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R S Johnson

Publications and source records attributed to R S Johnson.

At least 19 recordsLinked to original sources
PubMed1995

Mass spectrometric analysis of 21 phosphorylation sites in the internal repeat of rat profilaggrin, precursor of an intermediate filament associated protein.

Profilaggrin, a highly phosphorylated protein synthesized in mammalian cornified epithelia, is the precursor of filaggrin, a protein that is involved in aggregation of keratin during terminal differentiation. Possible functions for the phosphorylation include preventing premature aggregation of keratin, packing profilaggrin into a storage granule, association of other proteins with the granule, and/or regulating proteolytic processing of profilaggrin. As a first step in characterizing the phosphorylation of rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC and analyzed by ionspray mass spectrometry. Nine putative phosphopeptides were identified as those with masses 80 Da (or multiples of 80 Da) greater than the predicted unphosphorylated masses. The six that were phosphorylated to a high stoichiometry were analyzed further. Several multiply phosphorylated peptides underwent neutral loss of H3PO4 during collisional activation, complicating interpretation of the MS/MS spectra. In order to circumvent this problem, an alternative strategy was applied in which peptide mixtures were treated with Ba(OH)2, resulting in beta-elimination of H3PO4 and generation of dehydrated serine or threonine at the site of phosphorylation. Peptides containing dehydrated serine or threonine fragmented well, providing unequivocal identification of multiple phosphorylation sites in peptides as long as 39 amino acids. The phosphopeptides (with phosphorylated residues underlined) were GQQHSGHPQVYYYGVEETEDESDAQQGHHQQQQQQR, GGQAGSHSESEASGGQAGR, HTSRPEQSPDTAGR, GESPAGQQSPDR, EASASQSSDSEGHSGAHAGIGQGQTSTTHR, and GSSESQASDSEGHSDYSEAHTQGAHGGIQTSSQR.

Amino Acid Sequence↗
PubMed1995

Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS.

MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously [Alessi, D. R., et al. (1994) EMBO J. 13, 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S298 and Y300. Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25. Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase. Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300. Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle. The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions.

3T3 Cells↗
PubMed1995

Is weight gain after smoking cessation inevitable?

Weight gain after smoking cessation was studied in a naturalistic setting where (1) all smokers quit and (b) risk factors for postcessation weight gain were modified. Participants were 332 military recruits (227 men, 105 women), 86 of whom were smokers who quit during 6 weeks of basic training. Results showed no significant weight changes for smokers who quit. Pretest smoking rates and feat of weight gain were unrelated to changes in weight. Results suggest that an intensive program that limits access to alcohol and foods that are high in fat and that increases physical activity can attenuate weight gain after smoking cessation.

Adolescent↗
PubMed1995

Over-expression of the DM-20 myelin proteolipid causes central nervous system demyelination in transgenic mice.

We have created transgenic mice bearing varying copy numbers of a transgene coding for normal DM-20, the alternatively spliced quantitatively minor isoform of myelin proteolipid protein. Demyelination of the CNS occurs as a consequence of 70 copies of this transgene. Overt symptoms begin at approximately 3 months with a wobbling gait. Occasional seizures lasting a few seconds begin at 3-4 months. These symptoms progress in severity with age. Death occurs by 8-10 months. Myelination in 2-month-old animals, before the onset of any overt symptoms, appears morphologically normal at the electron microscopic level. However, the myelin in these 2-month-old animals has a reduced amount of the major myelin proteolipid protein and about three times as much DM-20 as normal animals. In 7-month-old animals that appear to be undergoing demyelination in the CNS, both the major myelin proteolipid protein and DM-20 are greatly reduced relative to the 2-month-old animal. Mice with 17 copies of the transgene also have a reduced amount of the major myelin proteolipid protein but appear to be otherwise normal and have normal life spans (> 2 yr). Mice with low copy numbers of the transgene (2-4 copies) appear to be unaffected and have normal life spans.

Alternative Splicing↗
PubMed1994

Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene.

