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.

Interaction Between Protein Kinase A (PKA) and Protein Phosphatase PP2A During Human Sperm Capacitation.

2012 ◽  
Vol 87 (Suppl_1) ◽  
pp. 448-448
Author(s):  
Patricio J. Morales ◽  
Kely Ordenes ◽  
Lidia Zuñiga ◽  
Emilce S. Diaz
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Phosphatase ◽  
Human Sperm ◽  
Sperm Capacitation ◽  
Kinase A

Protein Kinase A modulation by nitric oxide during human sperm capacitation

2018 ◽  
Author(s):  
Florentin-Daniel Staicu ◽  
CARMEN MATAS PARRA ◽  
Juan Carlos Martínez Soto
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Human Sperm ◽  
Sperm Capacitation ◽  
Kinase A

scholarly journals Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3501
Author(s):  
Héctor Zapata-Carmona ◽  
Lina Barón ◽  
Milene Kong ◽  
Patricio Morales
Keyword(s):  
Protein Kinase ◽  
Western Blot ◽  
Protein Kinase A ◽  
Human Sperm ◽  
Sperm Capacitation ◽  
Phosphorylation Pattern ◽  
Presence And Absence ◽  
Kinase A

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.

scholarly journals Fibronectin stimulates human sperm capacitation through the cyclic AMP/protein kinase A pathway

2015 ◽  
Vol 30 (9) ◽  
pp. 2138-2151 ◽  
Author(s):  
E. Martínez-León ◽  
C. Osycka-Salut ◽  
J. Signorelli ◽  
P. Pozo ◽  
B. Pérez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Human Sperm ◽  
Sperm Capacitation ◽  
Kinase A

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 Genetic Analysis of the Kinetochore DASH Complex Reveals an Antagonistic Relationship with the Ras/Protein Kinase A Pathway and a Novel Subunit Required for Ask1 Association

2005 ◽  
Vol 25 (2) ◽  
pp. 767-778 ◽  
Author(s):  
Ju-mei Li ◽  
Yumei Li ◽  
Stephen J. Elledge
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Chromosome Segregation ◽  
Biochemical Analysis ◽  
Genetic Interaction ◽  
Negative Regulator ◽  
Ras Protein ◽  
Sister Chromatids ◽  
Pka Pathway ◽  
Kinase A

ABSTRACT DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants. Ras2 negatively regulates DASH through the PKA pathway. Constitutive PKA activity caused by mutation of the negative regulator BCY1 is toxic to DASH mutants such as ask1 and dam1. In addition, we have discovered two novel subunits of DASH, Hsk2 and Hsk3 (helper of Ask1), which are microproteins of fewer than 75 amino acids, as dosage suppressors of ask1 mutants. These are essential genes that colocalize with DASH components on spindles and kinetochores and are present in the DASH complex. Mutants in hsk3 arrest cells in mitosis with short spindles and broken spindle structures characteristic of other DASH mutants. Hsk3 is critical for the integrity of the DASH complex because in hsk3 mutants the association of Dam1, Duo1, Spc34, and Spc19 with Ask1 is greatly diminished. We propose that Hsk3 acts to incorporate Ask1 into the DASH complex.

Sign in / Sign up

Export Citation Format

Share Document