Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.     

Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements

The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

Activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells by 2'-O-methylated poly (inosinic acid) . poly(cytidylic acid), Correlations with interferon-inducing activity

An oligonucleotide polymerase and a protein kinase which require double-stranded RNA (dsRNA) for activation are induced in HeLa cells by human fibroblast interferon. The polymerase synthesizes a series of oligonucleotides from ATP, whereas the kinase phosphorylates a polypeptide of Mr = 72,000 and the alpha subunit of initiation factor eIF-2. Partially or fully 2'-O-methylated derivatives of poly(inosinic acid) . poly(cytidylic acid) (rIn . rCn) were used to determine the structural requirements of dsRNA in the activation of these two enzymes. While fully methylated polymers failed to activate either enzyme, partially methylated polymers activated the enzymes in specific manners. The activation of the kinase by the rIn . rCn analogues was affected more severely by the level of methylation than was the activation of the polymerase. Moreover, fully methylated analogues blocked the activation of the kinase by rIn . rCn but not the activation of the polymerase. These observations are consistent with a biphasic model for enzyme activation similar to that proposed for interferon induction, which required the recognition of a relatively small region of rIn . rCn as the last step. Differences in the activation of the polymerase and kinase are explicable on the basis of the polymerase requirement for a smaller recognition region of the rIn . rCn duplex than the kinase. Dependence of polymerase activation on the level of methylation shows striking similarities with the interferon inducing activities of these analogues, suggesting a possible relationship between polymerase activation and interferon induction.     

Regulation of the immune response to tumor antigen. VIII. The effects of host specific anti-I-J antibodies on the immune response to tumors of different origin

The efficiency of in vivo therapy using alloantisera produced to interact specifically with I-J subregion encoded determinants has been investigated in two etiologically distinct syngeneic tumor systems, both of which have been shown to evoke suppressor T-cell host responses. Administration of 2 μl/day of anti-I-J^k alloantisera caused a significant reduction in the growth of the P815 methylcholanthrene (MCA)-induced mastocytoma or the 1316 ultraviolet (uv) radiation-induced fibrosarcoma in the syngeneic host. Inhibition of tumor growth with anti-I-J antibody treatment occurred in normal as well as in subcarcinogenically uv-treated hosts given the uv-induced 1316 fibrosarcoma, even though the normal host is capable of spontaneously rejecting the tumor graft in the absence of external manipulation. Evidence is also provided that the effects of anti-I-J antibody treatment are not due to direct interactions with the tumor cells, or to contaminating antiviral antibody activity within the antiserum. We have previously demonstrated the reduction of tumor growth in two antigenically distinct MCA-induced tumor systems (S1509a, SAI) using similar treatments. The data presented herein thus reinforce the possibility that such means of therapy may be beneficial to the treatment of a wide variety to tumor types where suppression represents a detrimental component of the host response, and may also provide some insight into the mechanisms underlying the effects of uv radiation on the immune response to tumor antigen.     

Inhibition of palatal epithelial cell death by altered protein synthesis.

Programmed cell death occurs in a defined region of the secondary palatal epithelium (medial edge) during its development in vivo or in vitro. The purpose of this study was to examine the effects of various inhibitors of macromolecular synthesis, particularly glycoprotein synthesis, on the death of palatal medial-edge epithelial cells in vitro. Rat palatal shelves explanted on gestational Day 15 and cultured singly showed medial-edge cell degeneration after 48 hr, whereas in the presence of 6-diazo-5-oxo-l-norleucine (DON), 2-deoxyglucose (DOG), or cycloheximide, cell death was prevented. This was in contrast to the lack of inhibition of cell death in the presence of actionomycin D or cytosine arabinoside. Selectively adding metabolites which bypass the effect of DON on various synthetic pathways demonstrated that the inhibition of cell death by DON was most likely due to a block in glucosamine formation. The synthesis and activity of various lysosomal enzymes were not affected by DON, whereas a marked decrease was observed with both cycloheximide and DOG. These results suggest that palatal epithelial cell death is not a passive event, but an active process requiring the programmed synthesis of specific proteins.     

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.