scholarly journals Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes.

1981 ◽  
Vol 91 (1) ◽  
pp. 167-174 ◽  
Author(s):  
R M Lewis ◽  
D L Nelson
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Excitable Membrane ◽  
Protein Kinase Activity ◽  
Paramecium Tetraurelia ◽  
Phosphoprotein Phosphatase ◽  
Endogenous Protein ◽  
Endogenous Substrates

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.

Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

1976 ◽  
Vol 35 (03) ◽  
pp. 635-642 ◽  
Author(s):  
M Steiner
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Qualitative Change ◽  
Protein Kinase Activity ◽  
Protein Substrates ◽  
Phosphoprotein Phosphatase ◽  
Progressive Loss ◽  
Platelet Membranes ◽  
Endogenous Protein ◽  
Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

2005 ◽  
Vol 288 (5) ◽  
pp. H2412-H2421 ◽  
Author(s):  
Markus Frederich ◽  
Li Zhang ◽  
James A. Balschi
Keyword(s):  
Protein Kinase ◽  
Metabolic Inhibitors ◽  
Protein Kinase Activity ◽  
Α Subunit ◽  
Rat Hearts ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

5′-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes

2011 ◽  
Vol 32 (2) ◽  
pp. 197-209 ◽  
Author(s):  
Yugo Tsuchiya ◽  
Fiona C. Denison ◽  
Richard B. Heath ◽  
David Carling ◽  
David Saggerson
Keyword(s):  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Calcium Signalling ◽  
Metabolic Stress ◽  
Protein Kinase Activity ◽  
Adult Rat ◽  
Protein Kinase C Inhibitor ◽  
Amp Activated Protein Kinase

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4 min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and β-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-β-D-ribofuranoside), agents that mimic ‘metabolic stress’. Inactivation of AMPK by adrenaline was abolished by 1 μM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK β-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (β-Ser24, β-Ser108 or β-Ser182) suggesting that another site(s) is targeted.

scholarly journals Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion.

1987 ◽  
Vol 105 (1) ◽  
pp. 181-189 ◽  
Author(s):  
M Momayezi ◽  
C J Lumpert ◽  
H Kersken ◽  
U Gras ◽  
H Plattner ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Fusion ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Current Finding ◽  
Phosphoprotein Phosphatase ◽  
Negative Results ◽  
Molecular Components

Since it had been previously shown that in Paramecium cells exocytosis involves the dephosphorylation of a 65-kD phosphoprotein (PP), we tried to induce exocytotic membrane fusion by exogenous phosphatases (alkaline phosphatase or calcineurin [CaN]). The occurrence of calmodulin (CaM) at preformed exocytosis sites (Momayezi, M., H. Kersken, U. Gras, J. Vilmart-Seuwen, and H. Plattner, 1986, J. Histochem. Cytochem., 34:1621-1638) and the current finding of the presence of the 65-kD PP and of a CaN-like protein in cell surface fragments ("cortices") isolated from Paramecium cells led us to also test the effect of antibodies (Ab) against CaM or CaN on exocytosis performance. Microinjected anti-CaN Ab strongly inhibit exocytosis. (Negative results with microinjected anti-CaM Ab can easily be explained by the abundance of CaM.) Alternatively, microinjection of a Ca2+-CaM-CaN complex triggers exocytosis. The same occurs with alkaline phosphatase. All these effects can also be mimicked in vitro with isolated cortices. In vitro exocytosis triggered by adding Ca2+-CaM-CaN or alkaline phosphatase is paralleled by dephosphorylation of the 65-kD PP. Exocytosis can also be inhibited in cortices by anti-CaM Ab or anti-CaN Ab. In wild-type cells, compounds that inhibit phosphatase activity, but none that inhibit kinases or proteases, are able to inhibit exocytosis. Exocytosis cannot be induced by phosphatase injection in a membrane-fusion-deficient mutant strain (nd9-28 degrees C) characterized by a defective organization of exocytosis sites (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). We conclude that exocytotic membrane fusion requires an adequate assembly of molecular components to allow for the dephosphorylation of a 65-kD PP and that this step is crucial for the induction of exocytotic membrane fusion in Paramecium cells. In vivo this probably involves a Ca2+-CaM-stimulated CaN-like PP phosphatase.

scholarly journals Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast

2000 ◽  
Vol 20 (16) ◽  
pp. 5858-5864 ◽  
Author(s):  
Gregory J. Reynard ◽  
William Reynolds ◽  
Rati Verma ◽  
Raymond J. Deshaies
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Multiple Substrates ◽  
Cell Extracts

ABSTRACT p13suc1 (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.

scholarly journals Testis-specific mak protein kinase is expressed specifically in the meiotic phase in spermatogenesis and is associated with a 210-kilodalton cellular phosphoprotein.

