Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme

Background

Mammalian sperm are not able to fertilize an oocyte immediately after ejaculation. In order to acquire fertilization competence, they must undergo a series of cellular changes collectively known as capacitation (Stival et al., 2016). This process takes place within the female reproductive tract but can be emulated in the laboratory by incubating spermatozoa in a defined medium containing Ca²⁺, albumin and HCO₃^-, i.e., the “capacitation medium” (Visconti et al., 1995). Both HCO₃^- and Ca²⁺ act synergistically on a soluble adenylyl cyclase (sAC), producing an elevation of intracellular cAMP levels. Among different targets, cAMP directly activates the Ser/Thr Protein Kinase A (PKA), which acts as a central player orchestrating capacitation signaling events (Buffone et al., 2014). Usually, two different approaches can be used to analyze its activity. One of them relies on western-blots, using commercially available antibodies that detect a consensus phosphorylation sequence of PKA (Krapf et al., 2010). However, the steady-state phosphorylation status of any protein depends on the relative activities of both kinases and phosphatases acting on it. Thus, this approach fails to directly analyze actual PKA activity. A second approach involves measuring in vitro PKA activity by direct quantification of ³²P incorporated into a peptidic substrate, in a controlled reaction mixture containing phosphatase inhibitors (Stival et al., 2018). This allows analysis of PKA independently of other factors that could modulate the phosphorylated state of the substrate. The chemical nature of the peptidic substrate named Kemptide, named after Dr Kemp, who first synthesized it in 1977 (Kemp et al., 1977), includes a phosphorylable serine residue and two arginine on positions -3 and -2, allowing high PKA specificity. In addition, the Kemptide possesses high relative positive charge which accounts for its strong binding to the negatively charged Whatman P81 cellulose paper, simplifying washing of excess radioactive material (Kemp et al., 1977). The enzyme PKA is composed by two regulatory and two catalytic subunits (Akamine et al., 2003; Zhang et al., 2012). Both types of subunits can be found in Triton X-100-soluble and –insoluble fractions of mouse sperm (Visconti et al., 1997). The activity of PKA within the soluble fraction increases during sperm capacitation. However, the activity of PKA that remains in the insoluble fraction does not change during the course of capacitation, and thus, acts as an excellent source of PKA for enzymatic studies (Visconti et al., 1997).

The protocol described herein is used to analyze the effect of agonists or antagonists on PKA activity, using mouse sperm as the source for the kinase, lowering costs while keeping high efficiency. However, this protocol can be easily adapted to analyze variations of PKA activity during the course of sperm capacitation, by using total unfractionated sperm extracts. In this regard, phosphorylation of the Kemptide reflects the given activity of PKA at any stage of capacitation.

Finally, other sources of PKA can also be used, such as purified PKA from plasmid expression.