scholarly journals Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation

2017 ◽  
Author(s):  
Céline Lecointre ◽  
Valérie simon ◽  
Clément kerneur ◽  
Frédéric Allemand ◽  
Aurélie Fournet ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Domain ◽  
Protein Tyrosine Phosphorylation ◽  
Dimerization Domain ◽  
Protein Kinase Domain ◽  
Protein Tyrosine ◽  
C Terminus ◽  
Self Association

ABSTRACTThe pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.

scholarly journals Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate

1993 ◽  
Vol 295 (3) ◽  
pp. 879-888 ◽  
Author(s):  
U Zor ◽  
E Ferber ◽  
P Gergely ◽  
K Szücs ◽  
V Dombrádi ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Tyrosine Phosphorylation ◽  
Phorbol Ester ◽  
Cellular Protein ◽  
Oxygen Species ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Ros Formation

We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.

lonomycin-stimulated Arachidonic Acid Release in Human Platelets: a Role for Protein Kinase C and Tyrosine Phosphorylation

1996 ◽  
Vol 76 (02) ◽  
pp. 248-252 ◽  
Author(s):  
Philip G Hargreaves ◽  
Susanne Jenner ◽  
Janet E Merritt ◽  
Stewart O Sage ◽  
Richard W Farndale
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Human Platelets ◽  
Protein Tyrosine Phosphorylation ◽  
Arachidonic Acid Release ◽  
Protein Tyrosine ◽  
Kinase C ◽  
Acid Release

SummaryCollagen (10-90 Μg/ml) and ionomycin (1 ΜM; a calcium iono-phore) each evoked rises in intracellular free calcium, protein kinase C activity and arachidonic acid release in human platelets, and as previously demonstrated for collagen, ionomycin (1 p,M) stimulated protein tyrosine phosphorylation. However, at lower concentrations (60 and 250 nM) ionomycin selectively mobilised calcium. Ro31-8220 (a selective inhibitor of protein kinase C) inhibited (by 50%) ionomycin-stimulated arachidonic acid release. Genistein (an inhibitor of protein tyrosine kinases) also reduced by 50% ionomycin-stimulated arachidonic acid release. In combination, genistein and Ro31-8220 abolished ionomycin-stimulated arachidonic acid release. These findings show 1) that a rise in calcium is not sufficient, and 2) the activation of both protein kinase C and protein tyrosine phosphorylation is necessary, for full ionomycin-stimulated arachidonic acid release in human platelets.

Metformin inhibits human spermatozoa motility and signalling pathways mediated by protein kinase A and tyrosine phosphorylation without affecting mitochondrial function

2019 ◽  
Vol 31 (4) ◽  
pp. 787 ◽  
Author(s):  
V. Calle-Guisado ◽  
L. Gonzalez-Fernandez ◽  
D. Martin-Hidalgo ◽  
L. J. Garcia-Marin ◽  
M. J. Bragado
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Superoxide Anion ◽  
Human Spermatozoa ◽  
Signalling Pathways ◽  
Sperm Function ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Superoxide Anion Generation ◽  
Mitochondrial Superoxide

Metformin is a leading antidiabetic drug that is used worldwide in the treatment of diabetes mellitus. This biguanide exerts metabolic and pleiotropic effects in somatic cells, although its invitro actions on human spermatozoa remain unknown. The present study investigated the effects of metformin on human sperm function. Human spermatozoa were incubated in the presence or absence of 10mM metformin for 8 or 20h, and motility was measured by computer-aided sperm analysis (CASA); other parameters were evaluated by flow cytometry. Metformin significantly reduced the percentage of motile, progressive and rapid spermatozoa and significantly decreased sperm velocity. Metformin did not affect viability, mitochondrial membrane potential (MMP) or mitochondrial superoxide anion generation of human spermatozoa at any time studied. However, metformin clearly inhibited the protein kinase (PK) A pathway and protein tyrosine phosphorylation at 8 and 20h, key regulatory pathways for correct sperm function. In summary, metformin treatment of human spermatozoa had a detrimental effect on motility and inhibited essential sperm signalling pathways, namely PKA and protein tyrosine phosphorylation, without affecting physiological parameters (viability, MMP, mitochondrial superoxide anion generation). Given the growing clinical use of metformin in different pathologies in addition to diabetes, this study highlights an adverse effect of metformin on spermatozoa and its relevance in terms of human fertility in patients who potentially could be treated with metformin in the future.

scholarly journals Elevated intracellular calcium concentration increases secretory processing of the amyloid precursor protein by a tyrosine phosphorylation-dependent mechanism

1996 ◽  
Vol 320 (3) ◽  
pp. 957-963 ◽  
Author(s):  
Magdalena A. PETRYNIAK ◽  
Richard J. WURTMAN ◽  
Barbara E. SLACK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Amyloid Precursor Protein ◽  
Tyrosine Phosphorylation ◽  
Precursor Protein ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Kinase C ◽  
Gf 109203X

Secretory cleavage of the amyloid precursor protein (APP), a process that releases soluble APP derivatives (APPs) into the extracellular space, is stimulated by the activation of muscarinic receptors coupled to phosphoinositide hydrolysis. The signalling pathways involved in the release process exhibit both protein kinase C- and protein tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337–8344]. The possibility that elevations in intracellular Ca2+ concentration initiate the tyrosine phosphorylation-dependent release of APPs was examined in human embryonic kidney cells expressing muscarinic m3 receptors. Inhibition of protein kinase C with the bisindolylmaleimide GF 109203X decreased the carbachol-evoked release of APPs by approx. 30%, as shown previously. The residual response was further decreased, in an additive manner, by the Ca2+ chelator EGTA, or by the tyrosine kinase inhibitor tyrphostin A25. The Ca2+ ionophore, ionomycin, like carbachol, stimulated both the release of APPs and the tyrosine phosphorylation of several proteins, one of which was identified as paxillin, a component of focal adhesions. The effects of ionomycin on APPs release and on protein tyrosine phosphorylation were concentration-dependent, and occurred over similar concentration ranges; both effects were inhibited only partly by GF 109203X, but were abolished by EGTA or by tyrosine kinase inhibitors. The results demonstrate for the first time that ionophore-induced elevations in intracellular Ca2+ levels elicit APPs release via increased tyrosine phosphorylation. Part of the increase in APPs release evoked by muscarinic receptor activation might be attributable to a similar mechanism.

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