β subunit lacking the N-terminal 30 amino acids. Moreover, our results suggest that 36 K-A protein is localized in the fibrous sheath of the principal piece of hamster spermatazoa. [Copyright &y& Elsevier]

In our previous paper [M. Fujinoki et al. (2001) Biomed. Res. 22, 45–58], we reported that two types of 36-kDa protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with the regulation of motility activation. In the present experiments, it was suggested that these two types of 36-kDa protein were phosphorylated in a cAMP-dependent manner associated with motility activation of hamster spermatozoa. Because the 36K-B protein was the most intensely phosphorylated in a cAMP-dependent manner, attempts were made to further characterize it. The 36K-B protein was assumed to be localized in the middle piece. The localization of the 36K-B protein was the same as that of the 36-kDa protein reported in our previous paper [Y. Si et al. (1999) Mol. Reprod. Dev. 52, 328–334]. In order to identify the 36K-B protein, it was analyzed by peptide mass finger printing and amino acid sequencing. The results suggested that the 36K-B protein was a pyruvate dehydrogenase E1 component β subunit and a component of the mitochondrial sheath of the middle piece. [ABSTRACT FROM PUBLISHER]

In response to stimulation of B-cells through cell surface IgM, the activity of the serine/ threonine protein phosphatase PP1, but not PP2A, was transiently decreased and reached a minimum 10–20 min after the stimulation. The decrease was more profound in the immature B-cell line WEHI-231, than in the mature B-cell line BAL-17. Under these conditions, PP1α, an isoform of PP1, showed unique alterations in the patterns of several spots with distinct isoelectic points in the Western blot after two-dimensional electropho-resis, whereas another isoform, PP1δ, did not show any alteration. PP1γ1 and PP1γ2 were not detected in B-cells. Similar alterations in these spots were observed in B-cells stimulated by PMA. When partially purified PP1 consisting of PP1α and PP1δ was incubated with [γ-32P]ATP and PKC, radioactive spots of PP1α could be detected, but no spot of PP1δ was detected. Because differences in sequence among PP1 isoforms are mostly restricted to their C-terminals, phosphorylation rates of the C-terminal peptides containing the PKC-phosphorylation motif were compared. The C-terminal peptide of PP1α is a better substrate for PKC than those of PP1γ1 and PP1γ2, and is phosphorylated at the serine residue corresponding to Ser-325 of PP1α. The corresponding C-terminal region of PP1δ does not contain the phosphorylation site. On the other hand, there was a large difference in subcellular distribution of PP1δ, but not PP1α, between immature and mature B-cells. From these results, it was strongly suggested that PP1α is involved, via phosphorylation by PKC, in the regulation of signal transduction in response to the stimulation of B-cells through cell surface IgM. [ABSTRACT FROM AUTHOR]

To elucidate the roles of serine/threonine protein phosphatases PP1 and PP2A in the morphological changes of B-lymphocytes during development and in immune responses, we investigated alterations of protein levels of catalytic subunits of PP1 and PP2A and regulatory subunits of PP1 including M130/M133, inhibitor-1 (I-1) and inhibitor-2 (I-2) in B-cell lines at different maturational stages and during their aggregation induced by phorbol myristate acetate (PMA). The protein levels of PPlδ and/or M130/M133 were significantly lower in B-cell lines without pseudopods, WEHI-231, BAL-17, Daudi, and CESS, than in those with pseudopods, Bcl.1, A20, M12, and SKW6.4, whereas the amounts of PP1α and PP2A were similar among them. During aggregation of A20 and CESS cells induced by PMA, an activator of PKC, the amount of PP1δ was progressively decreased, and this decrease was blocked by H7, an inhibitor of PKC. The amount of PP1α was constant under these conditions. Okadaic acid, an inhibitor of PP1 and PP2A, also induced aggregation of A20 cells at concentrations sufficient to inhibit PP1, but not at lower concentrations that inhibit PP2A alone. These results suggest that myosin light chain phosphatase composed of PP1δ and M130/M133 is involved in the maintenance and regulation of cytoskeletal structures in B-lymphocytes. [ABSTRACT FROM AUTHOR]

Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Recent work has revealed essential roles for Ser/Thr phosphatases, such as protein phosphatase-1 (PP1), in splicing, but how these phosphatases are regulated is largely unknown. We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155 (spliceosome-associated protein 155), an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap 155 dephosphorylation. C-terminally truncated NIPP1 (NIPP1-ΔC) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -ΔC resulted in slight and severe decreases in Sap155 hyperphosphorylation, respectively, and the latter was accompanied with inhibition of splicing. PP1 overexpression produced similar effects, whereas small interfering RNA-mediated NIPP1 knockdown enhanced Sap 155 hyper- phosphorylation upon okadaic acid treatment. NIPP1 did not inhibit but rather stimulated Sap 155 dephosphorylation by PP1 in vitro through facilitating Sap155/PP1 interaction. Further analysis revealed that NIPP1 specifically recognizes hyperphosphorylated Sap155 thorough its Forkhead-associated domain and dissociates from Sap155 after dephosphorylation by associated PP1. Thus NIPP1 works as a molecular sensor for PP1 to recognize phosphorylated Sap155. [ABSTRACT FROM AUTHOR]