PARENT SESSION

(W402) SERINE/THREONINE PHOSPHORYLATION BY PKA ASSOCIATED WITH AKAP4 DURING CAPACITATION.

Kaneto, Masako^{1, 2}, O'Brien, Deborah¹, Miki, Kiyoshi¹, ¹ University of North Carolina School of Medicine, Chapel Hill, NC² Developmental Research Laboratories, Shiongi & Co., Ltd., Osaka, Japan

ABSTRACT- Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins have not yet been identified as PKA substrates. The goal of these studies was to identify PKA substrate(s) in sperm, as a first step in clarifying how the PKA signaling pathway regulates sperm motility. Mouse sperm proteins were separated by SDS-PAGE, and analyzed by Western blotting using an antibody against the phosphorylated consensus peptide for PKA substrates (RXXpT or RRXpS). While several phosphorylated protein bands were detected, a protein with a relative molecular weight of 270,000 (p270) was the only protein detected that underwent capacitation-dependent serine/threonine phosphorylation. P270 was phosphorylated at the maximum level within one minute of incubation under capacitating conditions, while tyrosine phosphorylation required up to 2 hours incubation. Phosphorylation of p270 was dependent on the presence of bicarbonate in the medium. Dibutyryl cAMP combined with the phosphodiesterase inhibitor IBMX was able to mimic the effect of bicarbonate. The PKA inhibitor H-89 blocked this phosphorylation, and stearated Ht31, a permeable inhibitor of PKA binding to AKAPs, partially blocked p270 phosphorylation. Phosphorylated p270 (pp270) was insoluble in Triton X-100, but was solublized by sonication in an 8 M urea solution. Head/tail fractionation demonstrated that pp270 is localized in the tail. AKAP4, the major fibrous sheath protein, tethers regulatory subunits of PKA and is thought to regulate the phosphorylation of nearby proteins. Despite incubation under capacitating conditions, pp270 was not detected in sperm lacking AKAP4. These results suggest that p270 is a PKA substrate associated with AKAP4 and may be a component of the PKA signaling pathway that regulates sperm motility. This research was supported by NICHD/NIH through cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproductive Research.

KEY WORDS: PKA, sperm motility, AKAP4