Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha

J Biol Chem. 2000 Feb 18;275(7):4897-905. doi: 10.1074/jbc.275.7.4897.


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-Ialpha 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-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, 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-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha 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-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cattle
  • Cyclic GMP-Dependent Protein Kinase Type I
  • Cyclic GMP-Dependent Protein Kinases / metabolism*
  • DNA, Complementary
  • Gene Expression Regulation, Developmental
  • Male
  • Mice
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Binding
  • Saccharomyces cerevisiae / genetics
  • Signal Transduction
  • Spermatogenesis
  • Spermatozoa / enzymology*
  • Two-Hybrid System Techniques


  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • DNA, Complementary
  • Gkap1 protein, mouse
  • Cyclic GMP-Dependent Protein Kinase Type I
  • Cyclic GMP-Dependent Protein Kinases
  • Prkg1 protein, mouse

Associated data

  • GENBANK/AB033130