共查询到20条相似文献,搜索用时 15 毫秒
1.
Abstract: In this report, the phosphorylation sites of neurofilament protein of medium molecular mass (NF-M) by protein kinase FA /glycogen synthase kinase 3α (kinase FA /GSK-3α) were determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, HPLC, Edman degradation, and peptide sequencing. Kinase FA /GSK-3α phosphorylates NF-M predominantly on serine, residue. Three major tryptic phosphopeptide peaks were resolved by C18 reverse-phase HPLC. Edman degradation and peptide sequence analysis revealed that AKS(p)PVSK is the phosphorylation site sequence for the first major peak. When mapping with the amino acid sequence of neurofilament, we finally demonstrate Ser603 -Pro, one of the in vivo sites in NF-M, as the major site phosphorylated by kinase FA /GSK-3α. By using the same approach, we also identified the in vivo sites of Ser502 -Pro, Ser506 -Pro, and Ser666 -Pro as the other three major sites in NF-M phosphorylated by kinase FA /GSK-3α. Taken together, the results provide initial evidence that kinase FA /GSK-3α may represent a physiologically relevant protein kinase involved in the in vivo phosphorylation of NF-M. Because Ser502 , Ser506 , Ser603 , and Ser666 are all flanked by a carboxyl-terminal proline residue, the results provide further evidence that FA /GSK-3α may represent a proline-directed protein kinase involved in the structure-function regulation of the neuronal cytoskeletal system. 相似文献
2.
Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca2+ dependence to M-phase onset. A potential target of this Ca2+ dependence may be CaM kinase-II (the multifunctional [type II] Ca2+ /calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34cdc2 . We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34cdc2 , but again only after fertilization. These changes in CaMK-II activity and p34cdc2 -association after fertilization may ensure that Ca2+ signals are targeted to the M-phase machinery at the appropriate developmental times. 相似文献
3.
Phospholipid-Sensitive Ca2+ -Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein 总被引:1,自引:0,他引:1
R. Scott Turner C.-H. Jen Chou† Gonzalo J. Mazzei Philip Dembure ‡ J. F. Kuo 《Journal of neurochemistry》1984,43(5):1257-1264
Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP. 相似文献
4.
Benoit I. Giasson James A. Cromlish Eric S. Athlan Walter E. Mushynski 《Journal of neurochemistry》1996,66(3):1207-1213
Abstract: The activation of cyclic AMP-dependent protein kinase (PKA) in rat dorsal root ganglion (DRG) cultures increased phosphorylation of the low-molecular-mass neurofilament subunit (NFL) at a site previously identified as Ser55 but had no effect on neurofilament integrity. When PKA was activated in DRG cultures treated with 20–250 n M okadaic acid, neurofilament fragmentation was enhanced, and there was a corresponding increase in phosphorylation of NFL at a novel site. This site was also phosphorylated by PKA in vitro and was determined to be Ser2 by mass spectrometric analysis of the purified chymotryptic phosphopeptide. The PKA sites in NFL were dephosphorylated by the purified catalytic subunit of protein phosphatase-2A but not that of protein phosphatase-1, and phosphoserine-2 was a better substrate than phosphoserine-55. The phosphorylation and dephosphorylation of Ser2 and Ser55 in NFL may therefore be involved in the modulation of neurofilament dynamics through the antagonistic effects of PKA and protein phosphatase-2A. 相似文献
5.
6.
Shinya Matsuda Kyohei Kominato Shizuyo Koide-Yoshida Kenji Miyamoto Kinuka Isshiki Akihiko Tsuji Keizo Yuasa 《The Journal of biological chemistry》2014,289(26):18387-18400
PCTAIRE kinase 3 (PCTK3)/cyclin-dependent kinase 18 (CDK18) is an uncharacterized member of the CDK family because its activator(s) remains unidentified. Here we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP-dependent protein kinase (PKA). Using a pulldown experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1, although its activity was lower than that of CDK2. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser12, Ser66, and Ser109 and that PCTK3 activity significantly increased via phosphorylation at Ser12 by PKA even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced polymerized actin accumulation in peripheral areas and cofilin phosphorylation. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA and a physiological function of PCTK3. 相似文献
7.
8.
Tina K. Machu Jordan A. Firestone Michael D. Browning† 《Journal of neurochemistry》1993,61(1):375-377
Abstract: The γ2 subunit of the GABAA receptor (GABAA -R) is alternatively spliced. The long variant (γ2L ) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+ /calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ2L subunit has been shown to be necessary for ethanol potentiation of the GABAA -R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function. 相似文献
9.
