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Biomedical subjects

D A Jans

Publications and source records attributed to D A Jans.

At least 73 records · Page 4Linked to original sources
PubMed1998

Nuclear targeting of the serine protease granzyme A (fragmentin-1).

Cytolytic granule-mediated target cell killing is effected in part through synergistic action of the membrane-acting protein perforin and serine proteases such as granzymes A (GrA) or B (GrB). In the present study we examine GrA cellular entry and nuclear uptake in intact mouse myeloid FDC-P1 cells exposed to perforin using confocal laser scanning microscopy, as well as reconstitute GrA nuclear uptake in vitro. GrA alone was found to be able to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of perforin, it specifically accumulated in the cell nuclei, with maximal levels about 2.5 times those in the cytoplasm after 2. 5 hours. In vitro, GrA accumulated in the nucleus and nucleolus maximally to levels that were four- and sixfold, respectively, those in the cytoplasm. In contrast, the active form of the apoptotic cysteine protease CPP32 did not accumulate in nuclei in vitro. Nuclear/nucleolar import of GrA in vitro was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTPgammaS, but was dependent on exogenously added cytosol. Importantly, GrA was found to be able to accumulate in the nucleus of semi-intact cells in the presence of the nuclear envelope-permeabilizing detergent CHAPS, implying that the mechanism of nuclear accumulation was through binding to insoluble factors in the nucleus. GrB was found for the first time to be similar in this regard. The results support the contention that GrA and GrB accumulate in the nucleus through a novel nuclear import pathway, and that this is integral to induction of the nuclear changes associated with cytolytic granule-mediated apoptosis.

Adenosine Triphosphate↗
PubMed1997

Kinetic characterization of the human retinoblastoma protein bipartite nuclear localization sequence (NLS) in vivo and in vitro. A comparison with the SV40 large T-antigen NLS.

The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target beta-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTPgammaS. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.

Adenosine Triphosphate↗
PubMed1997

SV40 large tumor antigen nuclear import is regulated by the double-stranded DNA-dependent protein kinase site (serine 120) flanking the nuclear localization sequence.

Nuclear localization sequence (NLS)-dependent nuclear import of SV40 large tumor antigen (T-Ag) fusion proteins is regulated by phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase Cdc2 amino-terminal to the NLS (amino acids 126-132). Between the T-Ag CKII and Cdc2 sites is a site (Ser120) for the double-stranded DNA-dependent protein kinase (dsDNA-PK), which we show here for the first time to play a role in regulating T-Ag nuclear import. We replaced Ser120 by aspartic acid or alanine using site-directed mutagenesis and assessed the effects on nuclear transport kinetics both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in HTC rat hepatoma cells. Maximal nuclear accumulation of the Asp120 and Ala120 protein derivatives was approximately 40% and 70% reduced in vivo, respectively, compared with that of the wild type protein, and similarly reduced in vitro, although to a lesser extent. This implies that the dsDNA-PK site regulates the maximal level of nuclear accumulation, normally functioning to enhance T-Ag nuclear transport; the higher accumulation of the Asp120 protein compared with the Ala120 protein indicates that negative charge at the dsDNA-PK site is mechanistically important in regulating nuclear import. The Asp120 protein accumulated in the nucleus at a faster rate than the wild type protein, implying that phosphorylation at Ser120 may also regulate the nuclear import rate. CKII phosphorylation of the Asp120 protein in cytosol or by purified CKII was approximately 30% higher than that of the Ser120 and Ala120 proteins, while negative charge at the CKII site increased dsDNA-PK phosphorylation of Ser120 by approximately 80% compared with wild type, implying physical and functional interactions between the two phosphorylation sites. Quantitation of NLS recognition by the importin 58/97 subunits using an enzyme-linked immunosorbent assay indicated that while the Ala120 protein derivative had a binding affinity very similar to that of wild type, the Asp120 derivative showed 40% higher affinity. In vitro CKII phosphorylation increased importin binding by about 30% in all cases. These results imply that negative charge at the dsDNA-PK site may enhance nuclear import through increasing both NLS recognition by importin subunits, and phosphorylation at the CKII site, which itself also facilitates NLS recognition by importin 58/97.

