Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 7, Pages 4897-4905Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.7.4897
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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 cCK, we employed the yeast two-hybrid system using cGK-I alpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-I alpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-I beta, 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 ia 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-I alpha is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-I alpha and -I beta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-I alpha 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-I alpha stably associates with GKAP42 and that binding of cGMP to cGK-I alpha facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-I alpha and that cGK-I alpha may participate in germ cell develop ment through phosphorylation of Golgi-associated proteins such as GKAP42.
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