The principal substrate for the insulin and insulin-like growth factor-1 (IGF-1) receptors is the cytoplasmic protein insulin-receptor substrate-1 (IRS-1/pp185). After tyrosine phosphorylation at several sites, IRS-1 binds to and activates phosphatidylinositol-3'-OH kinase (PI(3)K) and several other proteins containing SH2 (Src-homology 2) domains. To elucidate the role of IRS-1 in insulin/IGF-1 action, we created IRS-1-deficient mice by targeted gene mutation. These mice had no IRS-1 and showed no evidence of IRS-1 phosphorylation or IRS-1-associated PI(3)K activity. They also had a 50 per cent reduction in intrauterine growth, impaired glucose tolerance, and a decrease in insulin/IGF-1-stimulated glucose uptake in vivo and in vitro. The residual insulin/IGF-1 action correlated with the appearance of a new tyrosine-phosphorylated protein (IRS-2) which binds to PI(3)K, but is slightly larger than and immunologically distinct from IRS-1. Our results provide evidence for IRS-1-dependent and IRS-1-independent pathways of insulin/IGF-1 signalling and for the existence of an alternative substrate of these receptor kinases.

Animals↗
PubMed1994

Identification and purification of a chicken brain neuroglia-associated protein.

The identification, purification, and biochemical characterization of specific markers for neuroglial cells in the central nervous system is an essential step toward a better understanding of the function of glial cells. This manuscript reports the identification and purification of a neuroglia-associated protein (NAP-185) with an apparent molecular mass of 185 kDa. While its expression is not restricted to the brain, it was first identified in a specific subpopulation of glial cells when chick brain stem sections were analyzed with an affinity-purified rabbit antiserum raised against the catalytic domain of the T-cell protein tyrosine phosphatase. This 185-kDa antigen was purified to apparent homogeneity and confirmed to be responsible for the neuroglial staining observed. In spite of its immunological relation to T-cell protein tyrosine phosphatase, purified NAP-185 failed to display tyrosine phosphatase activity. The primary sequence of five NAP-185-derived peptides shows that this protein has not yet been characterized and that it is possibly related to AP180, a clathrin-associated protein.

Amino Acid Sequence↗
PubMed1994

Heterogeneous N-acylation is a tissue- and species-specific posttranslational modification.

Heterogeneous N-terminal glycine acylation recently has been reported for two proteins involved in visual signal transduction. Similar N-acylations have typically involved only myristate; however, none of the previously examined proteins were isolated from retinas. To determine whether heterogeneous N-acylation is tissue-specific or protein sequence-specific, the N-terminal modifications of the catalytic subunit of cAMP-dependent protein kinase, partially purified from bovine retinas, heart, and brain tissues, were characterized. Using tandem mass spectrometry and liquid chromatography coupled directly to an electrospray mass spectrometer, we found only myristate at the N termini of catalytic subunits from brain and heart tissue, whereas the N termini of the retina-derived subunits were heterogeneously acylated in a manner similar to recoverin and transducin. Thus it appears that the nature of N-terminal glycine acylation is determined by the cell or tissue type in which it is located, and not by the sequence of the modified protein. We also examined the N-acylation of recoverin purified from human retinas, as well as transducin purified from frog retinas, to determine if heterogeneous acylation of retinal proteins is a uniquely bovine phenomenon. Interestingly, human recoverin was modified by the same family of fatty acids found on the bovine retinal proteins, while frog transducin was modified homogeneously not with myristate, but with a doubly unsaturated (C14:2) fatty acyl group.

Acylation↗
PubMed1994

Behavioral assessment of c-fos mutant mice.

