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.     

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.     

Decreased catalytic subunit mRNA levels and altered catalytic subunit mRNA structure in a cAMP-resistant Chinese hamster ovary cell line.

The mechanisms responsible for decreased levels of cAMP-dependent protein kinase activity in a mutant Chinese hamster ovary cell line have been examined. The cAMP-resistant Chinese hamster ovary 10260 cell line was found to possess only 20% of the cAMP-dependent protein kinase activity found in wild-type cells. The presence of decreased concentrations of the catalytic subunit in these cells was confirmed through binding studies using a radiolabeled, heat-stable inhibitor of the kinase. Cloned Chinese hamster ovary catalytic subunit cDNAs were isolated, characterized, and used as hybridization probes to examine the relative concentrations of catalytic subunit mRNAs in the wild-type and 10260 cell lines. A 40-50% decrease in the concentration of the mRNA for the C alpha isozyme of the catalytic subunit was observed in 10260 cells, as compared with wild-type. This decrease in catalytic subunit mRNA concentration probably accounts for a portion of the decreased kinase activity in the mutant cells. Further analysis of C alpha mRNA by polymerase chain reaction confirmed the decreased expression of C alpha mRNA in 10260 cells and further demonstrated the presence of two different species of C alpha mRNA in the 10260 cells. One species of C alpha cDNAs was indistinguishable from the wild-type cDNA, but the other species was shorter. Nucleotide sequence analysis of the amplified cDNAs led to the identification of a 191-base pair deletion in the shorter cDNA. Gene transfer studies using wild-type and 10260 C alpha cDNAs demonstrated that the longer cDNA from the 10260 cells produced wild-type activity, but the shorter cDNA was inactive. These studies suggest that at least two alterations in gene expression are responsible for decreased cAMP-dependent protein kinase activity in the 10260 cell line. One alteration results in an approximately 2-fold decrease in the concentrations of C alpha mRNA in the cells. The other change produces two species of C alpha mRNA; one of the C alpha mRNAs does not encode an active kinase.     

Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: Levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIα) was present in all the cells investigated, in contrast to the other R subunits (RIβ, RIIα, and RIIβ). Furthermore, three T cell lines and one B cell line expressed only RIα and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIα protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [³H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes. J. Cell. Physiol. 177:85–93, 1998. © 1998 Wiley-Liss, Inc.     

Cyclic AMP-dependent protein kinase isoenzymes in human myeloid leukemia (HL60) and breast tumor (MCF-7) cells

Combinations of retinoic acid (RA) and cAMP mediate many biological responses in a large variety of cell types. While the basis for the apparent synergistic effects of RA and cAMP are not clearly defined, it is likely that activation of PKA by cAMP is involved. However, literature reports concerning the identity of PKA isoforms in HL60 and MCF-7 cells are conflicting. The purpose of the present investigation is to identify PKA isoforms in HL60 and MCF-7 cells. Utilization of high-performance anion-exchange liquid chromatography, immunoblotting, and 8-azido-cAMP photoaffinity binding resulted in the finding that HL60 cells contain PKA types I alpha and II alpha, while MCF-7 cells contain PKA types I alpha, II alpha, and II beta. PKA type I alpha in both HL60 and MCF-7 cells eluted from columns as two well-separated peaks. One peak eluted at a low salt concentration in agreement with previous reports. The second HL60 PKA type I alpha peak eluted at a salt concentration intermediate between that eluting the first peak and that eluting PKA type II alpha and contained approximately 62% of the total RI alpha protein. However, the second MCF-7 PKA type I alpha peak contained approximately 66% of the total RI alpha protein and co-eluted with PKA types II alpha and II beta. This "contamination" of PKA type II fractions with PKA type I has led, in some cases, to interpretations that may need reevaluation.     

