adenosine-3--5--cyclic-phosphorothioate and herbimycin

The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Benzoquinones; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Genistein; Humans; In Vitro Techniques; Isoquinolines; Lactams, Macrocyclic; Leupeptins; Male; Oligopeptides; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein-Tyrosine Kinases; Rifabutin; Sperm Capacitation; Spermatozoa; Sulfonamides; Thionucleotides

Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1) family. We have used a human T-lymphotropic virus type 1 (HTLV-1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV-1 transcription following CD2 but not CD3 receptor cross-linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor-mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross-linking increased cAMP detected by competitive enzyme-linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross-linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post-translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp-cAMP-sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross-linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF-1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE-mediated transcription following ligand engagement (e.g. cell-to-cell contact).

Topics: Activating Transcription Factor 2; Benzoquinones; Blotting, Western; CD2 Antigens; CD3 Complex; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Electrophoresis; Enzyme Inhibitors; Human T-lymphotropic virus 1; Humans; Lactams, Macrocyclic; Lymphocyte Activation; Monocytes; Naphthalenes; Oligonucleotide Probes; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Receptor Cross-Talk; Rifabutin; Signal Transduction; Thionucleotides; Transcription Factors; Transcription, Genetic

The experiments reported herein have characterized the signaling pathway leading to stimulation of type I protein kinase A isozyme (PKA-I) activity during the early events of Ag receptor-mediated T cell activation. Inhibitor studies demonstrated that receptor-initiated activation of nonreceptor protein tyrosine kinases, phosphorylation and activation of phospholipase C-gamma 1, and activation of protein kinase C occur temporally and precede PKA-I activation. Bypass of both the TCR/CD3 complex and IL-1R and direct activation of protein kinase C by a phorbol ester can also activate PKA-I. To confirm that PKA-I activation via the TCR/CD3 complex and IL-1R requires antecedent protein tyrosine kinase-catalyzed phosphorylation of phospholipase C-gamma 1, we used wild-type and CD45-deficient (mutant J45.01) Jurkat T cell lines. Unlike wild-type Jurkat T cells, the absence of CD45 tyrosine phosphatase resulted in the failure of receptor-mediated activation of PKA-I activity and of IL-2 mRNA transcription in the mutant J45.01 Jurkat cell line. In conclusion, our data support the concept that a signal derived from ligand binding to both the TCR/CD3 complex and IL-1R receptor mediates rapid activation of the PKA-I isozyme in primary T lymphocytes by sequential activation of an intracellular pathway comprised of CD45 phosphatase/protein tyrosine kinase/polyphosphoinositide/Ca2+/protein kinase C pathway rather than via the conventional surface receptor/stimulatory G protein system.

Topics: Adult; Benzoquinones; Calcium; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Female; GTP-Binding Proteins; Humans; Interleukin-1; Interleukin-2; Isoenzymes; Lactams, Macrocyclic; Leukemia-Lymphoma, Adult T-Cell; Leukocyte Common Antigens; Lymphocyte Activation; Male; Membrane Proteins; Models, Biological; Muromonab-CD3; Phospholipase C gamma; Phospholipid Ethers; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Quinones; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Interleukin-1; Recombinant Proteins; Rifabutin; RNA, Messenger; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases