Use of PKA-mediated phenotypes for genetic and small-molecule screens in Schizosaccharomyces pombe

2013 ◽  
Vol 41 (6) ◽  
pp. 1692-1695 ◽  
Author(s):  
Ana Santos de Medeiros ◽  
Alexander Magee ◽  
Kyle Nelson ◽  
Liora Friedberg ◽  
Karolina Trocka ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Schizosaccharomyces Pombe ◽  
Protein Kinase A ◽  
Fission Yeast ◽  
Small Molecule ◽  
Sexual Development ◽  
Cyclic Nucleotide ◽  
Pka Pathway ◽  
Kinase A

PKA (protein kinase A) in the fission yeast Schizosaccharomyces pombe controls transcription of genes involved in metabolism, cell growth and sexual development. In the present review, we discuss phenotypes associated with either high or low PKA activity in the context of how they can be used to carry out genetic or small-molecule screens that affect components of the PKA pathway. Although our recent research has focused on the study of heterologously expressed cyclic nucleotide PDEs (phosphodiesterases), these same methods can be used to target other S. pombe proteins or their functionally equivalent orthologues that act in the PKA pathway.

scholarly journals The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

2010 ◽  
Vol 399 (2) ◽  
pp. 123-128 ◽  
Author(s):  
Yiwei Wang ◽  
Galina Gulis ◽  
Scott Buckner ◽  
P. Connor Johnson ◽  
Daniel Sullivan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Schizosaccharomyces Pombe ◽  
Protein Kinase A ◽  
Fission Yeast ◽  
Kinase A

scholarly journals Protein Kinase A Regulates Sexual Development and Gluconeogenesis through Phosphorylation of the Zn Finger Transcriptional Activator Rst2p in Fission Yeast

2002 ◽  
Vol 22 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Toru Higuchi ◽  
Yoshinori Watanabe ◽  
Masayuki Yamamoto
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Fission Yeast ◽  
Sexual Development ◽  
Zinc Finger Protein ◽  
Upstream Region ◽  
Reporter System ◽  
Negative Effect ◽  
Kinase A

ABSTRACT Protein kinase A (PKAi a cyclic AMP-dependent protein kinase) negatively regulates sexual development and gluconeogenesis in fission yeast by suppressing the transcription of ste11 required for the former and the transcription of fbp1 required for the latter. Here we show that Rst2p, a zinc finger protein that can bind to the upstream region of ste11 and fbp1 via the STREP motif, mediates the activity of PKA to transcription of these genes. A simple reporter system confirmed that PKA could cause its negative effect on transcription through the combination of Rst2p and STREP. Rst2p was phosphorylated by PKA in vitro at two consensus sequences on it. Substitution of the target threonine residues by alanine made the protein active even in the presence of high PKA activity. Rst2p underwent hyperphosphorylation in the medium lacking glucose, and PKA inhibited this hyperphosphorylation. Rst2p was mainly cytoplasmic under high PKA activity but was concentrated in the nucleus when this activity was lowered, suggesting that PKA might regulate ste11 and fbp1 negatively by excluding Rst2p from the nucleus. However, the shift of Rst2p localization was not perfect under physiological conditions, leaving the possibility that PKA inhibits Rst2p function in another way as well. Although the PKA-Rst2p-STREP pathway is apparently central to the regulation of ste11 and fbp1 transcription in accordance with nutritional conditions, some additional paths are likely to connect nitrogen to repression of ste11 and glucose to repression of fbp1. These paths may ensure the specificity between the type of nutrients in shortage and the type of genes to be expressed.

scholarly journals Cooperative Activation of Lipolysis by Protein Kinase A and Protein Kinase C Pathways in 3T3-L1 Adipocytes

Endocrinology ◽  
2004 ◽  
Vol 145 (11) ◽  
pp. 4940-4947 ◽  
Author(s):  
Katrin Fricke ◽  
Aleksandra Heitland ◽  
Erik Maronde
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathways ◽  
Hormone Sensitive Lipase ◽  
Glycerol Release ◽  
Kinase C ◽  
Lipolytic Effect ◽  
Pka Pathway ◽  
Kinase A

Abstract In the present study, we investigate the coherence of signaling pathways leading to lipolysis in 3T3-L1 adipocytes. We observe two linear signaling pathways: one well known, acting via cAMP and protein kinase A (PKA) activation, and a second one induced by phorbol 12-myristate 13-acetate treatment involving protein kinase C (PKC) and MAPK. We demonstrate that both the PKA regulatory subunits RIα and RIIβ are expressed in 3T3-L1 adipocytes and are responsible for the lipolytic effect mediated via the cAMP/PKA pathway. Inhibition of the PKA pathway by the selective PKA inhibitor Rp-8-CPT-cAMPS does not impair lipolysis induced by PKC activation, and neither PD98059 nor U0126, as known MAPK kinase inhibitors, changes the level of glycerol release caused by PKA activation, indicating no cross-talk between these two pathways when only one is activated. However, when both are activated, they act synergistically on glycerol release. Additional experiments focusing on this synergy show no involvement of MAPK phosphorylation and cAMP formation. Phosphorylation of hormone-sensitive lipase is similar upon stimulation of either pathway, but we demonstrate a difference in the ability of both PKA and the PKC pathway activation to phosphorylate perilipin, which in turn may be an explanation for the different maximal lipolytic effect of both pathways.

scholarly journals The activation of the chymotrypsin-like activity of the proteasome is regulated by soluble adenyl cyclase/cAMP/protein kinase A pathway and required for human sperm capacitation

2019 ◽  
Vol 25 (10) ◽  
pp. 587-600 ◽  
Author(s):  
Héctor Zapata-Carmona ◽  
Lina Barón ◽  
Lidia M Zuñiga ◽  
Emilce Silvina Díaz ◽  
Milene Kong ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Current Knowledge ◽  
Human Sperm ◽  
Adenyl Cyclase ◽  
Sperm Capacitation ◽  
Time Treatment ◽  
Proteasome Subunits ◽  
Pka Pathway ◽  
Kinase A

Abstract One of the first events of mammalian sperm capacitation is the activation of the soluble adenyl cyclase/cAMP/protein kinase A (SACY/cAMP/PKA) pathway. Here, we evaluated whether the increase in PKA activity at the onset of human sperm capacitation is responsible for the activation of the sperm proteasome and whether this activation is required for capacitation progress. Viable human sperm were incubated with inhibitors of the SACY/cAMP/PKA pathway. The chymotrypsin-like activity of the sperm proteasome was evaluated using a fluorogenic substrate. Sperm capacitation status was evaluated using the chlortetracycline assay and tyrosine phosphorylation. To determine whether proteasomal subunits were phosphorylated by PKA, the proteasome was immunoprecipitated and tested on a western blot using an antibody against phosphorylated PKA substrates. Immunofluorescence microscopy analysis and co-immunoprecipitation (IPP) were used to investigate an association between the catalytic subunit alpha of PKA (PKA-Cα) and the proteasome. The chymotrypsin-like activity of the sperm proteasome significantly increased after 5 min of capacitation (P < 0.001) and remained high for the remaining incubation time. Treatment with H89, KT5720 or KH7 significantly decreased the chymotrypsin-like activity of the proteasome (P < 0.001). IPP experiments indicated that PKA inhibition significantly modified phosphorylation of proteasome subunits. In addition, PKA-Cα colocalized with the proteasome in the equatorial segment and in the connecting piece, and co-immunoprecipitated with the proteasome. This is the first demonstration of sperm proteasome activity being directly regulated by SACY/PKA-Cα. This novel discovery extends our current knowledge of sperm physiology and may be used to manage sperm capacitation during assisted reproductive technology procedures.

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