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Biomedical subjects

G S McKnight

Publications and source records attributed to G S McKnight.

At least 19 recordsLinked to original sources
PubMed1995

Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.

Animals↗
PubMed1995

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

Base Sequence↗
PubMed1992

Mutations in the catalytic subunit of cAMP-dependent protein kinase result in unregulated biological activity.

Mutations were identified in the catalytic subunit (C) of the cAMP-dependent protein kinase (EC 2.7.1.37) that block inactivation by regulatory subunit (R) without compromising catalytic activity. Randomly mutagenized mouse C expression vectors were screened functionally for clones that stimulated gene induction in the presence of excess R. Point mutations in the C coding sequence were identified that result in a His----Gln substitution at amino acid 87 (His87Gln) and a Trp----Arg change at amino acid 196 (Trp196Arg). In contrast to wild-type C, both mutants retained partial activity in the presence of excess R isoform RI alpha, although only Trp196Arg retained partial activity in the presence of excess R isoform RII alpha. A C expression vector that included both mutations was fully active in promoting gene induction and was virtually unaffected by an 80-fold excess of either RI alpha or RII alpha. These results demonstrate that mutations at His-87 and Trp-196 alter R interactions with C at a site that is not involved in substrate recognition or enzymatic activity. In contrast to these randomly generated mutations, a site-specific alteration of the autophosphorylated Thr-197 to an Ala resulted in an 80% loss of biological activity and partial resistance to R inhibition. The location and proximity of His-87 and Trp-196 in the crystal structure of C suggest a surface domain that may interact with a region of R that is outside of the substrate/pseudosubstrate site.

Amino Acid Sequence↗
PubMed1992

Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells.

Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.

Adrenal Cortex↗
PubMed1992

Promoter for the regulatory type I beta subunit of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase directs transgene expression in the central nervous system.

Cyclic AMP-dependent protein kinase (cAPK) modulates synaptic transmission and influences memory and learning. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian cAPK, only the regulatory type I beta (RI beta) subunit is unique to nervous tissue. The requirement for RI beta in neurons is presently unknown. Previous studies demonstrate that holoenzyme containing RI beta activates at lower concentrations of cAMP compared to other forms of cAPK. Thus, neurons that induce RI beta expression may become more sensitive to subsequent hormonal signals and maintain more long-term phosphorylation events. To further elucidate the function of this novel protein, we have begun to investigate its gene. Here we report the isolation of the mouse RI beta promoter as determined by S1 nuclease analysis and transgenic mouse expression. A beta-galactosidase fusion gene containing 1.5 kilobases of 5'-nontranscribed RI beta DNA and 2 kilobases of intron 1 was expressed preferentially in the cortex and hippocampus of the brain and within the spinal cord. In addition to mimicking the location of endogenous RI beta expression, the transgene was activated at a similar time (embryonic day 11.5) during mouse fetal development. Isolation of the RI beta promoter will help identify the elements that direct transcription in a subset of neurons and illuminate the physiological conditions that may regulate RI beta expression. This promoter can also be used to target the expression of wild type and mutant cAPK subunit genes in order to investigate synaptic plasticity in animals.

Animals↗
PubMed1991

Hormonal activation of gene transcription in ras-transformed NIH3T3 cells overexpressing RII alpha and RII beta subunits of the cAMP-dependent protein kinase.

ras-Transformed NIH3T3 (R3T3) cells were transfected with expression vectors for the RII alpha and RII beta regulatory subunits of the type II isozyme of cAMP-dependent protein kinase, and the effects on gene activation by corticotropin-releasing factor (CRF) and prostaglandin E1 (PGE1) were analyzed. In RII alpha and RII beta-overexpressing cells, type II isozyme levels were increased, and type I isozyme levels were eliminated, demonstrating that both RII regulatory subunits compete efficiently with RI for catalytic subunit. The type II isozyme separated into three peaks on high performance liquid chromatography, referred to as A, B, and C. Western blot analysis strongly suggests that peak A and peak C correspond to holoenzymes containing RII beta and RII alpha, respectively. Overexpression of RII alpha resulted in the loss of peak A and a dramatic reduction in RII beta protein with no change in RII beta mRNA, indicating that the level of RII beta protein is controlled posttranscriptionally and that RII beta protein may become unstable when displaced from C. The role of type I and II kinases in transcriptional activation was investigated by comparing the response of control and RII expressing clones to site-selective cAMP analogs and the hormones, CRF and PGE1. The site-selective analogs demonstrated that either type I or type II kinase could activate the cAMP-responsive alpha-subunit promoter. The response to various concentrations of CRF or PGE1 was identical in control cells and transfected clones containing very little type I kinase. These experiments suggest that in the CRF and PGE1 response pathways leading to gene induction, the magnitude and sensitivity of the response are not influenced by the presence or absence of type I cAMP-dependent protein kinase.

