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

Abstract

Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of CAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all the human R (RIa, RIP, RIIa and RIIP) and C (Ca, CP and Cy) subunits in a wheat-germ extract.[35S]Methionine-labelled in-vitro-translated products were analyzed by SDSPAGE and revealed distinct protein bands with apparent molecular masses of 49 (RIaj,[54][55], 51 (RIIa) and 53 kDa (RIIP) for the R subunits. In vitro transcriptiordtranslation of the cDNAs for the C subunits of PKA gave proteins with molecular masses of approximately 40 kDa for all the different C subunits. Phosphorylation of RIIa and RIIP by the C subunit of PKA, revealed a distinct mobility shift of the RIIa subunit on one-dimensional SDSPAGE (51 -54 kDa), but not of RIIP (53 kDaj. Further characterization of the R subunits by two-dimensional SDS/PAGE revealed that RIa was more acidic than RIP, with PIS of 6.1 -6.0 and 6.4-6.2, respectively. Furthermore, the RIIa protein was more basic than RIIP, with PIS of approximately 5.4-5.3 and 5.3-5.1, respectively, All the in-vitrotranslated R subunits could be photoaffinity labelled by the CAMP-analog 8-azido-[32P]cAMP and were also detected by immunoprecipitation with subunit-specific antibodies.The CAMP-dependent protein kinase (PKAj is in its holoenzyme form an inactive tetrameric complex, composed of two regulatory (Rj and two catalytic (Cj subunits. Upon binding of four molecules of cAMP to the R dimer, active monomeric C subunits are released from the holoenzyme complex [1]. Free C subunits phosphorylate serine and threonine residues on specific protein substrates which mediate the effects of hormones acting via cAMP/PKA [2]. Two types of PKA (types I and 11) can be distinguished based on their order of elution from anion-exchange resins [3]. The different charge properties of the type-I and type-I1 holoenzymes are primarily due to differences in the R subunits (RI and RIT) [4]. During the last few years, a more complex picture of PKA subunits has emerged. 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]. This study concluded that the mobilities of human RIIa and RIIP on SDS/ PAGE are inverted compared to those of other species such as rat and bovine in the dephosphorylated form....

Development of Platelet Inhibition by cAMP during Megakaryocytopoiesis

“…Four isoforms of the PKA regulatory subunit (PKA-R) have been found and named the RI␣, RI␤, RII␣, and RII␤ isoforms, respectively. They have apparent molecular masses of 49 (PKA-RI␣), 54 -55 (PKA-RI␤), 51 (PKA-RII␣), and 53 kDa (PKA-RII␤) (19). PKA is activated by the binding of two cAMP molecules to each of the regulatory subunits, thereby releasing and activating the catalytic subunits.…”

Protein Kinase D Induces Transcription through Direct Phosphorylation of the cAMP-response Element-binding Protein

“…The empty expression vector pSR and the plasmid pSR C␣, containing the catalytic C␣ subunit from PKA, were the kind gifts from Dr. T. Jahnsen and Dr. K. Taskén (46). The GST-CREB plasmid (amino acids 1-261 of CREB) was a kind gift from Dr. Michael Comb (47).…”

Perspective: Transposable Elements, Parasitic Dna, and Genome Evolution

“…The C␥ gene lacks introns, contains a remnant of a poly(A) tail, and is flanked by direct repeats. Given that this gene can be translated in vitro (Foss et al 1994), is expressed in a highly regulated manner (Beebe et al 1992), and is conserved in monkeys and humans, it is very likely to produce a functional protein. As the start of transcription of this gene is likely to be located outside the retrotransposed portion of the gene (in an Alu element), it is likely that its tissue specificity arose because of borrowed signals from its new position (See Fig.…”