CLONING AND EXPRESSION OF A NOVEL CYCLIC GMP-DEPENDENT PROTEIN-KINASEFROM MOUSE-BRAIN

Synthetic oligonucleotides were used to amplify mouse brain cDNA seque nces homologous to conserved regions of known cGMP-dependent protein k inases, and two classes of products were identified. The first class ( CGKI) of amplification products contained approximately 1.0 kilobase ( kb) of DNA sequence between the oligonucleotide primers, and this sequ ence showed a high degree of homology (90% identity) with the known bo vine and human type I cDNA sequences for cGMP-dependent protein kinase . The second class (CGKII) of amplification products contained approxi mately 1.1 kb of DNA sequence between the oligonucleotide primers, and this sequence showed a much lower homology (65% identity) with the bo vine and human type I cDNA sequences. Northern blot analysis showed th at CGKI transcripts of 8.5 kb were abundant in brain and lung, whereas a 7-kb transcript could be detected in testis. CGKII transcripts of 6 kb were also abundant in brain and lung but could be detected at lowe r levels in kidney. The CGKII amplification product was used to screen a mouse brain cDNA library, and four overlapping cDNA clones were iso lated which comprised the entire CGKII coding region. The predicted CG KII protein consists of 761 amino acids and has a molecular mass of 87 kDa. The CGKII protein shows highest homology to the catalytic (66% a mino acid identity) and regulatory domains (45% identity) of bovine an d human CGKI. Little homology is observed at the amino terminus or in the region linking the regulatory and catalytic domains. An expression vector for mouse CGKII was constructed and transfected into COS-1 cel ls where it directed the expression of a protein kinase which was acti vated by cGMP with an apparent K(a) of 300 nM cGMP.