Changes in protein kinase activity within 30 min of induced differentiation in Friend erythroleukemia cells

1993 ◽  
Vol 71 (1-2) ◽  
pp. 1-6 ◽  
Author(s):  
C. C. Silliman ◽  
B. S. Beckman ◽  
James R. Jeter Jr.
Keyword(s):  
Cell Proliferation ◽  
Cell Differentiation ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Erythroleukemia Cells ◽  
Erythroleukemia Cell ◽  
Friend Erythroleukemia Cells ◽  
Endogenous Substrates

Induction of maturation in Friend erythroleukemia cells is accompanied by a programmed cessation in cell proliferation and a concomitant increase in hemoglobin production. To investigate the role of protein kinase activity in the initiation of Friend erythroleukemia cell differentiation, autoradiographs of one-dimensional, nondenaturing gels were prepared from Friend erythroleukemia cells either untreated or preincubated with dimethyl sulfoxide (DMSO) or aphidicolin for a 30-min period. Extracts were treated with cAMP or cGMP prior to electrophoresis and assayed for protein kinase activity in the presence of endogenous or exogenously added histone substrates. The data demonstrated differences in protein kinase activity following exposure of Friend erythroleukemia cells to either DMSO or aphidicolin for 30 min. These changes in enzyme activity varied with the treatment of the cells and the substrate used. Two sets of protein kinases, one responsive to cAMP and the other responsive to cGMP, were activated in control cultures. A different cAMP-responsive enzyme was active only in differentiating cells. Another protein kinase, activated by cGMP, was associated with both DMSO and aphidicolin treatment. All of these protein kinases had a histone substrate preference. One noncyclic nucleotide activated protein kinase phosphorylating endogenous substrates was associated only with DMSO-induced cells. These findings suggest a multifaceted role for protein kinases early in the maturation of Friend erythroleukemia cells.Key words: cell differentiation, cell proliferation, erythroleukemia, protein kinases, protein phosphorylation.

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.

Introduction to the Kinases in Diabetes Biochemical Society focused meeting: are protein kinases good targets for antidiabetic drugs?

2005 ◽  
Vol 33 (2) ◽  
pp. 339-342 ◽  
Author(s):  
M.P. Coghlan ◽  
D.M. Smith
Keyword(s):  
Insulin Resistance ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Whole Body ◽  
Protein Kinase Activity ◽  
Pharmacological Agents ◽  
Insulin Resistant ◽  
Insulin Mimetic ◽  
Glucose Homoeostasis

Insulin regulates whole-body glucose homoeostasis by modulating the activities of protein kinases in its target tissues: muscle, liver and fat. Defects in insulin's ability to modulate protein kinase activity lead to ‘insulin resistance’ or impaired insulin action. Insulin resistance in combination with defective insulin secretion from the pancreas results in the elevated blood glucose levels that are characteristic of diabetes mellitus. Pharmacological agents that selectively modulate protein kinase activities in insulin-resistant tissues may act either as insulin-sensitizing or insulin-mimetic drugs. Consistent with this, small molecule modulators of a number of protein kinases have demonstrated efficacy in animal models of insulin resistance and diabetes. Moreover, emerging data in humans suggest that marketed anti-diabetic agents may also act in part through modulating protein kinase activities. This meeting was convened to consider the potential to treat insulin resistance and Type II diabetes by modulating protein kinase activity.

scholarly journals Toxoplasma gondiiAttachment to Host Cells Is Regulated by a Calmodulin-like Domain Protein Kinase

2001 ◽  
Vol 276 (15) ◽  
pp. 12369-12377 ◽  
Author(s):  
Heidi Kieschnick ◽  
Therese Wakefield ◽  
Carl Anthony Narducci ◽  
Con Beckers
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Host Cells ◽  
Protein Kinase Activity ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases ◽  
Domain Protein

The role of calcium-dependent protein kinases in the invasion ofToxoplasma gondiiinto its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility ofToxoplasmatachyzoites and their attachment to host cells.In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases inToxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed inToxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detectedin vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasmamotility and host cell invasion.

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