Amino Acid Sequence↗
PubMed1997

Nuclear targeting of chlorin e6 enhances its photosensitizing activity.

Although photosensitizers, molecules that produce active oxygen species upon activation by visible light, are being extensively used in photodynamic therapy to treat cancer and other clinical conditions, problems include normal cell and tissue damage and associated side effects, which are attributable in part to the fact that cytotoxic effects are largely restricted to the plasma membrane. We have previously shown that the photosensitizer chlorin e6 has significantly higher photosensitizing activity when present in conjugates containing specific ligands and thus able to be internalized by receptor-expressing cells. In this study we use insulin-containing conjugates to which variants of the simian virus SV40 large tumor antigen nuclear localization signal (NLS) were linked to target chlorin e6 to the nucleus, a hypersensitive site for active oxygen species-induced damage. NLSs were either included as peptides cross-linked to the carrier bovine serum albumin or encoded within the sequence of a beta-galactosidase fusion protein carrier. The results for photosensitization demonstrate clearly for the first time that NLSs increase the photosensitizing activity of chlorin e6, maximally reducing the EC50 by a factor of over 2000-fold. This has wide-reaching implications for achieving efficient cell type-specific photodynamic therapy.

Amino Acid Sequence↗
PubMed1997

The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin.

The mechanism by which phosphorylation regulates nuclear localization sequence (NLS)-dependent nuclear protein import is largely unclear. Whereas nuclear accumulation of SV40 large tumor antigen (T-ag) fusion proteins is completely dependent on the T-ag NLS (amino acids 126-132), the rate of nuclear import is increased 50-fold by amino acid residues 111-125 and in particular a site for the protein kinase CK2 (CK2) at serine 111/112. Because the first step of nuclear protein import involves the binding of the NLS by an NLS-receptor complex such as the importin 58/97 heterodimer, we established a novel enzyme-linked immunosorbent assay to test whether NLS recognition is influenced by amino acids amino-terminal to the NLS and the CK2 site. We found that recognition of the T-ag NLS by importin 58/97 was enhanced 10-fold in the presence of amino acid residues 111-125 and strongly dependent on importin 97. A T-ag fusion protein in which the spacer between the CK2 site and the NLS was decreased showed 30% reduced binding by importin 58/97. Maximal nuclear accumulation of this protein was reduced by more than 50%, indicating the physiological importance of the correctly positioned CK2 site. Phosphorylation by CK2 increased the T-ag NLS binding affinity for importin 58/97 by a further 40%. We conclude that flanking sequences and in particular phosphorylation at the CK2 site are mechanistically important in NLS recognition and represent the basis of their enhancement of T-ag nuclear import. This study thus represents the first elucidation of the mechanistic basis of the regulation of nuclear protein import through phosphorylation within a phosphorylation-regulated NLS.

Amino Acid Sequence↗
PubMed1997

The cytokine interleukin-5 (IL-5) effects cotransport of its receptor subunits to the nucleus in vitro.

Interleukin (IL)-5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have recently shown that human (h) IL-5 both possesses a functional nuclear localization signal capable of targeting a heterologous protein to the nucleus and localises to the nucleus of intact receptor-expressing cells. In this study, the extracellular domains of the hIL-5 alpha- and beta-receptor subunits were expressed in baculovirus, fluorescently labelled and assayed for nuclear targeting in vitro in the absence and presence of IL-5. The beta-subunit, which lacks IL-5 binding activity, only accumulated in the nucleus in the presence of both the hIL-5 binding alpha-subunit and hIL-5. The IL-5-binding alpha-subunit showed similar results. IL-5 thus effected nuclear transport of its alpha- and beta-receptor subunits apparently through a 'piggy back' mechanism, raising the possibility that IL-5's nuclear signalling role may be to cotarget its receptor subunits to the nucleus. This is the first demonstration of nuclear protein piggy back transport in vitro.

