Abstract
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.
Highlights
The ubiquitin–proteasome system (UPS) is a major pathway for intracellular protein degradation [1,2]
The results show that the intensity of the phospho PRKA is composed of two catalytic subunits (PRKAC) band of sperm incubated in non-capacitating medium (NCM) (0 min) was lower than those incubated in capacitating medium (CM) (Figure 1A, p < 0.01)
We show that the proteasome degrades PRKAR1 in human sperm
Summary
The ubiquitin–proteasome system (UPS) is a major pathway for intracellular protein degradation [1,2]. In this system, substrates are typically marked for degradation by covalent linkages to multiple ubiquitin molecules. Once marked by polyubiquitin chains, proteins are rapidly degraded by the proteasome. Proteasome function is crucial for cellular viability [6,7]. The core of the proteasome, called 20S proteasome, is formed by two pairs of homologous rings, each containing seven subunits. The two outer rings contain α-type subunits (PSMA1-7), the function of which is to operate as a “gate” through which proteins enter the catalytic sites [8]. The β-subunits (PSMB1-7) form the two inner rings. Proteasomes are not static complexes, and the activity of the 20S proteasome is modulated by the binding of different regulatory complexes: 19S, PA28α/β, PA28γ, and PA200 [8,11,12]
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