Induction of the proto-oncogene c-fos has been associated with a number of neural and behavioral responses to acute stimuli. Behavioral characterization of mice containing a mutant c-fos allele created via homologous recombination-based gene targeting was performed to analyze the role of this protein in baseline neurological properties as well as paradigms that require neural adaptive responses. Performance of 9 out of 11 c-fos-deficient animals was impaired in the spatial version of the Morris water task. However, this poor performance in the spatial version of the task was highly correlated to their performance in the non-spatial version of the task which suggests that they have a behavioral impairment that interrupts their ability to perform adequately on both versions of the task with the same proficiency as wild-type and heterozygous litter mates. To examine learning impairments further, a simple left/right discrimination in a T-maze was used. Mutants were not impaired in this simple learning task. These results suggest that c-fos mutants have some behavioral impairments that interfere with evaluation of complex learning on the Morris water task, but because all genotypes could perform a simple discrimination task, it is clear that c-fos is not essential for this simpler form of learning and memory.

Animals↗
PubMed1994

Control of rhodopsin multiple phosphorylation.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor at multiple sites near the C-terminus by rhodopsin kinase and binding of a regulatory protein, arrestin. In the present study, the phosphorylation sites were examined in a partially reconstituted system under several experimental conditions. Initial phosphorylation sites were found to be 338Ser, 343Ser, and 334Ser based on analysis by mass spectrometry of proteolytic peptides from the C-terminus. The extent of phosphorylation was found to be limited by two mechanisms: (1) binding of arrestin to phosphorylated rhodopsin (one to three phosphate groups) appeared to prevent further phosphorylation (arrestin has also been observed to promote the initial phosphorylation of rhodopsin at 338Ser in rod outer segment homogenates); and (2) reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol prevented phosphorylation at more than three sites. We propose that previous observations of higher levels of rhodopsin phosphorylation may be the result of the removal of endogenous arrestin, or of exceeding the capacity of retinol dehydrogenase activity by intense bleaches (e.g., by exhausting endogenous NADPH).

Alcohol Oxidoreductases↗
PubMed1994

Characterization of a truncated form of arrestin isolated from bovine rod outer segments.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.

Amino Acid Sequence↗
PubMed1994

Mass spectrometric measurement of protein amide hydrogen exchange rates of apo- and holo-myoglobin.

Measurement of backbone amide hydrogen exchange rates can provide detailed information concerning protein structure, dynamics, and interactions. Although nuclear magnetic resonance is typically used to provide these data, its use is restricted to lower molecular weight proteins that are soluble at millimolar concentrations. Not subject to these limitations is a mass spectrometric approach for measuring deuterium incorporation into proteins that are subsequently proteolyzed by pepsin; the resulting peptide masses are measured using a flowing-fast atom bombardment ionization source (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). In the current study, amide deuterium incorporation for intact apo- and holo-myoglobin was measured using liquid chromatography coupled directly to an electrospray ionization (LC/MS) source. Electrospray ionization provided a more complete coverage of the protein sequence and permitted the measurement of deuterium incorporation into intact proteins. Tandem mass spectrometry was used to rapidly identify the peptic peptides. It was found that within 30 s, the amides in apo-myoglobin were 47% deuterated, whereas holo-myoglobin was 12% deuterated. Peptic digestion and LC/MS demonstrated that regions represented by peptic peptides encompassing positions 1-7, 12-29, and 110-134 were not significantly altered by removal of the heme. Likewise, destabilized regions were identified within positions 33-106 and 138-153.

Amides↗
PubMed1994

Topographic study of arrestin using differential chemical modifications and hydrogen/deuterium exchange.

Arrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P-Rho*). To study its conformational changes and regions interacting with P-Rho*, arrestin was subjected to (1) differential acetylation at lysine residues in the presence and absence of P-Rho*, and (2) amide hydrogen/deuterium exchange. Labeled protein was proteolysed and analyzed by mass spectrometry. Three Lys residues, 28, 176, and 211, were protected from acetylation in native arrestin, although they were not located in regions exhibiting slow amide hydrogen exchange rates. The presence of P-Rho* protected lysine 201 from acetylation and partially protected 14 other lysyl residues, including (2, 5), (163, 166, 167), (232, 235, 236, 238), (267, 276), (298, 300), and 367, where parentheses indicate lysine residues found within the same peptide. In contrast, in the C-terminal region of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P-Rho*. These data allowed us to identify functional regions in the arrestin molecule.

