Measurement of Cdk1/Cyclin B Kinase Activity by Specific Antibodies and Western Blotting

2016 ◽  
pp. 337-348 ◽  
Author(s):  
Cody W. Lewis ◽  
Ryan G. Taylor ◽  
Roy M. Golsteyn
Keyword(s):  
Western Blotting ◽  
Kinase Activity ◽  
Cyclin B ◽  
Specific Antibodies

A propagated wave of MPF activation accompanies surface contraction waves at first mitosis in Xenopus

1998 ◽  
Vol 111 (3) ◽  
pp. 385-393 ◽  
Author(s):  
D. Perez-Mongiovi ◽  
P. Chang ◽  
E. Houliston
Keyword(s):  
Kinase Activity ◽  
Direct Consequence ◽  
Time Lapse ◽  
Cyclin B ◽  
Triggering Mechanism ◽  
Microtubule Network ◽  
Regulatory Subunits ◽  
Vegetal Pole ◽  
Xenopus Egg

During the period of mitosis, two surface contraction waves (SCWs) progress from the animal to vegetal poles of the Xenopus egg. It has been shown that these SCWs occur in parallel with the activation of MPF and with its subsequent inactivation in the animal and vegetal hemispheres, suggesting that they are responses to propagated waves of MPF activity across the egg. We have analysed the mechanism of MPF regulation in different regions of the egg in detail in relation to SCW progression. The distributions of histone HI kinase activity and of Cdc2 and cyclin B (the catalytic and regulatory subunits of MPF) were followed by dissection of intact eggs following freezing and in cultured fragments separated by ligation. Cdc2 was found to be distributed evenly throughout the egg cytoplasm. Loss of phosphorylated (inactive) forms of Cdc2 coincided spatially with the wave of MPF activation, while cyclin B2 accumulation occurred in parallel in animal and vegetal regions. In ligated vegetal pole fragments no MPF activation or Cdc2 dephosphorylation were detectable. A wave of cyclin B destruction that occurred in concert with the second SCW was also blocked. Taken together these results indicate that the triggering mechanism for MPF activation requires components specific to the animal cytoplasm, acting via Cdc2 dephosphorylation, and that MPF activation subsequently propagates autocatalytically across the egg. SCW progression in the vegetal hemisphere was followed directly by time-lapse videomicroscopy of subcortical mitochondrial islands. The first SCW traversed the vegetal pole at the time of MPF activation in this region. Like MPF activation and inactivation, SCWs were blocked in the vegetal region by ligation. These observations reinforce the hypothesis that the first SCW is a direct consequence of the MPF activation wave. It may reflect depolymerisation of the subcortical microtubule network since it coincided exactly with the arrest of the microtubule-dependent movement of ‘cortical rotation’ and was related in direction in most eggs. The cyclin B destruction wave and associated cortical contraction of the second SCW may be localised downstream consequences of the MPF activation wave, or they may propagate independently from the animal cytoplasm.

scholarly journals Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

1990 ◽  
Vol 32 (5) ◽  
pp. 379-383 ◽  
Author(s):  
Anna Maria Simonsen Stolf ◽  
Eufrosina Setsu Umezawa ◽  
Bianca Zingales
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Chagas Disease ◽  
Western Blotting ◽  
Specific Antibodies ◽  
Internal Parasite ◽  
Sds Page ◽  
Blotting Technique ◽  
Simultaneous Identification ◽  
Acute Chagas Disease

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

The Reciprocal t(9;22) p96ABL/BCR fusion Protein Eenhances the Transformation Potential of BCR/ABL and Increases Therapy Resistance in Models of Ph+ Acute Lymphatic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3539-3539
Author(s):  
Anahita Rafiei ◽  
Afsar Mian ◽  
Anna Metodieva ◽  
Claudia Oancea ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Western Blotting ◽  
Fusion Proteins ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Therapy Resistance ◽  
Acute Lymphatic Leukemia ◽  
Lymphatic Leukemia

