In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

Tumor suppression and apoptosis of human prostate carcinoma mediated by a genetic locus within human chromosome 10pter-q11.

Prostate cancer is the second leading cause of male cancer deaths in the United States. Yet, despite a large international effort, little is known about the molecular mechanisms that underlie this devastating disease. Prostate secretory epithelial cells and androgen-dependent prostate carcinomas undergo apoptosis in response to androgen deprivation and, furthermore, most prostate carcinomas become androgen independent and refractory to further therapeutic manipulations during disease progression. Definition of the genetic events that trigger apoptosis in the prostate could provide important insights into critical pathways in normal development as well as elucidate the perturbations of those key pathways in neoplastic transformation. We report the functional definition of a novel genetic locus within human chromosome 10pter-q11 that mediates both in vivo tumor suppression and in vitro apoptosis of prostatic adenocarcinoma cells. A defined fragment of human chromosome 10 was transferred via microcell fusion into a prostate adenocarcinoma cell line. Microcell hybrids containing only the region 10pter-q11 were suppressed for tumorigenicity following injection of microcell hybrids into nude mice. Furthermore, the complemented hybrids undergo programmed cell death in vitro via a mechanism that does not require nuclear localization of p53. These data functionally define a novel genetic locus, designated PAC1, for prostate adenocarcinoma 1, involved in tumor suppression of human prostate carcinoma and furthermore strongly suggest that the cell death pathway can be functionally restored in prostatic adenocarcinoma.

Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.

Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice.

Translational control is a major form of regulating gene expression during gametogenesis and early development in many organisms. We sought to determine whether the translational repression of the protamine 1 (Prm1) mRNA is necessary for normal spermatid differentiation in mice. To accomplish this we generated transgenic animals that carry a Prm1 transgene lacking its normal 3' untranslated region. Premature translation of Prm1 mRNA caused precocious condensation of spermatid nuclear DNA, abnormal head morphogenesis, and incomplete processing of Prm2 protein. Premature accumulation of Prm1 within syncytial spermatids in mice hemizygous for the transgene caused dominant male sterility, which in some cases was accompanied by a complete arrest in spermatid differentiation. These results demonstrate that correct temporal synthesis of Prm1 is necessary for the transition from nucleohistones to nucleoprotamines.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway.

We report here that the activation of the interleukin 1 beta (IL-1 beta)-converting enzyme (ICE) family is likely to be one of the crucial events of tumor necrosis factor (TNF) cytotoxicity. The cowpox virus CrmA protein, a member of the serpin superfamily, inhibits the enzymatic activity of ICE and ICE-mediated apoptosis. HeLa cells overexpressing crmA are resistant to apoptosis induced by Ice but not by Ich-1, another member of the Ice/ced-3 family of genes. We found that the CrmA-expressing HeLa cells are resistant to TNF-alpha/cycloheximide (CHX)-induced apoptosis. Induction of apoptosis in HeLa cells by TNF-alpha/CHX is associated with secretion of mature IL-1 beta, suggesting that an IL-1 beta-processing enzyme, most likely ICE itself, is activated by TNF-alpha/CHX stimulation. These results suggest that one or more members of the ICE family sensitive to CrmA inhibition are activated and play a critical role in apoptosis induced by TNF.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression.

Anchorage-dependent cells that are prevented from attaching to an extracellular matrix substrate stop proliferating and may undergo apoptosis. Cell adhesion to a substrate is mediated by the integrin family of cell surface receptors, which are known to elicit intracellular signals upon cell adhesion. We show here that Chinese hamster ovary cells expressing the alpha 5 beta 1 integrin, which is a fibronectin receptor, do not undergo apoptosis upon serum withdrawal when the cells are plated on fibronectin. However, the alpha v beta 1 integrin, which is also a fibronectin receptor and binds fibronectin on the same RGD motif as alpha 5 beta 1, did not prevent apoptosis on fibronectin of the same cells. The cytoplasmic domain of the integrin alpha 5 subunit was required for the alpha 5 beta 1-mediated cell survival on fibronectin. The fibronectin-mediated survival effect appeared to be independent of the level of tyrosine phosphorylation of the focal adhesion kinase, which is induced by integrin-mediated cell attachment. The expression of the Bcl-2 protein, which counteracts apoptosis, was elevated in cells attaching to fibronectin through alpha 5 beta 1; cells attaching through alpha v beta 1 survived only if exogenous Bcl-2 was provided. Thus, alpha 5 beta 1, but not the closely related alpha v beta 1 integrin, appears to suppress apoptotic cell death through the Bcl-2 pathway.

Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe.

We describe a protein kinase, Shk1, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases. We provide genetic evidence for physical and functional interaction between Shk1 and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S. pombe. We further show that expression of the STE20 gene complements the shk1 null mutation and that Shk1 is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S. cerevisiae. Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shk1, Ste20, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.

Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

Nonneuronal expression of Rab3A: induction during adipogenesis and association with different intracellular membranes than Rab3D.

Rab3A is a small GTP-binding protein expressed predominantly in brain and neuroendocrine cells, in which it is associated with synaptic and synaptic-like vesicles, respectively. Here we report that adult mouse fat cells and 3T3-L1 adipocytes also express Rab3A mRNA and protein. They do not express synaptophysin, an abundant protein in synaptic vesicles or synaptic-like vesicles. The amount of Rab3A mRNA and protein, like that of the highly homologous isoform Rab3D, increases severalfold during differentiation of 3T3-L1 fibroblasts into mature adipocytes. In fat cells, most Rab3D and Rab3A protein is bound to membrane, irrespective of insulin addition. Rab3A and Rab3D are localized in different subcellular compartments, since about half of the Rab3A, but none of the Rab3D, is associated with a low-density organelle(s). Rab3D and Rab3A may be involved in different pathways of regulated exocytosis in adipocytes. Moreover, in adipocytes Rab3A may define an exocytic organelle that is different from synaptic vesicles or synaptic-like microvesicles found in neuronal and endocrine cells.