scholarly journals Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

1995 ◽  
Vol 15 (2) ◽  
pp. 661-670 ◽  
Author(s):  
R Bosser ◽  
M Faura ◽  
J Serratosa ◽  
J Renau-Piqueras ◽  
M Pruschy ◽  
...  
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Specific Protein ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Dependent Manner ◽  
Cell Nuclei ◽  
Ribonucleoprotein Particles ◽  
C Proteins

It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei.

Skeletal muscle phenotype signaling with ex vivo endurance-type dynamic contractions in rat muscle

2021 ◽  
Author(s):  
Jesper Emil Jakobsgaard ◽  
Jacob Andresen ◽  
Frank V. de Paoli ◽  
Kristian Vissing
Keyword(s):  
Skeletal Muscle ◽  
Translation Initiation ◽  
Ex Vivo ◽  
Contractile Activity ◽  
Specific Protein ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Metabolic Signaling ◽  
Dynamic Endurance

Skeletal muscle phenotype may influence the response sensitivity of myocellular regulatory mechanisms to contractile activity. To examine this, we employed an ex vivo endurance-type dynamic contraction model to evaluate skeletal muscle phenotype-specific protein signaling responses in rat skeletal muscle. Preparations of slow-twitch soleus and fast-twitch extensor digitorum longus skeletal muscle from 4-wk old female Wistar rats were exposed to an identical ex vivo dynamic endurance-type contraction paradigm consisting of 40 minutes of stretch-shortening contractions under simultaneous low-frequency electrostimulation delivered in an intermittent pattern. Phosphorylation of proteins involved in metabolic signaling and signaling for translation initiation was evaluated at 0, 1, and 4 hours after stimulation by immunoblotting. For both muscle phenotypes, signaling related to metabolic events was upregulated immediately after stimulation, with concomitant absence of signaling for translation-initiation. Signaling for translation-initiation was then activated in both muscle phenotypes at 1-4 hours after stimulation, coinciding with attenuated metabolic signaling. The recognizable pattern of signaling responses support how our ex vivo dynamic muscle contraction model can be utilized to infer a stretch-shortening contraction pattern resembling stretch-shortening contraction of in vivo endurance exercise. Moreover, using this model, we observed that some specific signaling proteins adhering to metabolic events or to translation initation exhibited phosphorylation changes in a phenotype-dependent manner, whereas other signaling proteins exhibited phenotype-independent changes. These findings may aid the interpretation of myocellular signaling outcomes adhering to mixed muscle samples collected during human experimental trials.

scholarly journals Determination of the amount of small messenger ribonucleic acid-containing particles free in liver cytoplasm

1975 ◽  
Vol 152 (1) ◽  
pp. 51-56 ◽  
Author(s):  
B M Mullock ◽  
R H Hinton
Keyword(s):  
Rat Liver ◽  
Ribonucleic Acid ◽  
Messenger Ribonucleic Acid ◽  
Ribonucleoprotein Particles

To assess the contribution made by mRNA-containing particles to the heterogeneity previously observed among rat liver 40S ribonucleoprotein particles, the amount of poly(A)-containing RNA in subribosomal particles was determined. RNA was labelled with orotate in vivo for 24h and then for 50min. Poly(A)-containing RNA was trapped on filters impregnated with poly(U). Very little poly(A)-containing RNA was found in conventionally prepared ribonucleoprotein particles after fractionation in sucrose. However, after preparation of ribonucleoprotein particles by sedimentation through 1 M-sucrose in the presence of 0.15M-KCl or by precipitation with Mg2+ as described by Leitin & Lerman [(1969) Biokhimiya 34, 839-849], amounts of poly(A)-containing RNA were similar to amounts of mRNA found by other workers in total ribonucleoprotein particles. Even in such preparations, less than 5% of the total rapidly labelled RNA in native subribosomal-particle fractions was mRNA. It seems that mRNA-containing particles make up only a very small part of the population of subribosomal particles in liver.

scholarly journals Purification of two distinct types of phosphoinositide-specific phospholipase C from rat liver. Enzymological and structural studies