Acetylation↗
PubMed1994

Pupillometric discrimination of insomniacs.

Unlike the multiple sleep latency test, pupillometry does not require sleep to test sleepiness, which may be an advantage in assessing insomniacs. Pupillometry was employed to study possible differences in daytime sleepiness between insomniacs and noninsomniacs. Thirty self-defined insomniacs and an equal number of noninsomniacs, age 30-50 yr, submitted to four 10-min pupillometry sessions distributed from early morning to bedtime. The sessions consisted of infrared television monitoring of the pupil in a totally dark, nonstimulating environment. Insomniacs exhibited significantly smaller pupil diameters than noninsomniacs, and this effect persisted in all sessions. However, the size of effect averaged about 0.5 mm, and this was judged to be a small magnitude. Heightened subjective sleepiness discriminated the insomniacs in only the midday session. The absence of polysomnography was a methodologically limiting factor. Theoretical and clinical implications of these data were discussed.

Adult↗
PubMed1994

Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein.

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

Amino Acid Oxidoreductases↗
PubMed1994

SPARC is a source of copper-binding peptides that stimulate angiogenesis.

SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113-130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo.

Amino Acid Sequence↗
PubMed1994

Effect of a null mutation of the c-fos proto-oncogene on sexual behavior of male mice.

Sexual behavior was observed in male mice that were homozygous for a null mutation of the c-fos proto-oncogene, as well as in heterozygous mutants and wild-type controls. The onset of mounting was slower and the subsequent mounting rate was significantly lower in homozygous mutants than in either group of controls. Even so, a similar percentage of males of each genotype achieved ejaculation, and ejaculation latencies were equivalent in these mice. Likewise, in males that intromitted, the intromission efficiency and the number of intravaginal thrusts/intromission were similar among the three genotypes. The nuclear protein product (Fos) of c-fos was visualized immunocytochemically in the brains of heterozygous male mice 1 h after they exhibited a series of mounts, with or without intromission, leading to an ejaculation. As in the male of several other rodent species, nuclear Fos immunoreactivity was augmented in neurons of limbic and midbrain regions thought to convey olfactory/vomeronasal and genital/somatosensory information, respectively, to the medial preoptic area following contact with an estrous female. One interpretation of our behavioral results is that in the absence of normal neuronal c-fos expression, sensory stimuli that impinge on the male brain during mating lose their ability to initiate a cascade of further gene transcription events that otherwise control the rate at which a male reorients towards and mounts an estrous female during an ejaculatory series. Alternatively, the c-fos null mutation may disrupt normal neural development, leading to a structural change that mediates the observed deficit in mounting capacity.

Amygdala↗
PubMed1994

Normal peripheral T-cell function in c-Fos-deficient mice.

The ubiquitous transcription factors Fos and Jun are rapidly induced in T cells stimulated through the T-cell antigen receptor and regulate transcription of cytokines, including interleukin 2, in activated T cells. Since positive and negative selection of thymocytes during T-cell development also depends on activation through the T-cell receptor, Fos and Jun may play a role in thymocyte development as well. Fos and Jun act at several regulatory elements in the interleukin 2 promoter, including the AP-1 and NFAT sites. Using antisera specific to individual Fos and Jun family members, we show that c-Fos as well as other Fos family members are present in the inducible AP-1 and NFAT complexes of activated murine T cells. Nevertheless, c-Fos is not absolutely required for the development or function of peripheral T cells, as shown by using mice in which both copies of the c-fos gene were disrupted by targeted mutagenesis. c-Fos-deficient mice were comparable to wild-type mice in their patterns of thymocyte development and in the ability of their peripheral T cells to proliferate and produce several cytokines in response to T-cell receptor stimulation. Our results suggest that other Fos family members may be capable of substituting functionally for c-Fos during T-cell development and cytokine gene transcription in activated T cells.

Animals↗