Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4beta2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated with ATP and either PKA or PKC. Both alpha4 and alpha4(336-597) were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the alpha4(336-597) fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and alpha4(336-597) were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amino acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP and the kinases. The phosphorylation of alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 microM and 160.8 +/- 26.8 microM, respectively, suggesting that Ser(368) was a better substrate for PKA than PKC.     

Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro.Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [³²P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and ³²P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 M of a synthetic peptide corresponding to the sequence around Ser¹⁰²⁹ of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser¹⁰²⁹ of the STaR/GC remains a candidate phosphorylation site by PKC.Abbreviations STa the heat-stable enterotoxin of E. coli, which has also been called ST-I and STp. The 18 amino acid variant was used throughout - PBS phosphate-buffered saline - PDB 4--12, 13-phorbol dibutyrate - ANP atrial natriuretic peptide - STaR/GC STa receptor/guanylyl cyclase, also called GC-C - PKC protein kinase C     

Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.     

Phosphorylation Sites Within α4 Subunits of α4β2 Neuronal Nicotinic Receptors: A Comparison of Substrate Specificities for cAMP-Dependent Protein Kinase (PKA) and Protein Kinase C (PKC)

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human 4 subunit of 42 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 M; Kemptide had a Km of 7.7 M. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 M and 2896 M, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 M; GS 1–8 had a Km of 2.1 M. VRCRSRSI had a comparative affinity for PKC with a Km of 327 M. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 M, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human 4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.     

Distribution of the extended family of protein kinase C isoenzymes in fetal organs of mice: an immunohistochemical study

Using isoenzyme-specific antisera, we have studied the distribution of protein kinase C isoforms in fetal mouse organs at the developmental age of 17 days. Two different sets of antibodies, produced by different manufacturers, were employed in this study. The specificity of the antisera was tested by immunoblotting experiments using whole fetal mouse extracts. Immunohistochemistry was carried out by means of an alkaline phosphatase-conjugated secondary antibody. Analysis of fetal mouse longitudinal cryostat sections stained with the antibodies demonstrated a distinct distribution of protein kinase C isoforms in the tissues. Protein kinase C- and C-I were present in all tissues examined, whereas the C-II isoform was absent in the lung and the liver. Protein kinase C- was identified in brain, spinal ganglia, and adrenal gland. The C- isoenzyme was abundantly expressed in spinal ganglia and in the smooth muscle cells of the bronchial wall. Antisera to C- and C- isoforms heavily stained liver, kidney, and spinal ganglia, whereas the C- isozyme was mainly detected in brain, stomach and kidney. Thus, protein kinase C-, C-I, C-II, C-, C- and C- were the isoforms present in many of the organs investigated. The two sets of antibodies gave slightly different results that might be ascribed to the different epitopes recognized by the antisera. One set of antisera was employed to investigate the distribution of the isoforms in selected organs from an earlier developmental age (15 days) and from adult animals. Both qualitative and quantitative differences were seen in comparison with the same organs from a 17-day fetus.     

A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

Qualitative and quantitative comparison of the distribution of phosphorylated and non-phosphorylated neurofilament epitopes within central and peripheral axons of adult hamster (Mesocricetus auratus)

Summary The distribution of phosphorylated and nonphosphorylated neurofilament epitopes was determined immunocytochemically in adjacent 2 m-thick sections of sciatic nerve, ventral root and spinal cord. Staining was scored as either intense, moderate or absent and the proportion of labeled axons was calculated for each category. Nearly all sciatic nerve and ventral root axons were immunoreactive with both antibodies against phosphorylated and non-phosphorylated neurofilaments and there were no significant differences in the number of intensely- or moderately-labeled axons. Within the spinal cord however, while the majority of large caliber axons was stained with both antibodies, there was a significant number of small caliber axons which stained only with antibodies against phosphorylated neurofilaments. These results show that phosphorylated and nonphosphorylated neurofilaments are extensively codistributed in CNS and PNS axons, and that in the CNS, staining intensity for non-phosphorylated epitopes is less in the smaller axons.     

