Molecular Cloning of a Tissue-Specific Protein Kinase (C_γ) from Human Testis—Representing a Third Isoform for the Catalytic Subunit of cAMP-Dependent Protein Kinase

Abstract

Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.

Molecular cloning of a second form of rac protein kinase.

“…Through this approach, many protein kinases have been shown to exist not only as a single enzyme but as groups of closely related proteins. The best-characterized examples of such families are protein kinase C (PKC)1 (reviewed by Nishizuka, 1988: see also Osada et al, 1990;Bacher et al, 1991) and cyclic AMP (cAMP)-dependent protein kinase (cAMP-PK) (reviewed by Edelman et al, 1987;Hanks et al, 1988; see also Beebe et al, 1990). Subfamilies have now been identified for both serine/threonine-and tyrosine-specific protein kinases, including src, abl, insulin receptor, calcium-calmodulin-dependent protein kinase, and cdc2/ CDC28 protein kinases (reviewed by Hanks, 1991).…”

“…LGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEA (Jones et al, 1991), human rac-PK,B, rabbit PKCa (Ohno etal., 1987), rabbit PKC-y (Ohno et al, 1987), rat PKCE (Ono et al, 1988), rat PKCR (Ono et al, 1988), murine PKCi7 (Osada et al, 1990), murine PKC-L (Bacher et al, 1991), bovine cAMP-PK Ca (Shoji et al, 1983), bovine cAMP-PK C,B (Showers and Maurer, 1986), human cAMP-PK Cy (Beebe et al, 1990), bovine cGMPdependent protein kinase (cGMP-PK) (Takio et al, 1984), and rat S6 kinase (Kozma etal., 1990) were used in a pairwise progressive alignment (Feinberg and Doolittle, 1987) using the algorithm of Needleman and Wunsch (1970) (PILEUP,GCG version 7.0,B). The dendrogram represents these pairwise alignments where the horizontal distance is a measure of the relatedness of the sequences.…”

Transmembrane signalling in eukaryotes: a comparison between higher and lower eukaryotes

“…Three different mammalian genes for the C subunit of PKA have been identified, C~, C a and C~. which are 80~o similar to each other [ 14]. The conserved protein kinase catalytic domain comprises most of the PKA C subunit, from amino acid residue 40 to 300 (see Fig.…”

Characterization of in‐vitro‐translated human regulatory and catalytic subunits of cAMP‐dependent protein kinases

“…Four different R subunits, cloned. cDNA clones for all the different R and C subunits from human testis have been isolated and sequenced in our laboratory [13,[16][17][18][19].We have previously identified and characterized the RIIa and RIIP subunits in rat tissues, showing apparent molecular masses of 54 kDa and 52 kDa, respectively [8,20, 211. However, characterization of purified RII proteins from human testis revealed subunit sizes of 51 kDa and 53 kDa, representing RIIa and RIIP, respectively [22].…”

“…In addition, two splice variants of C subunits, designated Cu2 [14] cloned. cDNA clones for all the different R and C subunits from human testis have been isolated and sequenced in our laboratory [13,[16][17][18][19].…”