Biomedical subjects

P D Adams

Publications and source records attributed to P D Adams.

12 recordsLinked to original sources

PubMed1997

Cloning and characterization of phorbol ester differentiation-resistant U937 cell variants.

Differentiation-resistant U937 cells were derived from parental U937 human promonocytic leukemia cells by selecting for a nonadherent phenotype in cell cultures continuously exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). Subsequent analysis indicated no differences between wildtype (wt) and resistant U937 cells with respect to protein kinase C (PKC) isozyme expression, activation, or down-modulation. The subcellular localization of PKCs is identical in wt and resistant cells with the exception of PKC beta 2, which no longer colocalizes with microtubules in the TPA-resistant cell lines. In contrast to wt-U937 cells, the resistant cells do not express beta 2-integrin adhesion molecules, cd11b and cd11c, on the cell surface following TPA treatment but do express cd11b and cd11c in intracellular vesicles. TPA stimulates the translocation of these vesicles to the cell surface in wt U937 cells but not in the resistant cells. These results suggest that events downstream of PKC activation may mediate cytoskeletal reorganization and beta 2-integrin transport to the cell surface in wt-U937 cells but not in the differentiation-resistant cells.

CD18 Antigens↗

PubMed1996

Improved prediction for the structure of the dimeric transmembrane domain of glycophorin A obtained through global searching.

A more global search method, using fewer assumptions, has been used to predict the structure of the dimeric transmembrane region of the protein glycophorin A. The resulting model significantly differs from that previously determined. In particular, the arrangement between the two transmembrane helices is now more symmetric resulting in improved interaction energies and an increased buried surface area. An increase in the van der Waals interaction energy due to tighter packing compensates for the loss of the interhelical hydrogen bond observed between Thr-87 of each helix in the previous model.

Computer Simulation↗

PubMed1996

The cellular effects of E2F overexpression.

The product of the retinoblastoma tumor-suppressor gene (RB) is a ubiquitously expressed, 105-kDa nuclear phosphoprotein (pRB). The pRB protein negatively regulates the cellular G1/S phase transition, and it is at this point in the cell cycle that it is thought to play its role as a tumor suppressor. The growth-inhibitory effects of pRB are exerted, at least in part, through the E2F family of transcription factors. This chapter reviews the insights into the mechanism of action of the E2F family members that have been obtained through overexpression studies. Studies in RB-/- SAOS-2 cells have provided evidence in support of the hypothesis that the E2F family members are negatively regulated by pRB and the related protein p130. In particular, the results obtained are consistent with the earlier biochemical data which suggested that E2F1 is regulated primarily by pRB, and E2F4 by p130. Results relating to p107 are also discussed. Consistent with the proposed role of pRB and E2F1 as coregulators of entry into S phase, experiments have demonstrated that overexpression of E2F1 is sufficient to override the cell cycle arrests caused by serum deprivation of fibroblasts or transforming growth factor-beta (TGF-beta) treatment of mink lung epithelial cells. However, at least in the case of the serum deprivation induced arrest, the ultimate result of E2F1 overexpression is death by p53-dependent apoptosis. In light of this and other data, a model is discussed as to how functional inactivation of pRB and p53 might cooperate to promote tumorigenesis. A number of studies have demonstrated the oncogenic potential of E2F family members, at least under certain conditions. This is, again, in keeping with the notion that these proteins play a critical role in controlling proliferation.

Animals↗

PubMed1996

Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors.

Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.

Amino Acid Sequence↗

PubMed1995

Transcriptional control by E2F.

Considerable evidence suggests that the E2F/DRTF1 family of transcription factors (hereafter referred to as 'E2F') plays a critical role in cell growth control. For example, the ability of several small DNA tumour viruses, such as SV40, adenovirus and human papillomavirus, to transform certain cells is tightly linked to their ability to deregulate E2F. Furthermore, E2F appears to directly regulate the transcription of a diverse set of genes implicated in DNA replication and cell growth control. Finally, a number of known cell cycle regulators, some of which are commonly mutated in human tumours, appear to exert their effects, at least in part, by altering E2F activity. Among these are pRb, p53, cdk-2, cdk-4 and certain cyclins.

Animals↗

PubMed1995

Computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban.

