Binding and Phosphorylation of a Novel Male Germ Cell-specific cGMP-dependent Protein Kinase-anchoring Protein by cGMP-dependent Protein Kinase Iα*

cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Iα as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinaseanchoring protein). Although the N-terminal region (amino acids 1–66) of cGK-Iα is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Iβ, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Iα is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-Iα and -Iβ, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Iα in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-Iα stably associates with GKAP42 and that binding of cGMP to cGK-Iα facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-Iα and that cGK-Iα may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.