Characterization of two mutants of the LLC-PK1 porcine kidney cell line affected in the catalytic subunit of the cAMP-dependent protein kinase

The catalytic (C) subunit activity of the cAMP-dependent protein kinase (cAMP-PK) from the mutant cell lines, FIB4 and FIB6, is only 10% compared with the parent cell line, LLC-PK1 [Jans and Hemmings (1986) FEBS Lett. 205, 127-131]. In order to understand the nature of the mutant phenotypes the cAMP-PK from parent and mutant cell lines was studied in more detail. Analysis of mutant cAMP-PK activity by ion-exchange chromatography revealed that kinase activity associated with type I holoenzyme of both FIB4 and FIB6 was only 5% parental, and the activity of the type II holoenzyme was about 20% parental. The type I regulatory (RI) subunits associated with the type I were also found to be reduced by 70-80% in both mutants, whereas the type II R subunit levels were similar to that of the parent. The residual kinase activity associated with the type I holoenzyme from FIB4 and FIB6 could not be activated by cAMP whereas the type II holoenzyme was activated by cAMP (Ka of 5.5 X 10(-8) M), and showed normal affinities for Kemptamide and ATP. A polyclonal antibody to the catalytic subunit was used to quantify the level of this protein in wild-type and mutant cells. This analysis showed that FIB4 and FIB6 had nearly normal levels of C subunit, suggesting that the C subunit synthesized by the mutants was mostly inactive. As both type I and type II cAMP-PK holoenzymes were abnormal, the most likely explanation of the mutant phenotype is a defect either in the structural gene for the C subunit or in an enzyme involved in its posttranslational processing. However, a second lesion affecting the RI subunit cannot be ruled out at this moment.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

Cellular concentrations of protein kinase A modulate prostaglandin and cAMP induction of alkaline phosphatase.

The relationship between the concentration of cAMP-dependent protein kinase (PKA) activity and the induction of alkaline phosphatase (AP) was examined in transfected L cell lines with altered PKA levels. C alpha 12 cells were generated by transfecting mouse L cells with an expression vector coding for the mouse C alpha catalytic subunit of PKA and were shown to contain 2.5-fold more PKA activity than L cells. RAB10 cells were generated by transfection with an expression vector for a mutant regulatory subunit and had 10-fold lower levels of PKA activity than L cells. AP induction by 8-chlorophenylthio-cAMP (CPT-cAMP) was found to be 2-fold greater in C alpha 12 cells than in L cells, while RAB10 cells lacked any induction of AP in response to CPT-cAMP. Northern blot and solution hybridization analyses of AP mRNA showed that induced AP mRNA levels were comparable in C alpha 12 and in L cells. Western blot analysis demonstrated that AP protein levels were greater in C alpha 12 cells and suggested that the increased AP protein level resulted from either increased stability of the AP protein or increased rate of translation of the AP mRNA. In contrast, Northern blot analysis of the RAB10 cells failed to detect AP mRNA after CPT-cAMP treatment and suggested that PKA is required for induction of AP mRNA. Stimulation of endogenous cAMP levels by treatment with prostaglandin E1 gave similar effects on AP activity as those seen with CPT-cAMP. These results indicate that cellular levels of PKA can determine the magnitude of cellular response to hormonal stimulation and also suggest that PKA can regulate AP gene expression at both the level of the AP mRNA and AP protein.     

Regulation of lung epithelial cell morphology by cAMP-dependent protein kinase type I isozyme

Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIalpha mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Calpha catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy.     

Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF. Mol. Reprod. Dev. 50:79–85, 1998. © 1998 Wiley-Liss, Inc.     

Oligonucleotide sequence-specific inhibition of gene expression, tumor growth inhibition, and modulation of cAMP signaling by an RNA-DNA hybrid antisense targeted to protein kinase A RIalpha subunit

