Regulation of the Human Enkephalin Promoter by Two Isoforms of the Catalytic Subunit of Cyclic Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase

Abstract

Cyclic AMP regulates a variety of cellular responses through activation of the catalytic subunit of cAMP-dependent protein kinase. The cDNAs for two protein isoforms of the catalytic subunit, C alpha and C beta, were placed into expression vectors, and their ability to stimulate cAMP-dependent transcription of the human enkephalin promoter was examined in transiently transfected CV-1 cells. Expression vectors for C alpha and C beta that were directed by the human cytomegalovirus promoter produced up to 350- and 200-fold increases in chloramphenicol acetyltransferase activity, respectively, when cotransfected with the ENKAT-12 reporter plasmid. Transcriptional activation was shown to be dependent upon functional kinase activity by point mutations in catalytic subunit vectors which eliminated activation. Transcriptional activation by C alpha and C beta was eliminated when the cAMP response elements (CREs) were deleted from the native enkephalin promoter, but activation was recovered when this region was replaced with an oligonucleotide containing two copies of the somatostatin CRE consensus TGACGTCA. C alpha expression vectors were found to produce 2-fold greater transcriptional activation than C beta expression vectors. These results were most likely due to the cellular kinase activity produced by the catalytic subunit expression vectors and did not appear to be dependent on CRE motif or substrate specificity. In vitro mutagenesis indicates that neither C alpha nor C beta requires N-terminal myristylation for transcriptional activation, but threonine-197 is critical to subunit function.

LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

“…The N-terminal sequence contains little sequence similarity to known proteins. The function of the N-terminus is unknown ; however, small N-terminal sequences of other kinases have been shown to be important in properly positioning the glycine-rich loop and stabilizing the enzyme [29,30]. Whereas the mLKB1 kinase domain shows high amino acid identity to the kinase domains of hLKB1 and XEEK1 (Figure 1), it is only distantly related to the kinase domains of other protein kinase families.…”

Cyclic AMP- and Cyclic GMP-dependent Protein Kinases Differ in Their Regulation of Cyclic AMP Response Element-dependent Gene Transcription

“…The resulting 2100-bp PCR fragment containing the coding region of murine cGKI␤ flanked by a BglII site and a BamHI site was digested with BglII and BamHI and ligated into the BglII site of pCMV.Neo (36) to create pCMV.mcGKI␤. During sequencing of cDNA clone mcGKI␤3D, a two-nucleotide deletion ( Fig.…”

Differential Regulation of Chromogranin B and Synapsin I Gene Promoter Activity by cAMP and cAMP‐Dependent Protein Kinase

“…The plasmid pCMVCa contained the cDNA of the catalytic subunit of PKA under control of the cytomegalovirus (CMV) immediate-early gene promoter (Huggenvik et al, 1991). This plasmid was a kind gift of Michael D. Uhler (University of Michigan, Ann Arbor, Michigan).…”