Structural and environmental constraints greatly simplify the folding problem for membrane proteins. Computational methods can be used in a global search to find a small number of chemically reasonable models within these constraints, such that a modest set of experimental data can distinguish among them. We show that, for phospholamban, the global search can be further simplified by reducing the problem to two-body, rather than many-body, interactions. This method of a constrained global search combined with experimental mutagenesis data yields a three-dimensional structure for this pentameric ion channel. The model is a left-handed symmetric homopentamer of alpha-helices with a well-defined channel, lined solely by hydrophobic residues.

Amino Acid Sequence↗

PubMed1995

Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution.

Improved refinement of the crystal structure of GroEL from Escherichia coli has resulted in a complete atomic model for the first 524 residues. A new torsion-angle dynamics method and non-crystallographic symmetry restraints were used in the refinement. The model indicates that conformational variability exists due to rigid-body movements between the apical and intermediate domains of GroEL, resulting in deviations from strict seven-fold symmetry. The regions of the protein involved in polypeptide and GroES binding show unusually high B factors; these values may indicate mobility or discrete disorder. The variability of these regions may play a role in the ability of GroEL to bind a wide variety of substrates.

Bacterial Proteins↗

PubMed1994

Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis.

E2F-1 is a transcription factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53. In contrast, coexpression of a naturally occurring loss-of-function RB mutant or wild-type p53 did not suppress the induction of apoptosis under these conditions. Thus, deregulated E2F-1 activity gives rise to proliferative and apoptotic signals. p53 appears to participate in the execution of the latter.

Animals↗

PubMed1994

Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel.

Phospholamban is a 52 amino acid calcium regulatory protein found as pentamers in cardiac SR membranes. The pentamers form through interactions between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed interactions between the transmembrane segments, using a chimeric protein construct in which staphylococcal nuclease (a monomeric soluble protein) is fused to the N-terminus of phospholamban. The chimera forms pentamers observable in SDS-PAGE, allowing the effects of mutations upon the oligomeric association to be determined by electrophoresis. The disruptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Residues of the same class lined up on faces of a 3.5 amino acids/turn helical projection, allowing the construction of a model of the interacting surfaces in which the helices are associated in a left-handed pentameric coiled-coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily form a left-handed coiled-coil helical bundle and have yielded molecular models for the interacting surfaces, the best of which is identical to that predicted by the mutagenesis. Residues lining the pore show considerable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channels. The cylindrical ion pore (minimal diameter of 2 A) appears to be defined largely by hydrophobic residues (I40, L43 and I47) with only two mildly polar elements contributed by sulfurs in residues C36 and M50.

Amino Acid Sequence↗

PubMed1994

A dimerization motif for transmembrane alpha-helices.

Specific helix-helix interactions inside lipid bilayers guide the folding and assembly of many integral membrane proteins and their complexes. We report here a pattern of 7 amino acids (LIxxGVxxGVxxT) which when introduced into several hydrophobic transmembrane alpha-helices promotes their specific dimerization. Dimerization is driven by interactions that are specific, dominated by the helix-helix interface, and involve no potentially ionizable groups. The motif may provide a useful tool for the functional analysis of such interactions in a variety of systems. Further, since this particular motif is rare, whilst specific helix association is not, many other such motifs may exist, which could permit sorting within complex membranes as well as guiding folding and oligomerization.

Amino Acid Sequence↗

PubMed1992

Activation of mitogen-activated protein (MAP) kinase by a MAP kinase-kinase.

Previously it has been shown that acute 12-O-tetradecanoylphorbol-13-acetate treatment of intact U937 cells results in activation of mitogen-activated protein (MAP) kinase and a MAP kinase activator. MAP kinase activator induces phosphorylation of MAP kinase on tyrosine and threonine residues, thereby activating MAP kinase. Here, experiments with the irreversible kinase inhibitor, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), show that MAP kinase activator is in fact a MAP kinase-kinase. Treatment of MAP kinase activator with FSBA results in complete inactivation. This inactivation is prevented by a 10-fold excess of ATP. Inactivation of MAP kinase by FSBA does not affect the extent of threonine/tyrosine phosphorylation induced by MAP kinase-kinase.

Adenosine↗

PubMed1991

TPA-induced activation of MAP kinase.

Threonine and tyrosine residue phosphorylation of a 42 kDa protein identified as mitogen-activated protein kinase (MAP kinase) was stimulated in extracts from TPA-pretreated cells. It is further shown that TPA pretreatment leads to the enhancement of an activity that will induce reactivation of dephosphorylated/inactivated MAP kinase. This TPA-induced activity induces the threonine and tyrosine phosphorylation of p42 in extracts from unstimulated cells.

Amino Acid Sequence↗