Animals↗
PubMed1997

A functional bipartite nuclear localisation signal in the cytokine interleukin-5.

Interleukin (IL)-5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have identified a bipartite nuclear localisation signal (NLS) within amino acids 95-111 of human IL-5 (hIL-5), also present in mouse IL-5 (mIL-5). hIL-5 and mIL-5 were labelled fluorescently, and nuclear uptake subsequent to membrane binding and internalisation by intact receptor expressing cells visualised and quantified using confocal laser scanning microscopy. hIL-5 and mIL-5 were shown to be transported to the nucleus in in vivo and in vitro nuclear protein import assays. The hIL-5 NLS was able to target a heterologous protein to the nucleus both in vivo and in vitro. Mutations within the proximal arm of the NLS abrogated nuclear targeting activity, confirming its bipartite nature. The results imply a nuclear signalling role for IL-5 additional to pathways linked to the membrane receptor system.

Amino Acid Sequence↗
PubMed1996

Nuclear transport of granzyme B (fragmentin-2). Dependence of perforin in vivo and cytosolic factors in vitro.

Cytotoxic T and natural killer cells are able to kill their target cells through synergistic action of the pore-forming protein perforin and the serine protease granzyme B, resulting in very distinctive nuclear changes typical of apoptosis. Whereas perforin acts at the membrane, granzyme B appears to be both capable of entering the cytoplasm of target cells and accumulating in isolated nuclei. In this study we examine nuclear transport of fluoresceinated granzyme B both in vivo in intact cells in the presence of perforin and in vitro in semi-permeabilized cells using confocal laser scanning microscopy. Granzyme B alone was observed to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of sublytic concentrations of perforin, however, it accumulated strongly in intact cell nuclei to levels maximally about 1.5 times those in the cytoplasm after about 2.5 h. In vitro nuclear transport assays showed maximal levels of nuclear and nucleolar accumulation of granzyme B of about 2.5- and 3-fold those in the cytoplasm. In contrast to signal-dependent nuclear accumulation of SV40 large tumor antigen (T-Ag) fusion proteins in vitro, nuclear/nucleolar import of granzyme B was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)). Similar to T-Ag fusion proteins, however, granzyme B nuclear and nucleolar accumulation was dependent on exogenously added cytosol. Specific inhibitors of granzyme B protease activity had no effect on nuclear/nucleolar accumulation, implying that proteolytic activity was not essential for nuclear targeting. The results imply that granzyme B (32 kDa) may be transported from the cytoplasm to the nucleus through passive diffusion and accumulate by binding to nuclear/nucleolar factors in a cytosolic factor-mediated process. Active and passive nuclear transport properties were normal in the presence of unlabeled granzyme B, implying that the nuclear envelope and pore complex are not granzyme B substrates.

Animals↗
PubMed1996

Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport.

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import.

Animals↗
PubMed1996

A consensus cAMP-dependent protein kinase (PK-A) site in place of the CcN motif casein kinase II site simian virus 40 large T-antigen confers PK-A-mediated regulation of nuclear import.

The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, together with the nuclear localization signal. Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin. To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts. Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy. In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest.

Amino Acid Sequence↗
PubMed1996

Localization of granzyme B in the nucleus. A putative role in the mechanism of cytotoxic lymphocyte-mediated apoptosis.

One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis.

Animals↗
PubMed1996

Regulation of protein transport to the nucleus: central role of phosphorylation.

