Human factor H-related protein 5 (FHR-5). A new complement-associated protein

A novel human plasma protein has been identified as a universal component of complement deposits, when complement is detected immunohistochemically in vivo. The protein is homologous to complement factor H and related proteins and has been designated factor H-related protein 5 (FHR-5). FHR-5 was identified by a monoclonal antibody raised using pathologic human glomerular preparations as the immunogen. FHR-5 was purified by affinity chromatography from complement-lysed erythrocytes, and the peptide sequence was obtained. The cDNA was cloned from a human liver library, and FHR-5 was deduced to be a protein containing 551 amino acids organized into nine short consensus repeat motifs. The short consensus repeats of FHR-5 show homology to Factor H and to other Factor H-related proteins, with some unique features demonstrated. Recombinant FHR-5, expressed in insect cells, was shown to bind C3b in vitro. The strong association of FHR-5 with tissue complement deposits in vivo suggests that this additional member of the Factor H family of proteins has a function in complement regulation.     

Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine

The reported cDNA structrre, of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olsonet al. Proc. Natl. Acad. Sci USA, 87: 2284–2288, 1990). The calculated Mr is 107, 534 whereas the estimate by SDS-PAGE is approximately 130, 000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors, in the cDNA sequence for non-muscle MLCK and that the NH₂-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH₂-terminally blocked. A CNBr peptide derived from smMLCK contains the NH₂-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating, that Met-1 is present. Using a limited thermolysin digest we isolated an NH₂-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH₂-terminal Met being blocked by actylation. The results demonstrate that the NH₂-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating, Met is not removed, but modified by -NH₂ acetylation of the translation product.     

Colon epithelial cell differentiation is inhibited by constitutive c-myb expression or mutant APC plus activated RAS

Blocked differentiation is a hallmark of cancer cells and the restoration of differentiation programs in vivo is an actively pursued clinical aim. Understanding the key regulators of cyto-differentiation may focus therapies on molecules that reactivate this process. c-myb expression declines rapidly when human colon cancer epithelial cells are induced to differentiate with the physiologically relevant short-chain fatty acid, sodium butyrate. These cells show increased expression of alkaline phosphatase and cytokeratin 8. Similarly, murine Immorto-epithelial cells derived from wild-type colon cells also show c-myb mRNA declines when induced to differentiate with sodium butyrate. Immorto-cells harboring a single APC mutation are indistinguishable from wild-type cells with regard to differentiation, while addition of activated RAS alone markedly enhances differentiation. In marked contrast, complete differentiation arrest occurs when both APC and RAS are mutated. Expression of MybER, a 4-hydroxytamoxifen-activatable form of c-Myb, blocks differentiation in wildtype and APC mutant Immorto-cell lines as well as LIM1215 human colon carcinoma cells. These data identify two pathways of oncogenic change that lead to retarded epithelial cell differentiation, one involving the presence of a single APC mutation in conjunction with activated RAS or alternatively constitutive c-myb expression.     

Effects of Opioid Peptides Containing the Sequence of Met5-Enkephalin or Leu5-Enkephalin on Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.     

AMP-activated protein kinase phosphorylation of endothelial NO synthase

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.     

The Akt kinase signals directly to endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.     

Analysis of androgen action on pituitary gonadotropin and prolactin secretion in ewes

To study the role of androgens in the control of gonadotropin and prolactin secretion in ther ewe, we have characterized androgen receptors in pituitary cytosol, and investigated the effect of androgens on pituitary hormone release in vivo and in vitro. High affinity, low capacity receptors, with an affinity for methyltrienolone (R1881) greater than 5 alpha-dihydrotestosterone (5 alpha-DHT) greater than testosterone (T) much greater than androstenedione (A4), estradiol-17 beta (E2) and progesterone (P), were identified in pituitary cytosol. Addition of 1 nM 5 alpha-DHT, but not A4, inhibited luteinizing hormone (LH) release from pituitary cells in vitro, induced by 10(10) to 10(-7) M luteinizing hormone releasing hormone (LHRH). The release of follicle-stimulating hormone (FSH) with 10(-9) M LHRH was inhibited when cells were incubated with 1 nM 5 alpha-DHT. 5 alpha-DHT had no effect when higher or lower doses of LHRH were used. In ovariectomized ewes, neither an i.v. injection of 1 mg, nor intracarotid injections of up to 1 mg, 5 alpha-DHT affected plasma LH, FSH or prolactin levels, despite dose-related increases in plasma 5 alpha-DHT levels. Daily or twice daily i.m. injections of 5 mg 5 alpha-DHT in oil did not affect LH or FSH levels, but daily injections of 20 mg significantly reduced plasma LH levels within 4 days and plasma FSH levels within 6 days. Thus, despite the presence of androgen receptors in the ewe pituitary, we conclude that androgens per se are of minimal importance in the regulation of pituitary LH, FSH and prolactin secretion in the ewe. The low binding affinity of A4 and the lack of its effect on hormone secretion in vitro suggests that A4 may act as an estrogen precursor rather than an androgenic hormone. The function of the pituitary androgen receptor remains to be established.     

Phosphorylation at the cyclin-dependent kinases site (Thr85) of parathyroid hormone-related protein negatively regulates its nuclear localization.

Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr85 immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr85 to Ala85 resulted in nuclear accumulation of PTHrP, while mutation to Glu85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10,34,35) for PTHrP.