3T3 Cells↗
PubMed1991

A constitutively active holoenzyme form of the cAMP-dependent protein kinase.

The major function of the regulatory (R) subunit of the cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R.C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RII) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RII and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RII in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RII and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RII could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RII.cAMP was increased by addition of the C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RII subunit may interact with the C subunit in a cAMP-dependent manner.

Amino Acid Sequence↗
PubMed1991

Cyclic AMP second messenger systems.

Cells carefully regulate the generation and destruction of cAMP using diverse families of adenylate cyclases and phosphodiesterases. Genes for several cyclases have now been cloned, giving structural information about the enzymes and providing access to the remaining members of this family. A much larger family of phosphodiesterases has been uncovered and the regulatory properties of both the cyclases and phosphodiesterases provide diverse mechanisms to modulate intracellular cAMP. Most of the actions of cAMP are mediated through phosphorylation of substrates of the cAMP-dependent protein kinases. Recent progress has helped define the pathway between cAMP and the activation of gene transcription.

Adenylyl Cyclases↗
PubMed1991

Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.

Animals↗
PubMed1990

Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides.

Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties. We have used recombinant techniques to express and purify the type I regulatory subunits, RI alpha and RI beta, the catalytic subunits C alpha and C beta, and then reconstituted holoenzymes with the various combinations of R and C subunits. The ability of the subunits to form inactive holoenzymes and then to be activated in the presence of cyclic nucleotides was examined. Holoenzymes containing C beta had essentially the same activation properties exhibited by C alpha holoenzymes. However, the presence of the neural form of RI, RI beta, led to formation of a holoenzyme which was activated at a 3-7-fold lower concentration of cyclic nucleotides compared to holoenzymes containing RI alpha. Expression of the RI beta protein in discrete regions of the central nervous system may provide a mechanism for increasing the sensitivity of the kinase to what would otherwise be subthreshold levels of stimulation. Two mutant forms of RI beta were constructed that converted the RI beta sequence to that of RI alpha at position 98 (RI beta Ala) or positions 98 and 99 (RI beta Ala/Ile). These sequences form part of a pseudosubstrate site thought to interact with the C subunit. Wild type and mutant R subunits were combined in vitro with purified bovine C subunits and half maximal activation constants (Ka) were determined with cyclic nucleotides. Holoenzymes containing RI beta Ala and RI beta Ala/Ile gave Ka values which were higher than wild type RI beta, with the double mutant shifting toward the Ka value of RI alpha holoenzymes by about 30%. These results suggest that amino acid differences in the pseudosubstrate site may account for some, but not all, of the increased sensitivity to cyclic nucleotides exhibited by RI beta.

Amino Acid Sequence↗
PubMed1990

The S49 Kin- cell line transcribes and translates a functional mRNA coding for the catalytic subunit of cAMP-dependent protein kinase.

The S49 mouse lymphoma mutant cell line Kin- is resistant to the cytotoxic effects of elevated cAMP levels, has no detectable cAMP-dependent protein kinase activity, and has depressed levels of cAMP-binding regulatory subunits. We demonstrate that although the Kin- cell line lacks detectable catalytic subunit protein, these cells express wild-type levels of mRNA for both C alpha and C beta catalytic subunit isoforms. Translation of C alpha mRNA appears to be normal in the Kin- cell, based on the observation that C alpha mRNA associates with large polyribosomes in both wild-type and Kin- cells. We cloned the C alpha cDNA from Kin- cells and show that its transient expression in another cell type leads to activation of a cAMP-sensitive luciferase reporter gene, suggesting that functional C alpha protein is made. In addition to having catalytic activity, the C alpha subunit from Kin- cells is inhibited in the presence of mouse RI alpha regulatory subunit, indicating that formation of the holoenzyme complex is normal. We suggest that the mutation responsible for the Kin- phenotype is in a cellular component that directly or indirectly causes Kin- catalytic subunit protein to be degraded rapidly.

