scholarly journals In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction

Reproduction ◽  
2007 ◽  
Vol 134 (3) ◽  
pp. 425-433 ◽  
Author(s):  
Yadira Bastián ◽  
Armando Zepeda-Bastida ◽  
Salvador Uribe ◽  
Adela Mújica
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Embryonic Development ◽  
Protein Kinase A ◽  
Signal Transducer ◽  
Acrosomal Reaction ◽  
Specific Proteins ◽  
Spatio Temporal ◽  
And Migration ◽  
Kinase A

A role for sperm-specific proteins during the early embryonic development has been suggested by a number of recent studies. However, little is known about the participation of transcription factors in that stage. Here, we show that the signal transducer and activator of transcription 1 (Stat1), but not Stat4, was phosphorylated in response to capacitation and the acrosomal reaction (AR). Moreover, Stat1 phosphorylation correlated with changes in its localization: during capacitation, Stat1 moved from the cytoplasm to the theca/flagellum fraction. During AR, Stat1 phosphorylation increased again. In addition, blocking protein kinase A (PKA) and PKC during capacitation abolished both phosphorylation and migration of Stat1. Our results show tight spatio–temporal rearrangements of Stat1, suggesting that after fertilization Stat1 participates in the first rounds of transcription within the male pronucleus.

scholarly journals Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 361
Author(s):  
Han-Yu Wang ◽  
Chun-Hsiang Lin ◽  
Yi-Ru Shen ◽  
Ting-Yu Chen ◽  
Chia-Yih Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Primary Cilia ◽  
Cell Growth And Migration ◽  
Filament Dynamics ◽  
Gtp Binding ◽  
Cilia Formation ◽  
And Migration ◽  
Septin Filament ◽  
Kinase A

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

scholarly journals A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation

2020 ◽  
Vol 295 (10) ◽  
pp. 3316-3329
Author(s):  
Tung O. Chan ◽  
Roger S. Armen ◽  
Santosh Yadav ◽  
Sushrut Shah ◽  
Jin Zhang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Airway Smooth Muscle Cells ◽  
Activation Loop ◽  
Allosteric Sites ◽  
And Migration ◽  
Kinase A ◽  
In Cells

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it “constitutively” phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

scholarly journals Protein Kinase A and Sch9 Cooperatively Regulate Induction of Autophagy in Saccharomyces cerevisiae

2007 ◽  
Vol 18 (10) ◽  
pp. 4180-4189 ◽  
Author(s):  
Tomohiro Yorimitsu ◽  
Shadia Zaman ◽  
James R. Broach ◽  
Daniel J. Klionsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factors ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Nutrient Sensing ◽  
Regulatory Mechanisms ◽  
Eukaryotic Cells ◽  
Tor Kinase ◽  
Kinase A

Autophagy is a highly conserved, degradative process in eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that the protein kinase A (PKA) and Sch9 signaling pathways regulate autophagy cooperatively in yeast. Autophagy is induced in cells when PKA and Sch9 are simultaneously inactivated. Mutant alleles of these kinases bearing a mutation that confers sensitivity to the ATP-analogue inhibitor C3-1′-naphthyl-methyl PP1 revealed that autophagy was induced independently of effects on Tor kinase. The PKA–Sch9-mediated autophagy depends on the autophagy-related 1 kinase complex, which is also essential for TORC1-regulated autophagy, the transcription factors Msn2/4, and the Rim15 kinase. The present results suggest that autophagy is controlled by the signals from at least three partly separate nutrient-sensing pathways that include PKA, Sch9, and TORC1.

scholarly journals Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles.

1994 ◽  
Vol 179 (3) ◽  
pp. 931-942 ◽  
Author(s):  
D Chen ◽  
E V Rothenberg
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Interleukin 2 ◽  
Structural Basis ◽  
Dominant Negative Mutant ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Kinase A

Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein-DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus.

scholarly journals Monitoring of cAMP Synthesis and Degradation in Living Cells

Physiology ◽  
2006 ◽  
Vol 21 (2) ◽  
pp. 86-92 ◽  
Author(s):  
Viacheslav O. Nikolaev ◽  
Martin J. Lohse
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cyclic Nucleotide ◽  
Living Cells ◽  
Cellular Effects ◽  
Cyclic Nucleotide Gated Channels ◽  
Camp Synthesis ◽  
Spatio Temporal ◽  
Synthesis And Degradation ◽  
Kinase A

cAMP is an important second messenger with a plethora of cellular effects and biological roles. To monitor and visualize cAMP in intact living cells, electrophysiological and fluorescent methods have been developed based on activation of all three types of cAMP effectors: protein kinase A, cyclic nucleotide-gated channels, and exchange protein directly activated by cAMP. In this review, we describe and compare these techniques in terms of their robustness, sensitivity and spatio-temporal resolution.

scholarly journals Functional Roles of Protein Kinase A (PKA) and Exchange Protein Directly Activated by 3′,5′-Cyclic Adenosine 5′-Monophosphate (cAMP) 2 (EPAC2) in cAMP-Mediated Actions in Adrenocortical Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (5) ◽  
pp. 2151-2161 ◽  
Author(s):  
Linda Aumo ◽  
Marte Rusten ◽  
Gunnar Mellgren ◽  
Marit Bakke ◽  
Aurélia E. Lewis
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Steroid Hormones ◽  
Steroid Hormone ◽  
Regulatory Protein ◽  
Adrenocortical Cancer ◽  
Hormone Production ◽  
Genes Encoding ◽  
And Migration ◽  
Kinase A

In the adrenal cortex, the biosynthesis of steroid hormones is controlled by the pituitary-derived hormone ACTH. The functions of ACTH are principally relayed by activating cAMP-dependent signaling pathways leading to the induction of genes encoding enzymes involved in the conversion of cholesterol to steroid hormones. Previously, protein kinase A (PKA) was thought to be the only direct effector of cAMP. However, the discovery of the cAMP sensors, exchange proteins directly activated by cAMP (EPAC1 and 2), has led to a reevaluation of this assumption. In the present study, we demonstrate the occurrence of the EPAC2 splicing variant EPAC2B in adrenocortical cancer cells. Immunocytochemistry demonstrated that EPAC2B is localized predominantly in the nucleus. EPAC2B is functional because it activates Rap1 in these cells. Using the cAMP analogs 8-p-chlorophenylthio-2′-O-methyl-cAMP and N6-benzoyl-cAMP, which specifically activate EPAC1/2 and PKA, respectively, we evaluated the contribution of these factors in steroid hormone production, cell morphology, actin reorganization, and migration. We demonstrate that the expression of cAMP-inducible factors involved in steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 11A1 and 17, and nerve growth factor-induced clone B) and the cAMP-induced biosynthesis of steroid hormones (cortisol and aldosterone) are mediated by PKA and not by EPAC2B. In contrast, both PKA- and EPAC-specific cAMP analogs induced cell rounding, loss of stress fibers, and blocked migration. Taken together, the presented data confirm PKA as the central cAMP mediator in steroid hormone production and reveal the involvement of EPAC2B in cAMP-induced effects on cytoskeleton integrity and cell migration.

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