Identification of protein kinase C and its potential substrate in Entamoeba histolytica

1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phospholipids and diacylglycerol, and specifically bound tritiated phorbol ester at an apparent KD of 9 nM. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phosphoprotein of mol. wt 68,000 was identified. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.     

A short peptide is a protein kinase C (PKC) alpha-specific substrate

The purpose of this study was to find protein kinase C (PKC) isozyme-specific peptides. A peptide library containing 1772 sequences was designed using Scansite and screened by MALDI-TOF MS and kinase activity assays for PKC isozyme-specificity. A peptide (Alphatomega; H-FKKQGSFAKKK-NH(2)) with high specificity for PKC alpha relative to other isozymes was identified. The peptide was phosphorylated to a greater extent by tissue lysates from B16 melanoma, HepG2, and human breast cancer, which had higher levels of activated PKC alpha, when compared to normal skin, liver, and human breast tissue lysates, respectively. Moreover, addition of Ro-31-7549, an inhibitor with great specificity for PKC alpha, to the phosphorylation reaction caused a dose-dependent reduction in phosphorylation, but no inhibition was identified with the addition of rottlerin and H-89. These results show that this peptide has great potential as a PKC alpha-specific substrate.     

Neuron-specific protein F1/GAP-43 shows substrate specificity for the beta subtype of protein kinase C

We determined whether the beta or gamma protein kinase C (PKC) subtypes implicated in long-term potentiation (LTP) selectively regulates protein F1 phosphorylation. Purified bovine PKC subtypes and recombinant PKC subtypes activated by phosphatidylserine (PS) and calcium were tested for their relative ability to phosphorylate purified rat protein F1 (a.k.a. GAP-43). After equalizing enzyme activity against histone, the recombinant beta II PKC phosphorylated protein F1 to a 6 fold greater extent than the recombinant gamma PKC. Bovine beta I PKC phosphorylated protein F1 to a 3 fold greater extent than bovine gamma PKC. Even when PS was replaced by lipoxin B4, which can selectively increase gamma PKC activity, beta I PKC was still superior to gamma PKC in phosphorylating protein F1. Taken together with previous cellular studies of brain showing parallel levels of expression of beta PKC mRNA and protein F1 mRNA, the present results make it attractive to propose that beta PKC regulates protein F1 phosphorylation during the development of synaptic plasticity.     

Action of amyloid beta-protein on protein kinase C activity

Amyloid beta-protein (A beta), the major protein of cerebrovascular and plaque amyloid in Alzheimer disease, is considered a primary factor in the pathology of this disease. The effect of synthetic A beta (1-40) on the activity of protein kinase C (PKC) was studied with histones for a substrate in a mixed micellar assay, and with calmodulin-depleted soluble brain proteins in a liposomal system. We report here that A beta affects PKC activity in a biphasic manner. An initial stimulation of PKC was noted at low concentrations of A beta (less than 2.5 microM); while PKC-inhibition was observed in a concentration-dependent manner at higher concentrations of A beta. The in vitro phosphorylation of 20, 47, and 87 kDa brain proteins (known PKC substrates) was significantly reduced by 60 microM A beta. The role of 20 kDa in memory storage, of 87 kDa in neurotransmission and neurosecretory processes, and of 47 kDa in long-term potentiation or memory is well recognized, and A beta is known to have both neurotrophic and neurotoxic effects. Since PKC plays an important role in neuronal function, it is suggested that dual modulation of PKC by A beta may be linked to its neurotrophic and neurotoxic effects. We propose that at low concentrations A beta, by stimulating PKC, may contribute to neurites generation; and at higher concentrations A beta, by inhibiting PKC activity, might lead first to memory impairment, and then to neuronal loss.     