Abstract Abstract 3539 The t(9;22)(q34;q11) is a balanced translocation. The cytogenetic correlate of der22 is the so-called Philadelphia chromosome (Ph). Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. BCR/ABL is a constitutively activated kinase which induces the leukemic phenotype by the aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The BCR/ABL kinase activity is efficiently targeted by tyrosin-Kinase inhibitors such Imatinib, Nilotinib, or Dasatinib. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients suffering from chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). In our previous studies we have shown the leukemogenic potential of the ABL/BCR fusion proteins. To further disclose the role of ABL/BCR proteins, mainly p96ABL/BCR, in the transformation process induced by BCR/ABL and the leukemogenesis of Ph+ ALL, we co-expressed p96ABL/BCR and p185BCR/ABL retrovirally in the IL-3 dependent murine Ba/F3 pro-lymphocytic cell line. p96ABL/BCR and p185BCR/ABL were expressed from P2A peptide-linked multicistronic retroviral vectors, which allow the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The effect of p96ABL/BCR on the kinase activity of p185BCR/ABL, was assessed by the rate of autophosphorylation at Y245 and Y412, the BCR/ABL-dependent substrate phosphorylation (CrkL, Bcr) and by the activation of down-stream signaling pathway (Stat, Erk,) determined by Western blotting. Proliferation of the cells was assessed by growth curve and XTT assays upon withdrawal of IL-3. As classical transformation assays we performed focus formation assays (loss of contact inhibition) and colony formation in semi-solid medium (support independent growth) in untransformed Rat-1 fibroblasts. The p96ABL/BCR expression in primary Ph+ ALL patient derived long term cultures (PDLTCs) was targeted by retrovirally transduced shRNA. The efficient targeting of p96ABL/BCR was confirmed by western blotting. Here we report that p96ABL/BCR i.) p96ABL/BCR enhanced not only the autophosphorylation of p185BCR/ABL at Y245, but also the activation of all the downstream signaling pathways; ii.) p96ABL/BCR by itself did not transform Rat-1 cells but impressively increased the number of colonies and foci induced by p185BCR/ABL in Rat-1 cells; iii.) p96ABL/BCR increased the proliferation of p185BCR/ABL-positive Ba/F3 cells; iv.)p96ABL/BCR reduced the responsiveness to TKI in p185BCR/ABL positive Ba/F3 cells; v.) targeting the p96ABL/BCR by shRNA decreased the proliferation of Ph+PDLTCs by the induction of apoptosis and increased their sensitivity towards kinase inhibitors (Imatinib, Nilotinib) and the allosteric inhibition by GNF-2 directed against p185BCR/ABL. Taken together these data suggest that p96ABL/BCR plays an important role in the determination of the leukemic phenotype and the therapy resistance of Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

Cdc7 inhibition as a novel approach for pancreas cancer therapy.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15059-e15059
Author(s):  
Mark G. Frattini ◽  
Lucia Regales ◽  
Ruth Santos ◽  
Diana Carrillo
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Dna Damage ◽  
Cell Viability ◽  
Cell Cycle Arrest ◽  
Western Blotting ◽  
Kinase Activity ◽  
S Phase ◽  
Cycle Arrest ◽  
Cdc7 Kinase

e15059 Background: Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, 43,920 people will be diagnosed and 37,390 people will die of this disease. 95% of tumors reveal loss of the p16 protein, a regulator of the G1 to S phase transition. Cdc7 is a conserved kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Downregulation of Cdc7 kinase activity resulted in slowing of S-phase and cell cycle arrest followed by accumulation of DNA damage. Cdc7 has been shown to be over-expressed in many different tumors including the majority of solid and liquid tumors. In our laboratory a novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be efficacious in cell based cytotoxicity assays and multiple animal models of cancer. Methods: We have examined the efficacy of Cdc7 kinase inhibition as a therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in Capan-1, BxPC3, and PANC-1 cell lines. These cells were treated with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS), and western blotting. Results: Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. Conclusions: We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease.

scholarly journals Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

1995 ◽  
Vol 15 (12) ◽  
pp. 7143-7151 ◽  
Author(s):  
K S Lee ◽  
Y L Yuan ◽  
R Kuriyama ◽  
R L Erikson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Maximum Level ◽  
Specific Protein ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
M Phase ◽  
Nih 3T3

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro.

scholarly journals Mechanisms of p34cdc2 regulation.

1993 ◽  
Vol 13 (3) ◽  
pp. 1675-1685 ◽  
Author(s):  
S Atherton-Fessler ◽  
L L Parker ◽  
R L Geahlen ◽  
H Piwnica-Worms
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Cyclin B ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Maximal Inhibition

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

scholarly journals Mitotic Effects of a Constitutively Active Mutant of the Xenopus Polo-Like Kinase Plx1

1999 ◽  
Vol 19 (12) ◽  
pp. 8625-8632 ◽  
Author(s):  
Yue-Wei Qian ◽  
Eleanor Erikson ◽  
James L. Maller
Keyword(s):  
Xenopus Laevis ◽  
Aspartic Acid ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Interphase Nuclei ◽  
Aspartic Acid Residue ◽  
Early Embryos ◽  
M Phase ◽  
Egg Extracts

ABSTRACT During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a “trigger” kinase for the activation of Cdc25C during the G2/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.

scholarly journals A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.

1991 ◽  
Vol 112 (4) ◽  
pp. 523-533 ◽  
Author(s):  
G Dessev ◽  
C Iovcheva-Dessev ◽  
J R Bischoff ◽  
D Beach ◽  
R Goldman
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Kinase Activity ◽  
Nuclear Lamina ◽  
Cyclin B ◽  
Protein Kinase Activity ◽  
Nuclear Lamin ◽  
Single Protein ◽  
M Phase

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.

scholarly journals Activation of the Xenopus cyclin degradation machinery by full-length cyclin A

1996 ◽  
Vol 109 (5) ◽  
pp. 1071-1079 ◽  
Author(s):  
C. Jones ◽  
C. Smythe
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cell Types ◽  
Cyclin B1 ◽  
Full Length ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
Nuclear Envelope Breakdown

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin. A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.

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