Abstract: The Na+ /Ca2+ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na+ /Ca2+ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na+ /Ca2+ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na+ /Ca2+ exchange activity and prolonged cytosolic Ca2+ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na+ /Ca2+ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na+ /Ca2+ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na+ /Ca2+ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na+ /Ca2+ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion. 相似文献
10.
Staurosporine Activates a 60,000 Mr Protein Kinase in Bovine Chromaffin Cells That Phosphorylates Myelin Basic Protein In Vitro 总被引:2,自引:2,他引:0
Biljana Pavlovi-urjanev Anne L. Cahill Robert L. Perlman 《Journal of neurochemistry》1993,61(2):697-703
Abstract: Bovine chromaffin cells contain a family of renaturable protein kinases. One of these, a 60,000 Mr kinase (PK60) that phosphorylated myelin basic protein in vitro, was activated fourfold when cells were treated with the protein kinase inhibitor Staurosporine. Because staurosporine inhibits protein kinase C, the role of this kinase in the regulation of PK60 activity was investigated. Fifty nanomolar Staurosporine produced half-maximal inhibition of protein kinase C activity in chromaffin cells, whereas ∼225 n M Staurosporine was required to induce half-maximal activation of PK60. Other protein kinase C inhibitors, H-7 and K-252a, did not mimic the effect of Staurosporine on PK60 activity. Chromaffin cells have three protein kinase C isoforms: α, ε, and ζ. Prolonged treatment with phorbol esters depleted the cells of protein kinase C α and ε, but not ζ. Neither activation nor depletion of protein kinase C affected the basal activity of PK60. Moreover, Staurosporine activated PK60 in cells depleted of protein kinase C α and e; thus, Staurosporine appeared to activate PK60 by a mechanism that does not require these protein kinase C isoforms. Incubation of cell extracts with Staurosporine in vitro did not activate PK60. Incubation of these extracts with adenosine 5'-O-(3-thiotriphosphate), however, caused a twofold activation of PK60. Although this suggests that PK60 activity is regulated by phosphorylation, the mechanism by which Staurosporine activates PK60 is not known. Staurosporine has been reported to promote neurite outgrowth from chromaffin cells. The role of PK60 in mediating the effects of Staurosporine on chromaffin cell function remains to be determined. 相似文献
11.
Akihiko Yamaguchi Masakane Yamashita Michiyasu Yoshikuni Yasuo Hotta Paul Nurse Yoshitaka Nagahama 《Development, growth & differentiation》1991,33(6):625-632
We have taken advantage of the synchrony of meiotic prophase I in Lilium microsporocytes to investigate the presence and involvement in four stages of meiotic prophase I (leptotene, zygotene, pachytene, and diplotene) of the p34cdc2 H1 histone kinase, a component of MPF and a key participant in division control in other eukaryotes. H1 kinase activity showed a peak pattern during meiotic prophase I with the highest kinase activity at pachytene. A monoclonal antibody directed against a highly conserved region of p34cdc2 (termed the 'PSTAIR') recognized three major protein forms by immunoblotting. The highest level of the fastest-migrating form was observed at pachytene, coinciding with the highest activity of H1 kinase. Both the proteins recognized by the anti-PSTAIR antibody and H1 histone kinase activity were retained on beads conjugated with p13suc1 , a protein known to physically associate with p34cdc2 . These observations suggest that p34cdc2 or protein(s) highly homologous to p34cdc2 is a component of Lilium H1 histone kinase and plays a role in regulating meiotic prophase I. 相似文献
12.
Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca2+ / phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo-daltons) showed an absolute requirement for the presence of both Ca2+ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca2+ /phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca2+ /phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca2+ /phos-pholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca2+ /phospholipid-dependent protein kinase in chromaffin cell function. 相似文献
13.
Summary The p34cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34cdc2 (each ca. 60 % identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with trypthophan renders the resulting mutant protein p80cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34cdc2 function. Five acidic amino acids have also been mutated within p34cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34cdc2 enzyme. 相似文献
14.