1993 ◽  
Vol 13 (7) ◽  
pp. 4146-4156 ◽  
Author(s):  
A Jinno ◽  
K Tanaka ◽  
H Matsushime ◽  
T Haneji ◽  
M Shibuya
Keyword(s):  
Protein Kinase ◽  
Cellular Protein ◽  
Kinase Assay ◽  
Soluble Form ◽  
Protein Kinase Activity ◽  
Cell Fractionation ◽  
Testicular Germ Cell ◽  
Fusion Products

The mak gene encodes a new protein kinase distantly related to cdc2 kinase, and its transcripts are expressed exclusively in testicular germ cells at and after meiosis (H. Matsushime, A. Jinno, N. Takagi, and M. Shibuya, Mol. Cell. Biol. 10:2261-2268, 1990). In this study, we prepared a series of antibodies against synthetic peptides and fusion products of the mak gene and characterized the subcellular localization, protein kinase activity, and association with other cellular proteins of Mak. Mak products were identified as 66- and 60-kDa proteins that specifically appeared in rat testes after puberty. Testicular germ cell fractionation revealed that Mak products were most abundant in the fraction of the late pachytene stage and that their levels were dramatically decreased in postmeiotic haploid cells. Mak products were localized mostly in the cytoplasm as a soluble form. [35S]methionine labelling demonstrated that Mak products were associated with a 210-kDa cellular protein; in an in vitro kinase assay with immunoprecipitates of Mak products, the 210-kDa cellular protein was efficiently phosphorylated on serine and threonine residues. Furthermore, in a testicular cell culture system with 32Pi, the 210-kDa molecule associated with Mak was phosphorylated in vivo on serine and threonine residues. These results strongly suggest that the Mak complex may play a role in meiosis during spermatogenesis and that a phosphorylated 210-kDa protein is one of the physiological substrates for this protein kinase.

Testis-specific mak protein kinase is expressed specifically in the meiotic phase in spermatogenesis and is associated with a 210-kilodalton cellular phosphoprotein

1993 ◽  
Vol 13 (7) ◽  
pp. 4146-4156
Author(s):  
A Jinno ◽  
K Tanaka ◽  
H Matsushime ◽  
T Haneji ◽  
M Shibuya
Keyword(s):  
Protein Kinase ◽  
Cellular Protein ◽  
Kinase Assay ◽  
Soluble Form ◽  
Protein Kinase Activity ◽  
Cell Fractionation ◽  
Testicular Germ Cell ◽  
Fusion Products

The mak gene encodes a new protein kinase distantly related to cdc2 kinase, and its transcripts are expressed exclusively in testicular germ cells at and after meiosis (H. Matsushime, A. Jinno, N. Takagi, and M. Shibuya, Mol. Cell. Biol. 10:2261-2268, 1990). In this study, we prepared a series of antibodies against synthetic peptides and fusion products of the mak gene and characterized the subcellular localization, protein kinase activity, and association with other cellular proteins of Mak. Mak products were identified as 66- and 60-kDa proteins that specifically appeared in rat testes after puberty. Testicular germ cell fractionation revealed that Mak products were most abundant in the fraction of the late pachytene stage and that their levels were dramatically decreased in postmeiotic haploid cells. Mak products were localized mostly in the cytoplasm as a soluble form. [35S]methionine labelling demonstrated that Mak products were associated with a 210-kDa cellular protein; in an in vitro kinase assay with immunoprecipitates of Mak products, the 210-kDa cellular protein was efficiently phosphorylated on serine and threonine residues. Furthermore, in a testicular cell culture system with 32Pi, the 210-kDa molecule associated with Mak was phosphorylated in vivo on serine and threonine residues. These results strongly suggest that the Mak complex may play a role in meiosis during spermatogenesis and that a phosphorylated 210-kDa protein is one of the physiological substrates for this protein kinase.

scholarly journals Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

1986 ◽  
Vol 6 (6) ◽  
pp. 2033-2040 ◽  
Author(s):  
H Piwnica-Worms ◽  
D R Kaplan ◽  
M Whitman ◽  
T M Roberts
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Shuttle Vector ◽  
T Antigen ◽  
Protein Kinase Activity ◽  
Polyomavirus Middle T Antigen ◽  
Nih 3T3 ◽  
Middle T Antigen

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.

scholarly journals The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain

2006 ◽  
Vol 27 (5) ◽  
pp. 1581-1591 ◽  
Author(s):  
Pauline Douglas ◽  
Xiaoping Cui ◽  
Wesley D. Block ◽  
Yaping Yu ◽  
Shikha Gupta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Kinase Domain ◽  
Dependent Protein

ABSTRACT The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

scholarly journals Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase

2013 ◽  
Vol 33 (6) ◽  
Author(s):  
James M. J. Dickson ◽  
Woo-Jeong Lee ◽  
Peter R. Shepherd ◽  
Christina M. Buchanan
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Cell Signalling ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
His Tag ◽  
The Impact

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.

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