Nuclear protein transport is integral to eukaryotic cell processes such as differentiation, transformation, and the control of gene expression. Although the targeting role of nuclear localization signals (NLSs) has been known for some time, more recent results indicate that NLS-dependent nuclear protein import is precisely regulated. Phosphorylation appears to be the main mechanism controlling the nuclear transport of a number of proteins, including transcription factors such as NFkappaB, c-rel, dorsal, and SWI5 from yeast. Cytoplasmic retention factors, intra- and intermolecular NLS masking, and NLS masking by phosphorylation are some of the mechanisms by which phosphorylation specifically regulates nuclear transport. Even nuclear localization of the archetypal NLS-containing simian virus 40 large tumor antigen (T-ag) is regulated, namely by the "CcN motif," which comprises the T-ag NLS ("N") determining ultimate subcellular destination, a casein kinase II site ("C") 13 amino acids NH2-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site ("c") adjacent to the NLS regulating the maximal level of nuclear accumulation. The CcN motif appears to be a special form of phosphorylation-regulated NLS (prNLS), where phosphorylation at site(s) close to the NLS specifically regulates NLS function. The regulation of nuclear transport through phosphorylation and prNLSs appears to be common in eukaryotic cells from yeast and plants to higher mammals.

Amino Acid Sequence↗
PubMed1996

In vivo chromatin structure of the murine interleukin-5 gene region: a new intact cell system.

The study of chromatin involvement in the regulation of gene expression has traditionally required the isolation of nuclei. However, cell fractionation techniques are subject to redistribution of proteins during the isolation procedure, which prevents rigorous physiologically relevant analysis. To eliminate the need to isolate nuclei and to analyze chromatin structures in vivo in response to agents regulating murine interleukin-5 (IL-5) gene activation, we have established a novel lysolecithin permeabilized intact cell system for suspension cell types, in this case T cells. Nuclear integrity of permeabilized cells is demonstrated by nuclear transport assays using confocal laser scanning microscopy. Results are identical in unstimulated and stimulated T cells, indicating that the chromatin structure after activation is not the result of gross alterations in nuclear protein transport properties. Potential new IL-5 gene regulatory regions are identified by DNase I hypersensitivity mapping. Our lysolecithin permeabilized intact cell system is amenable to physiologically relevant analysis of responses to signaling pathways at the level of chromatin, nuclear protein translocation and possibly other cellular functions in a variety of suspension and adherent cell types.

Animals↗
PubMed1995

Cyclin-dependent kinase site-regulated signal-dependent nuclear localization of the SW15 yeast transcription factor in mammalian cells.

Control over the nuclear transport of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation, transformation and signal transduction. The Saccharomyces cerevisiae TF SWI5 is excluded from the nucleus in a cell cycle-dependent fashion, mediated by phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear entry occurs in G1. beta-galactosidase fusion proteins carrying SWI5 amino acids 633-682, including the nuclear localization sequence (NLS: Lys-Lys-Tyr-Glu-Asn-Val-Val-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro- Arg-Lys655) were analyzed for subcellular localization in appropriate temperature-sensitive yeast strains blocked in G1 or G2/M using indirect immunofluorescence, and for nuclear import kinetics in living rat hepatoma or Vero African green monkey kidney cells microinjected with fluorescently labeled bacterially expressed protein and quantitative confocal laser microscopy. Cell cycle-dependent nuclear localization in yeast was both NLS and cdk site-dependent, whereby mutation of the cdk site serines (Ser646 and Ser664) to alanine resulted in constitutive nuclear localization. In mammalian cells, the SWI5 fusion proteins were similarly transported to the nucleus in an NLS-dependent fashion, while the mutation to Ala of the cdk site serines increased the maximal level of nuclear accumulation from about 1- to over 8-fold. We suggest that phosphorylation at the cdk sites inhibits nuclear transport of SWI5, consistent with our previous observations for the inhibition of SV40 large tumor antigen nuclear transport by phosphorylation by the cdk cdc2. The results indicate for the first time that a yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of regulatory signals for protein transport to the nucleus.

Amino Acid Sequence↗
PubMed1995

Insulin-mediated intracellular targeting enhances the photodynamic activity of chlorin e6.

