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.

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 Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3′:5′-phosphate-dependent protein kinase in vivo

1977 ◽  
Vol 168 (2) ◽  
pp. 307-310 ◽  
Author(s):  
P J England
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Troponin I ◽  
Protein Kinase Activity ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Parallel Increase ◽  
Dependent Protein

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.

scholarly journals Platelet-activating factor induces protein kinase activity in the particulate fraction of human neutrophils

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 159-165 ◽  
Author(s):  
JC Gay ◽  
ES Stitt
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Platelet Activating Factor ◽  
Particulate Fraction ◽  
Human Neutrophils ◽  
Protein Kinase Activity ◽  
Resting Cells ◽  
Initial Exposure ◽  
Calcium Dependent ◽  
Dependent Protein

Abstract Platelet-activating factor (PAF) is a proinflammatory lipid that has both platelet- and phagocyte-stimulating properties. Because several known activators of calcium-, phospholipid-dependent protein kinase (protein kinase c, PKC) also stimulate neutrophil responses and because neutrophil stimuli such as phorbol diesters and the chemotactic peptide f-Met-Leu-Phe are reported to increase protein kinase activity in neutrophil (PMN) particulate fractions, we investigated the effect of PAF on neutrophil protein kinase activities. In neutrophils exposed to 10(-6) mol/L PAF, cytosolic PKC activity was 521 +/- 38 pmol 32P/10(7) PMN/min (mean +/- SEM), which was not significantly lower than cystolic activity in buffer-treated controls (558 +/- 32 pmol 32P/10(7) PMN/min, n = 14). PAF-exposed cells exhibited a concomitant rise in protein kinase activity associated with the particulate fraction with 53 +/- 4 pmol 32P/10(7) PMN/min compared with 32 +/- 2 pmol in control cells (n = 14). Particulate protein kinase activity was independent of the presence of calcium and phospholipid in the assay medium. The specific PKC inhibitor H-7 inhibited particulate protein kinase activity, however, which suggested that the enzyme activity assayed in this fraction may be PKC in a constitutively activated form. The increase in particulate protein kinase activity induced by PAF required the presence of cytochalasin B, was detectable within 5 seconds of exposure to PAF, and was not reversed by washing the cells free of extracellular PAF after initial exposure. Although PAF did not have a direct effect on PKC activity from cytosolic fractions from resting cells, the increase in particulate protein kinase activity induced by PAF was inhibited when the cells were first depleted of calcium by incubation with Quin 2. These results suggest that PAF induces an increase in particulate protein kinase activity in neutrophils by a calcium- dependent mechanism and that the induction of membrane-associated protein kinase activity may be involved in neutrophil-stimulating actions such as superoxide production, which occur at higher concentrations of PAF.

scholarly journals Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase

2002 ◽  
Vol 368 (1) ◽  
pp. 243-251 ◽  
Author(s):  
Pauline DOUGLAS ◽  
Gopal P. SAPKOTA ◽  
Nick MORRICE ◽  
Yaping YU ◽  
Aaron A. GOODARZI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Activity ◽  
Catalytic Subunit ◽  
Protein Sequencing ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

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