Monocytes differentiation upon treatment with a peptide corresponding to the C-terminus of activated T cell-expressed Tirc7 protein

Atp6v0a3 gene encodes for two alternative products, Tirc7 and a3 proteins, which are differentially expressed in activated T cells and resorbing osteoclasts, respectively. Tirc7 plays a central role in T cell activation, while a3 protein is critical for osteoclast-mediated bone matrix resorption. Based on the large body of evidences documenting the relationships between T cells and osteoclasts, we hypothesized that the extracellular C-terminus of Tirc7 protein could directly interact with osteoclast precursor cells. To address this issue, we performed the molecular cloning of a mouse Atp6v0a3 cDNA segment encoding the last 40 amino acids of Tirc7 protein, and we used this peptide as a ligand added to mouse osteoclast precursor cells. We evidenced that Tirc7-Cter peptide induced the differentiation of RAW264.7 cells into osteoclast-like cells, stimulated an autocrine/paracrine regulatory loop potentially involved in osteoclastic differentiation control, and strongly up-regulated F4/80 protein expression within multinucleated osteoclast-like cells. Using a mouse bone marrow-derived CD11b(+) cell line, or total bone marrow primary cells, we observed that similarly to Rankl, Tirc7-Cter peptide induced the formation of TRACP-positive large multinucleated cells. At last, using mouse primary monocytes purified from total bone marrow, we determined that Tirc7-Cter peptide induced the appearance of small multinucleated cells (3-4 nuclei), devoid of resorbing activity, and which displayed modulations of dendritic cell marker genes expression. In conclusion, we report for the first time on biological effects mediated by a peptide corresponding to the C-terminus of Tirc7 protein, which interfere with monocytic differentiation pathways.     

A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC

A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a Km of 11 microM and Vmax of 24 micromol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the Km values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.     

Characterization of the specific CD4+ T cell response against the F protein during chronic hepatitis C virus infection

Background

The hepatitis C virus (HCV) Alternate Reading Frame Protein (ARFP or F protein) presents a double-frame shift product of the HCV core gene. We and others have previously reported that the specific antibodies against the F protein could be raised in the sera of HCV chronically infected patients. However, the specific CD4⁺ T cell responses against the F protein during HCV infection and the pathological implications remained unclear. In the current study, we screened the MHC class II-presenting epitopes of the F protein through HLA-transgenic mouse models and eventually validated the specific CD4⁺ T cell responses in HCV chronically infected patients.

Methodology

DNA vaccination in HLA-DR1 and-DP4 transgenic mouse models, proliferation assay to test the F protein specific T cell response, genotyping of Chronic HCV patients and testing the F-peptide stimulated T cell response in the peripheral blood mononuclear cell (PBMC) by in vitro expansion and interferon (IFN)- γ intracellular staining.

Principal Findings

At least three peptides within HCV F protein were identified as HLA-DR or HLA-DP4 presenting epitopes by the proliferation assays in mouse models. Further study with human PBMCs evidenced the specific CD4⁺ T cell responses against HCV F protein as well in patients chronically infected with HCV.

Conclusion

The current study provided the evidence for the first time that HCV F protein could elicit specific CD4⁺ T cell response, which may provide an insight into the immunopathogenesis during HCV chronic infection.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits a HeLa protein kinase that phosphorylates an RNA polymerase II-derived peptide

A protein kinase that phosphorylates Lys(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4, a synthetic peptide homologous to the evolutionarily-conserved, tandemly-repeated heptapeptide sequence at the C-terminus of the large subunit of eukaryotic RNA polymerase II, has been detected in HeLa cell extracts and chromatographic fractions therefrom. The enzyme, which phosphorylates serine principally, can be distinguished from previously described major protein kinases which phosphorylate the peptide poorly, if at all. It is inhibited by the nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Results suggest that human placental RNA polymerase II is phosphorylated at the C-terminus of the large subunit by the partially-purified protein kinase and that the phosphorylation is also sensitive to the nucleoside analog.     

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189–193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120–150) with the E.R.-associated domain (a.a.1–50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP. Electronic Supplementary MaterialThe online version of this article (doi: ) contains supplementary material, which is available to authorized users.