Photodynamic therapy has been applied quite extensively over the last few years, whereby the activation of photosensitizers by light causes the production of reactive oxygen species such as singlet oxygen, which is cytotoxic. The goal of this study was the enhancement of the photodynamic activity of photosensitizers through their delivery to specific, sensitive intracellular compartments of target cells. We synthesized a BSA-insulin-chlorin e6 conjugate that bound specifically to the insulin receptors (EC50, 1 nM) of the human hepatoma cell line PLC/PRF/5 and could be internalized by receptor-mediated endocytosis. Photodynamic activity, as assessed by various tests, indicated EC50s at about 100 times lower concentrations of conjugate compared to free chlorin e6 itself; and lower doses of irradiation were necessary to activate the conjugate compared to free chlorin e6. Inhibition of endocytosis of the conjugate abrogated the enhanced photodynamic activity of the conjugate above that of free chlorin e6. Endocytosis and subsequent localization around and in the cell nucleus of the BSA-insulin-chlorin e6 conjugate could be visualized using both FITC-labeled conjugate and 2',7'-dichlorofluorescin diacetate, a fluorescent indicator of the production of active oxygen species due to chlorin e6 activation. It was concluded that photodynamic activity of the conjugate is higher than that of free chlorin e6 through its receptor-mediated delivery into sensitive intracellular compartments.

Carcinoma, Hepatocellular↗
PubMed1995

Interaction of the GTP-binding protein Gi2 with a protein kinase A-like kinase in mouse fibroblasts.

We have previously shown that the GTP-binding protein, Gi2 of mouse Balb/c3T3 cells is linked to a serine kinase which phosphorylates the alpha-subunit of Gi itself. In this report we show that Gi is coupled to a second protein kinase. This kinase does not phosphorylate G but phosphorylates another protein bound non-covalently to G. Phosphorylation of the Gi-linked protein induces its release from Gi. Kinase activity is slightly enhanced by GTPyS, suggesting that this kinase may be physiologically regulated by Gi. In an attempt to identify the kinase we have examined the effect of peptide substrates and inhibitors on kinase activity. We found that the protein kinase A inhibitory peptide, PK1 5-24, inhibited the kinase activity, but at concentrations above those usually required to block protein kinase A. The protein kinase A substrate peptide, kemptide, acted as a substrate of the kinase, and was an inhibitor of the phosphorylation of the Gi-linked protein. However, a protein kinase A, catalytic subunit antibody failed to react with any proteins linked to Gi., A protein kinase C inhibitory peptide had no effect on phosphorylation of the Gi-linked protein. Thus, the identity of this kinase has not been resolved, but it may form part of the signalling system of activated Gi in fibroblasts.

Animals↗
PubMed1994

Lateral mobility of the antagonist-occupied V2 vasopressin receptor in membranes of renal epithelial cells.

The lateral mobility of membrane integral receptors has been implicated as playing a significant role in signal transduction. The adenylate cyclase-coupled vasopressin V2 receptor has been shown to be highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature using a fluorescent vasopressin agonist, with lateral mobility of the V2 receptor proposed to play a role in both adenylate cyclase activation and ligand induced receptor internalization and down-regulation. This study reports the synthesis and characterization of two new fluorescent antagonists [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1,D-Tyr2,Ile4,Lys9(N6-fluoresceinylaminothiocarbonyl )]AVP (FL-AVP-anta) and [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1,D-Tyr2,Ile4,Lys9(N6-tetramethylrhodamylaminothioca rbonyl)]AVP (TR-AVP-anta) for the V2 receptor. The latter was used to determine the parameters of lateral mobility of the V2 receptor in the non-activated antagonist-occupied form. Using fluorescence photobleaching techniques, results were largely comparable to those for agonist-occupied receptor, indicating high mobility at 37 degrees C. Antagonistic properties of the V2 receptor ligands are apparently not related to decreased receptor lateral mobility. Photobleaching measurements, however, did show that in contrast to V2 agonist, V2 antagonist did not induce receptor immobilization due to aggregation with time at 37 degrees C, indicating that this could be of mechanistic importance in the internalization process.

Amino Acid Sequence↗