Decreased expression of the type I isozyme of cAMP-dependent protein kinase in tumor cell lines of lung epithelial origin

A spontaneous transformant derived from a mouse lung epithelial cell line exhibited decreased cAMP-dependent protein kinase (PKA) activity. DEAE column chromatography demonstrated that this was caused by specific loss of the type I PKA isozyme (PKA I). Western immunoblot analysis indicated that indeed several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenic lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines. PKA II regulatory subunit protein differed only slightly among cell lines and showed no conspicuous trend between normal and neoplastic cells. The decrease in RI was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography. Northern blot analysis using RI alpha and C alpha cDNA probes showed that the levels of RI alpha and C alpha mRNAs paralleled their intracellular protein concentrations; neoplastic cell lines contained significantly less RI alpha and C alpha mRNAs than the normal cell line. The decreased expression of both RI and C subunits therefore results in a net decrease of PKA I in neoplastic lung cells, an isozymic difference which may account for the differential effects of cAMP analogs on cell growth and differentiation in normal and neoplastic cells.     

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.     

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.     

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.     

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.     

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.     

Differential expression and subcellular localization for subunits of cAMP-dependent protein kinase during ram spermatogenesis

The expression of mRNAs for the RI alpha, RII alpha, and C alpha subunits of cAMP-dependent protein kinase has been studied in different ram germ cells. The sizes of the specific RI alpha, RII alpha, and C alpha mRNAs, observed in germ cells were 1.6, 2.0, and 2.6 kb, respectively. RI alpha and C alpha mRNAs were mainly expressed in primary spermatocytes. A postmeiotic expression predominating in early spermatids was unique to RII alpha mRNA. The location of RI, RII alpha, and C subunits in well-defined organelles of ram spermatids and epididymal sperm was assessed by immunogold electron microscopy. In spermatids, RI, RII alpha, and C were essentially present in the forming acrosome and, to a lesser extent, in the nucleus. During sperm epididymal maturation, the protein kinases disappeared from the acrosome and were detected in a variety of sperm functional areas, such as the tip of the acrosome, the motility apparatus, and the membrane network. The present study on subunits of cAMP-dependent protein kinase supports the concept that specific functions are attached to the different subunits in that it shows differential expression and differential subcellular localization in germ cells.