Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase.

Abstract

ABSTRACTmRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 animino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using singlestranded RNA probes prepared with SP6 polymerase.Many hormones exert their effects on cellular metabolism and gene expression by regulating the intracellular level of cAMP, which in turn regulates the activity of cAMPdependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) (1-3). The holoenzyme, consisting of two identical regulatory (R) and two catalytic (C) subunits, dissociates when each of the R subunits binds two cAMP molecules (3). The C subunit phosphorylates serine and threonine residues in substrate proteins involved in mediating the biological response to hormonal stimulation. Although many of the effects of cAMP-dependent protein kinase on cellular metabolism involve phosphorylation of substrate proteins by the C subunit (3), there is evidence that the free R subunits also may play a direct role in the regulation of cellular functions (4, 5).There are at least three types of cAMP-dependent protein kinase: type I, which is the predominant form in bovine skeletal muscle; type II, which is the major form in bovine heart; and a neural type II, which is found in bovine brain (2,6). The difference between the various types ofprotein kinase lies in the type of R subunit associated with C subunit. Although three forms of the C subunit have been characterized by isoelectric focusing, C subunits from both types of cAMP-dependent protein kinase have otherwise similar physical and enzymatic properties (1, 7). In addition, the structure of the R and C subunits appears to have been conserved over the course of evolution, since a functional enzyme can be reconstituted using yeast C subunit and bovine R subunit (8).The C subunit of cAMP-dependent protein kinase has been studied extensively. The complete amino acid sequence of the bovine heart C subunit has been published (9), and some of the amino acid residues that form the C site have been identified by photoaffinity labeling (10, 11). These active-site residues include a lysine at position 72, which oc...

Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation

“…A targeting vector containing three loxP sites was created to allow tissue-specific expression of a C␣M120A mutation that confers sensitivity to inhibition by 1NM-PP1. The targeting vector also contains a neomycin phosphotransferase cassette (NEO) and a minigene containing exons 5-10 of the C␣ cDNA followed by a poly(A) addition site from the human growth hormone gene (29). The presence of NEO allows positive selection of transfected ES cells in culture and the minigene was designed to allow expression of wild-type C␣ in vivo in C␣LoxM120A animals (Fig.…”

Structural insights into mis-regulation of protein kinase A in human tumors

“…Overexpression of the PKA C subunit indeed contributes to disease, as discovered with copynumber gains for PRKACA in some cases of adrenal hyperplasia (8). In FL-HCC, a significant increase in C-subunit levels may favor formation of additional type-I holoenzyme complexes as increases in levels of RI, but not RII, are known to compensate for increased C-subunit levels (26). In the context of cancer, type I rather than type II holoenzymes are known to be associated with growth stimulation (27).…”

Mouse models of altered protein kinase A signaling

“…Of these two isoforms, there also exist splice variants that have similarities to those found in humans. The Ca subunit consists of two splice variants defined as Ca1 and Ca2, also referred to as Ca-s (Uhler et al 1986a,b, San Agustin & Witman 2001. Although the Ca1 isoform is expressed ubiquitously in mice, the expression of Ca2 is restricted to male germ cells, and it was shown that Ca2 is the only catalytic subunit of PKA expressed in mature sperm (Desseyn et al 2000).…”

“…Although the Ca1 isoform is expressed ubiquitously in mice, the expression of Ca2 is restricted to male germ cells, and it was shown that Ca2 is the only catalytic subunit of PKA expressed in mature sperm (Desseyn et al 2000). Cb, on the other hand, has three splice variants -Cb1, Cb2, and Cb3 (Uhler et al 1986a,b, Guthrie et al 1997. Cb1 is ubiquitously expressed, albeit at lower levels than Ca1, however, Cb2 and Cb3 expressions are limited to the brain.…”