Cellular compartmentalization of protein kinase activity during the cell cycle

1987 ◽  
Vol 65 (12) ◽  
pp. 1070-1079
Author(s):  
Anna A. Fabisz-Kijowska ◽  
Katherine Lumley-Sapanski ◽  
Margaret S. Halleck ◽  
Robert A. Schlegel
Keyword(s):  
Cell Cycle ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Kinase Activity ◽  
Culture Cells ◽  
Nuclear Extracts ◽  
Interphase Cells ◽  
Cellular Compartmentalization ◽  
Mitotic Cells

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.

scholarly journals Detection of protein kinase activity specifically activated at metaphase-anaphase transition.

1996 ◽  
Vol 132 (4) ◽  
pp. 635-641 ◽  
Author(s):  
M Sekimata ◽  
K Tsujimura ◽  
J Tanaka ◽  
Y Takeuchi ◽  
N Inagaki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Glioma Cells ◽  
Cell Types ◽  
Protein Kinase Activity ◽  
Cleavage Furrow ◽  
Wide Range ◽  
Interphase Cells ◽  
Cycle Dependent ◽  
Mitotic Cells

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle-dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.

scholarly journals The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle.

1991 ◽  
Vol 11 (8) ◽  
pp. 4045-4052 ◽  
Author(s):  
S Garrett ◽  
M M Menold ◽  
J R Broach
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
S Phase ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Dependent Protein

Null mutations in the gene YAK1, which encodes a protein with sequence homology to known protein kinases, suppress the cell cycle arrest phenotype of mutants lacking the cyclic AMP-dependent protein kinase (A kinase). That is, loss of the YAK1 protein specifically compensates for loss of the A kinase. Here, we show that the protein encoded by YAK1 has protein kinase activity. Yak1 kinase activity is low during exponential growth but is induced at least 50-fold by arrest of cells prior to the completion of S phase. Induction is not observed by arrest at stages later in the cell cycle. Depending on the arrest regimen, induction can occur either by an increase in Yak1 protein levels or by an increase in Yak1 specific activity. Finally, an increase in Yak1 protein levels causes growth arrest of cells with attenuated A kinase activity. These results suggest that Yak1 acts in a pathway parallel to that of the A kinase to negatively regulate cell proliferation.

scholarly journals Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14.

1994 ◽  
Vol 5 (3) ◽  
pp. 273-282 ◽  
Author(s):  
S Kornbluth ◽  
B Sebastian ◽  
T Hunter ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Schizosaccharomyces Pombe ◽  
Protein Kinases ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Dual Specificity ◽  
Detergent Extraction

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.

scholarly journals Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

1982 ◽  
Vol 2 (2) ◽  
pp. 199-206 ◽  
Author(s):  
T D Gilmore ◽  
K Radke ◽  
G S Martin
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Defective Mutant ◽  
Sarcoma Virus

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

scholarly journals S183. ABNORMALITIES IN SERINE/THREONINE SIGNALING NETWORKS IN SEVERE NEUROPSYCHIATRIC ILLNESS: DEVELOPMENT OF A PIPELINE TO EXPLORE ABNORMALITIES OF KINASE ACTIVITY AND IDENTIFY NOVEL TREATMENT STRATEGIES FOR SCHIZOPHRENIA

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S107-S108
Author(s):  
Robert McCullumsmith ◽  
Khaled Alganem ◽  
Nicholas Henkel ◽  
Abdul Hammoud ◽  
Rammohan Shukla ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Biological Pathways ◽  
Signaling Networks ◽  
Protein Kinase Activity ◽  
Drug Candidates ◽  
Group 10 ◽  
Disease Signatures ◽  
Neuropsychiatric Illness

