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.     

Revertants of a trans-dominant S49 mouse lymphoma mutant that affects expression of cAMP-dependent protein kinase

Phenotypic revertants were isolated from an S49 mouse lymphoma tissue culture cell mutant that lacks cAMP-dependent protein kinase (cA-PK) activity (kin-). The mutant phenotype is trans-dominant and results from a lesion that probably lies outside the cA-PK subunit structural genes. The nature of the event that produces the kin- phenotype is unknown. However, the mechanism that is responsible for its behavior is genetically encoded because: spontaneous revertants arise at low frequency; reversion frequency is increased by mutagen treatment; mutagen-specific classes of revertant phenotypes are induced; and some revertants are temperature-sensitive for expression of cA-PK subunit polypeptides. Additional evidence is provided that argues against structural lesions in cA-PK catalytic (C) subunits as explanatory of the kin- phenotype. Kin- cells do not express an immunologically detectable C polypeptide, whereas C expression is restored in revertant cells. Revertants in which phenotype and cA-PK activity levels are only partially restored to that of wild-type cells contain a commensurately reduced amount of C polypeptide. Finally, the structure of C polypeptide in partial revertants is unaltered from that of wild-type C. The evidence supports the hypothesis that the kin- lesion defines a regulatory gene responsible for setting intracellular levels of cA-PK C subunit expression.     

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.     

Kinase-negative mutants of S49 mouse lymphoma cells carry a trans-dominant mutation affecting expression of cAMP-dependent protein kinase.

Kinase-negative mutants of S49 mouse lymphoma cells are pleiotropically negative for all known cAMP-mediated responses of S49 cells and yield cell extracts which are deficient in cAMP binding activity and devoid of cAMP-dependent protein kinase activity. In hybrids between kinase-negative and wild-type cells, the mutant phenotype is dominant: the tetraploid hybrids have reduced cAMP-binding activity and undetectable cAMP-dependent kinase activity. The mutant phenotype is attributable to neither a soluble inhibitor of kinase catalytic subunit, nor a defective kinase regulatory subunit acting as an inhibitor, nor a defective catalytic subunit which sequesters regulatory subunits in inactive complexes. We propose that these mutants carry trans-dominant lesions in a regulatory locus responsible for setting intracellular levels of kinase expression.     

A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the H118N C subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the alpha4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression.     

The causal relationship between mutations in cAMP-dependent protein kinase and the loss of adrenocorticotropin-regulated adrenocortical functions.

The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.     

Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase

We have used a previously characterized mouse cDNA clone for the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M. D., Carmichael, D. F., Lee, D. C., Chrivia, J. C., Krebs, E. G., and McKnight, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1300-1304), which we designate C alpha, to isolate cDNA clones coding for a second isoform of the C subunit, C beta. C alpha cDNA clones hybridize to a 2.4-kilobase mRNA on Northern blots whereas C beta cDNA clones detect a 4.3-kilobase mRNA. Nucleotide sequence comparison between C alpha and C beta cDNA clones shows that the C beta cDNA codes for a protein which shows 91% identity with C alpha. Determination of mRNA levels for C beta in various tissues shows that it is most highly expressed in brain although it is detectable in all tissues examined. The presence of two genes coding for the C subunit of cAMP-dependent protein kinase may explain past reports of heterogeneity in C subunit protein preparations.     

Recovery of cyclic nucleotide regulation in protein-kinase-defective adrenal cells through somatic cell fusion

A mutant cell line (designated Kin-8), isolated from the Y1 mouse adrenocortical tumor cell line on the basis of its resistance to growth-inhibition by 8-bromoadenosine 3', 5'-monophosphate (8BrcAMP), was resistant to the steroidogenic effects of the cyclic nucleotide analog and did not round up in the presence of 8BrcAMP as did responsive Y1 adrenal cells. In Kin-8 cells, the mutation to cyclic nucleotide resistance was associated with a defective type 1 cAMP-dependent protein kinase activity, suggesting an obligatory role for the enzyme in the regulation of these adrenal functions. In this study, the Kin-8 mutant was fused with a rat glioma cell line (C6) in order to analyze the genetic behavior of the protein kinase mutation in somatic cell hybrids. The growth of C6 glial cells was inhibited by 8BrcAMP and its cAMP-dependent protein kinase responded normally to cAMP. In addition, C6 cells had no capacity for steroidogenesis nor did they round up when treated with 8BrcAMP. In Kin-8 X C6 hybrids, the protein kinase mutation seemed to behave recessively. The growth of hybrid cells was inhibited by 8BrcAMP and the protein kinase responded to cAMP over a normal range. Kin-8 X C6 hybrids, when treated with 8BrcAMP, exhibited steroidogenic activities which were greater than the activity measured in either fusion partner and which had lower ED50 values for 8BrcAMP. In addition, Kin-8 X C6 hybrids rounded up in the presence of 8BrcAMP, a morphologic change unlike that seen with either fusion partner. The effects of 8BrcAMP on the steroidogenic activity and morphology of Kin-8 X C6 hybrids was reminiscent of the effects of the cyclic nucleotide on cAMP-responsive, parental Y1 adrenal cells. These results suggest that cell fusion provided a normal cAMP-dependent protein kinase for Kin-8 cells and led to the recovery of a cAMP-responsive adrenal phenotype. type. These results provide additional evidence in support of an obligatory role for cAMP-dependent protein kinase in the regulation of adrenal steroidogenesis, cell division, and cell shape.     

Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival

The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (alpha/beta), whereas three gene families (B, B', and B') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aalpha subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aalpha RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aalpha triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aalpha cDNAs rescued RNAi-induced loss of the C subunit, and Aalpha point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Abeta and Aalpha mutants capable of binding to at least one family of regulatory subunits were able to delay Aalpha RNAi-induced death of PC12 cells. However, only the expression of wild-type Aalpha restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aalpha subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.     

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.     

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.     

mRNA from mutant Y1 adrenal cells directs the synthesis of altered regulatory subunits of type 1 cAMP-dependent protein kinase

The molecular basis for altered cyclic AMP-dependent protein kinase activity was examined in three different mutant clones (Kin-1, Kin-7, and Kin-8) derived from the Y1 mouse adrenocortical cell line. Parental Y1 cells and the Kin mutants were labeled with L-[35S] methionine and the regulatory subunit of the type 1 cAMP-dependent protein kinase isozyme (RI) was immunoprecipitated from each clone with a specific guinea pig antiserum. When analyzed by electrophoresis on isoelectric focusing gels, the immunoprecipitates from mutant clones exhibited parental forms of RI plus an additional acidic variant form which likely accounted for altered cAMP-dependent protein kinase activity. Poly(A+) RNA was isolated from Y1 and Kin mutant cells and was translated in a cell-free, reticulocyte lysate system in the presence of L-[35S]methionine. The RI synthesized from poly(A+) RNA was immunoprecipitated from the translation mixture and analyzed on isoelectric focusing gels. The poly(A+) RNA from the Kin mutant clones directed the synthesis of parental and acidic variant forms of RI. These results suggest that the altered electrophoretic forms of RI arise from mutations in one of two RI genes rather than from post-translational modifications of the protein. The coexistence of parental and variant forms of RI in the Kin mutants indicate that the mutations are codominant.     

Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197.

We recently found, using cultured mouse cell systems, that newly synthesized catalytic (C) subunits of cyclic AMP-dependent protein kinase undergo a posttranslational modification that reduces their electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels and activates them for binding to a Sepharose-conjugated inhibitor peptide. Using an Escherichia coli expression system, we now show that recombinant murine C alpha subunit undergoes a similar modification and that the modification results in a large increase in protein kinase activity. Threonine phosphorylation appears to be responsible for both the enzymatic activation and the electrophoretic mobility shift. The phosphothreonine-deficient form of C subunit had reduced affinities for the ATP analogs p-fluorosulfonyl-[14C]benzoyl 5'-adenosine and adenosine 5'-O-(3-thiotriphosphate) as well as for the Sepharose-conjugated inhibitor peptide; it also had markedly elevated Kms for both ATP and peptide substrates. Autophosphorylation of C-subunit preparations enriched for this phosphothreonine-deficient form reproduced the changes in enzyme activity and SDS-gel mobility that occur in intact cells. A mutant form of the recombinant C subunit with Ala substituted for Thr-197 (the only C-subunit threonine residue known to be phosphorylated in mammalian cells) was similar in SDS-polyacrylamide gel electrophoresis mobility and activity to the phosphothreonine-deficient form of wild-type C subunit. In contrast to the wild-type subunit, however, the Ala-197 mutant form could not be shifted or activated by incubation with the phosphothreonine-containing wild-type form. We conclude that posttranslational autophosphorylation of Thr-197 is a critical step in intracellular expression of active C subunit.     

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.     

The alpha4-containing form of protein phosphatase 2A in liver and hepatic cells

The Ser/Thr phosphatase PP2A is a set of multisubunit enzymes that regulate many cellular processes. In yeast, the PP2A regulatory subunit Tap42 forms part of the target of rapamycin (TOR) signaling pathway that links nutrient and energy availability to cell growth. The physiological intersection between the mammalian orthologs of Tap42 and TOR, alpha4 and mTOR, has not been fully characterized. We used two in vivo models of liver growth in the rat, late gestation fetal development and regeneration after partial hepatectomy, to explore the regulation of the alpha4-containing form of PP2A. The alpha4/PP2A catalytic subunit (alpha4/PP2A-C) complex was present in both fetal and adult liver extracts. There was a trend towards higher levels of alpha4 protein in fetal liver, but the complex was more abundant in adult liver. Fractionation of extracts by ion exchange chromatography and transient transfection of the AML12 mouse hepatic cell line indicated that alpha4 associates with PP2A-C but that these complexes have low catalytic activity with both peptide and protein substrates. alpha4 was able to associate with forms of PP2A-C that were both methylated and non-methylated at the carboxy-terminus. The mTOR inhibitor rapamycin did not block the formation of alpha4/PP2A-C in liver or hepatic cells, nor did it appear to modulate PP2A activity. Furthermore, sensitivity to the growth inhibitory effects of rapamycin among a panel of hepatic cell lines did not correlate with levels of alpha4 or alpha4/PP2A-C. Our results indicate that the yeast Tap42/TOR paradigm is not conserved in hepatic cells.