Zhenhai Yu Xiaoping Zhao Liangqian Huang Teng Zhang Fajun Yang Lei Xie Shaoli Song Ping Miao Li Zhao Xiaoguang Sun Jianjun Liu Gang Huang 《The Journal of biological chemistry》2013,288(49):35406-35416
Pyruvate kinase M2 (PKM2) is a key player in the Warburg effect of cancer cells. However, the mechanisms of regulating PKM2 are not fully elucidated. Here, we identified the protein-serine/threonine kinase PIM2, a known oncogene, as a novel binding partner of PKM2. The interaction between PIM2 and PKM2 was confirmed by multiple biochemical approaches in vitro and in cultured cells. Importantly, we found that PIM2 could directly phosphorylate PKM2 on the Thr-454 residue, resulting in an increase of PKM2 protein levels. Compared with wild type, PKM2 with the phosphorylation-defective mutation displayed a reduced effect on glycolysis, co-activating HIF-1α and β-catenin, and cell proliferation, while enhancing mitochondrial respiration of cancer cells. These findings demonstrate that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target. 相似文献
15.
Abstract: The regulatory role of A2A adenosine receptors in P2 purinoceptor-mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2- p -(2-carboxyethyl)-phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680), a specific agonist of the A2A adenosine receptor, the extracellular ATP-evoked rise in cytosolic free Ca2+ concentration ([Ca2+ ]i ) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5-trisphosphate production evoked by ATP were not affected by CGS-21680 treatment. However, ATP-evoked Ca2+ influx was inhibited following CGS-21680 stimulation. The CGS-21680-mediated inhibition occurred independently of nifedipine-induced inhibition of the [Ca2+ ]i rise. The CGS-21680-induced inhibition was completely blocked by reactive blue 2. The CGS-21680 effect was mimicked by forskolin and dibutyryl-cyclic AMP and blocked by Rp -adenosine 3',5'-cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A2A adenosine receptors leads to inhibition of P2 purinoceptor-mediated Ca2+ influx through ATP-gated cation channels and involves protein kinase A. 相似文献
16.
Abstract: Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca2+ /calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II α and β subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the a and β subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (≤25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death. 相似文献
17.
Mireille Ehret Christopher D. Cash Michel Hamon Michel Maitre 《Journal of neurochemistry》1989,52(6):1886-1891
Tryptophan hydroxylase is activated in a crude extract by addition of ATP and Mg2+. This activation is reversible and requires in addition both Ca2+ and calmodulin. Thus, phosphorylation by an endogenous calmodulin-dependent protein kinase has long been suspected. Now that we have prepared a specific polyclonal antibody to rat brain tryptophan hydroxylase, we have been able to prove that this hypothesis is correct. After incubation of purified tryptophan hydroxylase with Ca2+/calmodulin-dependent protein kinase together with [gamma-32P]ATP, Mg2+, Ca2+, and calmodulin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting of the enzymes onto nitrocellulose sheets, we could label the band of tryptophan hydroxylase by the antiserum and the peroxidase technique and show by autoradiography that 32P was incorporated into this band. By measuring the radioactivity, we calculated that about 1 mol of phosphate was incorporated per 8 mol of subunits of the enzyme (2 mol of native enzyme). Because the concentration of ATP which we employed (50 microM) gives about half-maximal activation in crude extract compared to saturating ATP conditions (about 1 mM), this result indicates that the incorporation of at least 1 mol of phosphate/mol of tetramer of native tryptophan hydroxylase is required for maximal activation. 相似文献
18.
The activities of Ca2+/calmodulin (CaM)-dependent, Ca2+/phospholipid-dependent, and cyclic AMP-dependent protein kinases (CaM-KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM-KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM-KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM-KII 50-kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM-KII in the pellet. Thus, the loss of CaM-KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM-KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM-KII activity parallels neuronal damage associated with ischemia; down-regulation of CaM-KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM-KII phosphorylation. 相似文献
19.
The p34cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34cdc2, as well as several of the proteins that interact with and regulate p34cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34cdc2 protein is entirely absent and is replaced by its human functional homologue p34CDC2, We have used this strain to analyse aspects of the function of the human p34CDC2 protein genetically. We show that the function of the human p34CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80cdc25 that it responds to altered levels of both the mitotic inhibitor p1072331 and the p34cdc2-binding protein p13suc1, and is lethal in combination with the mutant B-type cyclin p56cdc13-117. In addition, we demonstrate that the human p34CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34CDC2 proteins in fission yeast. 相似文献
20.
Hideyuki Yamamoto Nobuaki Maeda Michio Niinobe Eishichi Miyamoto Katsuhiko Mikoshiba† 《Journal of neurochemistry》1989,53(3):917-923
Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A-kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献