Abstract Background Abnormalities of cellular signaling are well characterized in neuropsychiatric illnesses, including schizophrenia. Changes in signaling pathways reflect the underlying genetic, environmental, and epigenetic perturbations driving disease phenotypes. A shortcoming of most signaling studies is a focus on one or a few protein kinases at a time, a limitation since protein kinases work in networks with other kinases, phosphatases, and regulatory molecules to effect signaling events. We addressed this challenge by employing a kinome array platform that simultaneously measures protein kinase activity at hundreds of reporter peptide substrates. We then developed a novel bioinformatics pipeline to identify protein kinase nodes, signaling networks, upstream biological pathways, and drug candidates that “reverse” kinomic disease signatures. Methods Postmortem DLPFC brain samples from subjects with schizophrenia (n = 20 per group, 10 males and 10 females per group), were compared to age, PMI and pH matched control subjects (n = 20 per group, 10 males and 10 females per group) using the Pamgene12 serine/threonine kinome array chip. Samples were pooled by diagnosis and gender, and run in triplicate. The R-shiny app KRSA was created to automate assignment of kinases, perform permutation analyses, identify biological pathways, and connect to iLINCs for identification of drugs that reverse kinomic disease signatures. We also performed targeted confirmation studies using specific kinase activity assays, QPCR, and western blot analyses. Results We identified unique and common kinase nodes for each diagnostic group. Several of the nodes (for example AKT) are well characterized in schizophrenia, while others have not previously been identified (such as AMPK). We used AMPK KD cultures and AMPK KO brain tissues to demonstrate the validity if the kinome array for this protein kinase. We used standard kinase activity assays for AMPK and found decreased activity for AMPK (P < 0.05). We also found decreased expression of transcripts for the regulatory subunits of AMPK (P < 0.05). We identified several unique biological pathways, as well as candidate drugs, associated with the disease signature in schizophrenia. Discussion Our results confirm well characterized signaling defects in severe neuropsychiatric illness, and identify novel signaling nodes for further study. Confirmation studies for AMPK kinase show significant changes in expression and activity of this kinase, suggesting perturbation of energy sensing and production pathways in schizophrenia. Bioenergetic pathways may be targeted by myriad mechanisms, and we identified several drug candidates that might help restore this pathway in afflicted persons. Overall our novel workflow and pipeline provides a promising new avenue for understanding the complex signaling perturbations found in brain diseases and may provide new leads for developing treatments for schizophrenia and other cognitive disorders.

Biochemical changes accompanying enhanced cardiac contractility by ionophore A23187

1985 ◽  
Vol 249 (6) ◽  
pp. H1204-H1210 ◽  
Author(s):  
J. J. Murray ◽  
P. W. Reed ◽  
J. G. Dobson
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Ionophore A23187 ◽  
Phosphorylase Activity ◽  
Dependent Protein Kinase ◽  
Camp Content ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

We have reported that the divalent cation ionophore A23187, like the beta-adrenergic agonist isoproterenol, increased the force of contraction and rate of relaxation and shortened the duration of contraction of papillary muscles isolated from guinea pigs. A23187 produced a fall in resting tension and decreased the contracture tension of K +/- depolarized muscles, as did isoproterenol. In the present studies, isoproterenol produced a concentration-dependent, rapid, and sustained increase in the cyclic AMP (cAMP) content of papillary muscle. In contrast, A23187 had no detectable effect on cAMP levels, even in the presence of the phosphodiesterase inhibitor, papaverine. Neither drug, at concentrations maximal for contractile effects, altered cyclic GMP (cGMP). Isoproterenol increased the cAMP-dependent protein kinase activity ratio, whereas A23187 did not change the activity of this enzyme. However, both A23187 and isoproterenol produced a concentration-dependent increase in phosphorylase activity. Concentrations of A23187 or isoproterenol that enhanced contractility maximally increased the alkali-labile phosphate (by ca. 35%) but were without effect on the acid-labile, alkali-stable phosphate in the total acid precipitable protein. Contractile effects of isoproterenol, which reflect activated Ca2+ uptake, and the increase in phosphorylase activity produced by this agent are believed to be due to an increase in cAMP with subsequent activation of cAMP-dependent protein kinases and phosphorylation of proteins. A23187 may produce similar contractile effects without an increase in cAMP or cAMP-dependent protein kinase activity by activating other protein kinases and/or inhibiting phosphoprotein phosphatases, most likely by its effects on intracellular calcium.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

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