Protein Kinase A modulation by nitric oxide during human sperm capacitation

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

Biology of Reproduction ◽

10.1093/biolreprod/87.s1.448 ◽

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

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Evidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase A activation during human sperm capacitation

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gau073 ◽

2014 ◽

Vol 20 (11) ◽

pp. 1054-1066 ◽

Cited By ~ 31

Author(s):

M. A. Battistone ◽

A. Alvau ◽

A. M. Salicioni ◽

P. E. Visconti ◽

V. G. Da Ros ◽

...

Keyword(s):

Protein Kinase ◽

Tyrosine Kinase ◽

Protein Kinase A ◽

Tyrosine Phosphorylation ◽

Human Sperm ◽

Sperm Capacitation ◽

Tyrosine Kinase 2 ◽

Kinase A

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

Cells ◽

10.3390/cells10123501 ◽

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.

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