1988 ◽  
Vol 256 (2) ◽  
pp. 453-459 ◽  
Author(s):  
O Nakanishi ◽  
Y Homma ◽  
H Kawasaki ◽  
Y Emori ◽  
K Suzuki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phospholipase C ◽  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Homogenate ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].

scholarly journals Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

2007 ◽  
Vol 28 (4) ◽  
pp. 1313-1325 ◽  
Author(s):  
Meredith E. K. Calvert ◽  
Kristin M. Keck ◽  
Celeste Ptak ◽  
Jeffrey Shabanowitz ◽  
Donald F. Hunt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
S Phase ◽  
Bud Formation ◽  
Evolutionarily Conserved ◽  
Assembly Factor ◽  
And Function ◽  
Cycle Regulation

ABSTRACT In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.

Inhibition of Casein Kinase 2 Impairs Wnt Signaling and Cell Survival in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2050-2050
Author(s):  
Christina Wu ◽  
Fitzgerald S Lao ◽  
Emily Nan ◽  
Hongying Li ◽  
Michael Y. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wnt Signaling ◽  
Conflicts Of Interest ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Lymphocytic Leukemia ◽  
Similar Reduction ◽  
Gene Assay

Abstract The oncogenic Wnt pathway is aberrantly activated in most CLL clones, and hence is an attractive target for therapy. The casein kinase 2 (CK2) enzyme is an established positive regulator of Wnt signaling. The inhibitor Silmitasertib, also known as CX-4945, is a nanomolar inhibitor of CK2. It has been reported that CK2 is overexpressed in CLL. Here we have investigated the effects of CX-4945 on WNT signaling in primary CLL cells. We confirmed that CX-4945 displayed in vitro cytotoxic activity toward CLL cells at very low µM concentration, as previously reported by others. However, at least 2-3 fold higher concentration of CX-4945 was required to achieve a similar toxicity against normal PBMC. Previously, our laboratory has successfully utilized a short-term CLL "parking" model in immunodeficient RAG/gamma chain knock out (RG-KO) mice to evaluate the in vivo efficacy and potential toxicity of anti-CLL agents. CX-4945 at dosages of 0.3-10 mg/kg was administered by oral gavage daily for 6 days to mice injected i.p. with 10 million CLL cells. These dosages of drug were well tolerated, and potently inhibited CLL persistence in the xenotransplanted mice. In a reporter gene assay, CX-4945 dose-dependently inhibited Wnt target gene expression. Furthermore, inhibition of dishevelled-2 (Dvl-2) protein expression was observed in primary CLL patient samples treated with 3-10 µM CX-4945 for 4-16 hours. Similar reduction in p-GSK3b(S9) protein was also observed. Quantitative RT-PCR also confirmed down regulation of b-catenin gene expression in primary CLL patient samples treated with 10 µM CX-4945 for 4h. Further molecular analyses of predictive or correlative biomarkers is ongoing using Nanostring PanCancer multipathway gene analysis. In a preliminary study, we found that CX-4945 perturbed the expression of multiple genes implicated in CLL development and survival. In summary, the CK2 inhibitor CX-4945 inhibited Wnt signaling and CLL survival, and displayed oral activity in mice. CK2 inhibitors are thus potential therapeutic agents for CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of Human Herpesvirus 8 ORF59 Protein (PF-8) and Mapping of the Processivity and Viral DNA Polymerase-Interacting Domains

2000 ◽  
Vol 74 (23) ◽  
pp. 10920-10929 ◽  
Author(s):  
Szeman Ruby Chan ◽  
Bala Chandran
Keyword(s):  
Dna Polymerase ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Dependent Manner ◽  
Cell Nuclei ◽  
Viral Dna ◽  
Herpesvirus 8 ◽  
Barr Virus

ABSTRACT Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118–126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.

scholarly journals Phosphorylation of Rat Liver Mitochondrial Glycerol-3-phosphate Acyltransferase by Casein Kinase 2

2005 ◽  
Vol 280 (20) ◽  
pp. 19527-19534 ◽  
Author(s):  
Thomas M. Onorato ◽  
Sanjoy Chakraborty ◽  
Dipak Haldar
Keyword(s):  
Rat Liver ◽  
Casein Kinase ◽  
Casein Kinase 2
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