The primary mediator of cAMP action in mammalian cells is cAMP-dependent protein kinase (PKA). There are two types of PKA, type I (PKA-I) and type II (PKA-II), which share a common catalytic subunit but contain distinct regulatory subunits, RI and RII, respectively. Evidence suggests that increased expression of RIalpha/PKA-I correlates with neoplastic cell growth. Here, we show that sequence-specific oligonucleotide inhibition of RIalpha expression results in inhibition of growth and modulation of cAMP signaling in cancer cells. The antisense promoted growth inhibition in a time-dependent, concentration-dependent, and sequence-dependent manner in human cancer cells in monolayer culture, and it inhibited colony formation in soft agar and tumor growth in nude mice. Among the cancer cells are LS-174T, HCT-15, and Colo-205 colon carcinoma cells; A-549 lung carcinoma cells; LNCaP prostate adenocarcinoma cells; Molt-4 leukemia cells; and Jurkat T lymphoma cells. Northern blot and immunoprecipitation analyses revealed that the growth inhibitory effect of the antisense correlated with a decrease in RIalpha expression at both the mRNA and protein levels. Pulse-chase experiments revealed that the antisense-directed inhibition of RIalpha expression resulted in compensatory changes in expression of the isoforms of R and C subunits and cAMP signaling in a cell type-specific manner. These results demonstrate that cAMP is ubiquitous in the regulation of cell growth and that the antisense oligonucleotide, which inhibits the synthesis of the RIalpha subunit of PKA, can be targeted to a single gene for treatment of cancer in a variety of cell types.     

A kinase-negative mutant of S49 mouse lymphoma cells is defective in posttranslational maturation of catalytic subunit of cyclic AMP-dependent protein kinase.

Kinase-negative mutants of S49 mouse lymphoma cells, which lack detectable catalytic (C) subunit of cyclic AMP-dependent protein kinase, nevertheless contain cytoplasmic mRNAs for the two major forms of C subunit, C alpha and C beta. Investigation of the metabolism of C subunits in wild-type and mutant cells was undertaken to identify the step(s) at which C subunit expression was defective in kinase-negative cells. [35S]methionine-labeled C subunits from cytosolic fractions of wild-type S49 cells or C subunit-overexpressing cell lines were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after purification by either affinity chromatography using a peptide inhibitor of C subunit as the ligand or immunoadsorption with an anti-C subunit antiserum. Immunoadsorption revealed electrophoretic forms of C alpha and C beta subunits that migrated faster than those detected in affinity-purified samples; this unexpected heterogeneity suggested that functional activation of C subunit may require posttranslational modification. Immunoadsorption of cytosolic fractions from wild-type cells labeled for various times with [35S]methionine revealed an additional posttranslational maturation step. The bulk of immunoadsorbable C subunit label in cells pulse-labeled for 5 min or less was in an insoluble fraction from which it could be solubilized with a detergent-containing buffer; solubilization of the newly synthesized material proceeded over an incubation period of about 10 min. The primary defect in kinase-negative cells appeared to be in this solubilization step, since about equal C subunit radioactivity was found in detergent extracts of wild-type and kinase-negative cells but very little was found in mutant cytosols. I speculate that an accessory factor required for proper folding of newly synthesized C subunit in defective in the kinase-negative cells.     

Protein kinase C activation selectively increases mRNA levels for one of the regulatory subunits (RI alpha) of cAMP-dependent protein kinases in HT-29 cells

We have examined the effect of the protein kinase C activator, TPA, on mRNA levels for subunits of cAMP-dependent protein kinases in the human colonic cancer cell line HT-29, subline m2. Messenger RNA for the regulatory subunit, RI alpha, of cAMP-dependent protein kinases was shown to be present and regulated by TPA. Other mRNAs for subunits of cAMP-dependent protein kinases (RI beta, RII alpha, RII beta, C alpha, C beta) were also present in these cells, but revealed no or only minor changes upon TPA stimulation. When HT-29 cells were cultured in the presence of 10 nM TPA for various time periods, a biphasic response was observed in RI alpha mRNA levels with a maximal increase (approximately 4 fold) after 24 hours. TPA stimulated RI alpha mRNA increased in a concentration-dependent manner and maximal response (4-8 fold) was seen at 3-10 nM. The TPA-induced increase in RI alpha mRNA was not obtained when cells were incubated with TPA together with the protein kinase C inhibitors, staurosporine or H7. The cAMP-analog 8-CPTcAMP alone induced RI alpha mRNA levels 50% more than TPA. Combined treatment with TPA (10 nM) and 8-CPTcAMP (0.1 mM) gave an increase in RI alpha mRNA similar to TPA. These results demonstrate an interaction between the protein kinase C pathway and mRNA levels for the RI alpha subunit of cAMP-dependent protein kinases in HT-29 cells.     

Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.