Chemical synthesis of phosphopeptides using the arylthio group for protection of phosphate: application to identification of cdc2 kinase phosphorylation sites

2009 ◽  
Vol 46 (2) ◽  
pp. 106-112 ◽  
Author(s):  
YOSHIHITO UENO ◽  
SHINGO MAKINO ◽  
MASATOSHI KITAGAWA ◽  
SUSUMU NISHIMURA ◽  
YOICHI TAYA ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
Kinase Phosphorylation

THE DYNACTOME: INVESTIGATING DYNAMIC KINASE NETWORKS IN CELLULAR DECISION-MAKING

2008 ◽  
Vol 31 (4) ◽  
pp. 22
Author(s):  
Jonathan So ◽  
Kelly Elder ◽  
Anna Dai ◽  
Claus Jorgensen ◽  
Rune Linding ◽  
...  
Keyword(s):  
Spectrometric Analysis ◽  
Phosphorylation Sites ◽  
Complex Samples ◽  
Downstream Signaling ◽  
Through Flow ◽  
Colorectal Cancer Cells ◽  
Induced Apoptosis ◽  
Cell Phenotypes ◽  
Kinase Phosphorylation ◽  
Kinase Expression

Networks of kinases play a role in the transmission and integration of signals from the membrane to the nucleus. We aim to elucidate kinase phosphorylation and interaction partners in these networks through the immuno-precipitation and mass spectrometric analysis of a representative set of 100 Flag-tagged kinases stably expressed in human colorectal cancer cells. The goal is to generate a comprehensive set of interactions and dynamic phosphorylation sites which correlate with cell phenotypes such as apoptosis and proliferation. The techniques of mass-spectrometry have allowed for the identification of proteins and their phosphorylation sites in complex samples. Various labeling methods such as iTRAQ has enabled the relative quantification of these sites as afunction of time (White et al. PNAS, 2007). However, kinases usually work in the context of particular signaling stimuli. We aim to characterize the role of these over-expressed kinases in the context of Trail-induced apoptosis. This isparticularly relevant to tumorigenesis in that many cancers are resistant to apoptosis and recombinant Trail therapies are currently undergoing clinical trials. We present assays to correlate the proliferative ability and sensitivity to apoptosis of various stable cell lines with kinase expression levels through flow cytometry. We also present efforts to trace downstream signaling through the monitoring of MAP kinase phosphorylation using a high-throughput bead array.

scholarly journals Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

2001 ◽  
Vol 21 (17) ◽  
pp. 5767-5777 ◽  
Author(s):  
Amit Vas ◽  
Winnie Mok ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Safety Net ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
Origin Recognition ◽  
Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

cdc2 Kinase Phosphorylation of Desmin at Three Serine/Threonine Residues in the Amino-Terminal Head Domain

1993 ◽  
Vol 190 (3) ◽  
pp. 927-934 ◽  
Author(s):  
M. Kusubata ◽  
Y. Matsuoka ◽  
K. Tsujimura ◽  
H. Ito ◽  
S. Ando ◽  
...  
Keyword(s):  
Cdc2 Kinase ◽  
Amino Terminal ◽  
Head Domain ◽  
Kinase Phosphorylation

scholarly journals Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis

2001 ◽  
Vol 12 (1) ◽  
pp. 239-250 ◽  
Author(s):  
Shigeko Yamashiro ◽  
Hueylan Chern ◽  
Yoshihiko Yamakita ◽  
Fumio Matsumura
Keyword(s):  
Cho Cells ◽  
Kinase Activity ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Cdc2 Kinase ◽  
Effective Inhibition ◽  
M Phase ◽  
Phase Progression ◽  
Phase Entry

Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division ofXenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.

scholarly journals The Anaphase Promoting Complex Targeting Subunit Ama1 Links Meiotic Exit to Cytokinesis during Sporulation inSaccharomyces cerevisiae

2009 ◽  
Vol 20 (1) ◽  
pp. 134-145 ◽  
Author(s):  
Aviva E. Diamond ◽  
Jae-Sook Park ◽  
Ichiro Inoue ◽  
Hiroyuki Tachikawa ◽  
Aaron M. Neiman
Keyword(s):  
Cell Division ◽  
Plasma Membranes ◽  
Leading Edge ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Prospore Membrane ◽  
A Cell ◽  
Essential Function ◽  
Kinase Phosphorylation

Ascospore formation in yeast is accomplished through a cell division in which daughter nuclei are engulfed by newly formed plasma membranes, termed prospore membranes. Closure of the prospore membrane must be coordinated with the end of meiosis II to ensure proper cell division. AMA1 encodes a meiosis-specific activator of the anaphase promoting complex (APC). The activity of APCAma1is inhibited before meiosis II, but the substrates specifically targeted for degradation by Ama1 at the end of meiosis are unknown. We show here that ama1Δ mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1Δ mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1Δ mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APCAma1functions to coordinate the exit from meiosis II with cytokinesis.

scholarly journals SMRT ‐mediated co‐shuttling enables export of class II a HDAC s independent of their CaM kinase phosphorylation sites

2012 ◽  
Vol 124 (1) ◽  
pp. 26-35 ◽  
Author(s):  
Francesc X. Soriano ◽  
Sangeeta Chawla ◽  
Paul Skehel ◽  
Giles E. Hardingham
Keyword(s):  
Class Ii ◽  
Phosphorylation Sites ◽  
Cam Kinase ◽  
Kinase Phosphorylation
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