Animals↗
PubMed1990

Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase.

Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway.

Biological Transport↗
PubMed1990

Subunits of cyclic adenosine 3',5'-monophosphate-dependent protein kinase show differential and distinct expression patterns during germ cell differentiation: alternative polyadenylation in germ cells gives rise to unique smaller-sized mRNA species.

Cyclic AMP (cAMP) and cAMP-dependent protein kinases (PKAs) are believed to be involved in the regulation of essential spermatozoal functions, such as motility, epididymal maturation, capacitation, and the acrosome reaction. In this study, we document the presence of significant mRNA levels for 5 different PKA subunits (RI alpha, RI beta, RII alpha, RII beta, and C alpha) in germ cells and demonstrate differential expression patterns for these subunits during spermatogenesis. Messenger RNAs for RI (RI alpha and RI beta) and C alpha appear to be induced at premeiotic germ cell stages, whereas mRNAs for RII (RII alpha and RII beta) are first expressed at haploid stages. The individual PKA subunits may convey specific functions in developing germ cells and mature sperm. The present study, furthermore, demonstrates the presence of unique smaller-sized mRNAs in germ cells compared with somatic cells. Specific, truncated forms of RI alpha, RII alpha, RII beta, and C alpha mRNAs appear to be selected in the germ cells. Our data suggest this to be due to the use of alternative polyadenylation site signals. The selection of shorter mRNA species, with higher stability, may be essential for the delayed translation observed in spermatids. This may ensure certain levels of mRNA for translation at late spermatid stages, after cessation of transcription.

Adolescent↗
PubMed1989

Overexpression of the type II regulatory subunit of the cAMP-dependent protein kinase eliminates the type I holoenzyme in mouse cells.

Mammalian tissues and cell lines express two major types of cAMP-dependent protein kinase, PKA-I and PKA-II, which can be distinguished at the molecular level by the presence of either type I or type II regulatory subunits in the holoenzyme. An expression vector for the mouse type II regulatory subunit (RII alpha) was transfected into ras-transformed NIH3T3 (R3T3) cells, which contain approximately equal amounts of both holoenzymes, PKA-I and PKA-II. In RII alpha-overexpressing R3T3 cells, PKA-II levels were increased, and the level of PKA-I declined. The decrease in PKA-I was dependent on the amount of RII alpha expressed, and at high levels of RII alpha expression, PKA-I was completely eliminated. In contrast, overexpression of the type I regulatory subunit (RI alpha) did not alter PKA isozyme levels. We propose that competition between RII alpha and RI alpha for a limited pool of catalytic subunit results in preferential assembly of PKA-II and that significant amounts of PKA-I are formed only if catalytic subunit is present in excess of the RII alpha subunit. The PKA-I isozyme, which is absent in untransformed 3T3 cells, is not essential for the transformed phenotype of R3T3 cells. RII alpha-overexpressing R3T3 cells that are devoid of PKA-I continued to exhibit a transformed phenotype including anchorage-independent growth. Overexpression of RII alpha provides a genetic approach that may prove useful in demonstrating specific functions for the two PKA isozymes in cAMP-dependent signal transduction pathways.

Animals↗
PubMed1989

A mutation in the catalytic subunit of protein kinase A prevents myristylation but does not inhibit biological activity.

The catalytic of subunit of cAMP-dependent protein kinase is acylated at its NH2 terminus with myristic acid. This type of modification is thought to mediate the association of proteins with lipid bilayers, yet the catalytic subunit shows no preferential binding with membranes. We investigated the role of C subunit myristylation using a cDNA expression vector in which the acylated NH2-terminal Gly was mutagenized to Ala. Protein synthesized in NIH 3T3 cells from this modified gene did not incorporate [3H]myristate. However, the kinase activity of this altered C subunit on a synthetic substrate was not diminished, nor was its ability to form holoenzyme with regulatory subunit. Non-myristylated C subunit also regulated several biological processes occurring in specific subcellular compartments; mutant C subunit stimulated dramatic cell shape changes controlled by the cytoskeleton, restored steroidogenesis in the mitochondria of defective adrenocortical cells, and effectively induced the transcription of genes in the nucleus. These results suggest that myristylation is nonessential for C subunit conformation and enzyme activation, and is not required for C subunit interaction with other proteins in regions where C is thought to localize upon activation. The purpose for NH2-terminal myristylation of this important signal-transducing enzyme remains an enigma.

Animals↗