The Localization and Activity of cAMP-dependent Protein Kinase Affect Cell Cycle Progression in Thyroid Cells*

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases (PKAs), typically determined by their specific regulatory subunits. We describe changes in the cAMP signal transduction pathway during cell cycle progression in synchronized rat thyroid cells. Both PKA type II (PKAII) localization and nuclear cAMP signaling are significantly modified during G0 and G1-S transitions. G1 is characterized by PKA activation and amplified cAMP signal transduction. This is associated with a decrease in the concentration of RI and RII regulatory subunits and enhanced anchoring of PKAII to the Golgi-centrosome region. Just prior to S, the cAMP pathway is depressed. Up-regulation of the pathway by exogenous cAMP in G1 inhibited the subsequent decay of the Cdk inhibitor p27 and delayed the onset of S phase. Forced translocation of endogenous PKAII to the cytosol down-regulated cAMP signaling, advancing the timing of p27 decay and inducing premature exit from G1. These data indicate that membrane-bound PKA amplifies the transduction of cAMP signals in G1 and that the length of G1is influenced by cAMP-PKA.

The growth and differentiation of several cell types is controlled by cAMP (1,2). In eukaryotes, cAMP binds the regulatory subunit of cAMP-dependent protein kinases (PKA). 1 This releases the catalytic subunit (C-PKA), which phosphorylates a wide variety of substrate proteins. A fraction of the C-PKA migrates to the nucleus and phosphorylates nuclear proteins and transacting factors. Phosphorylated transcriptional factors activate the transcription of several genes (3)(4)(5).
cAMP also drives the cell cycle of thyroid cells. Thyrotropin (TSH), a pituitary hormone, binds to and stimulates a specific G s -coupled receptor on the membrane of thyroid cells. Stimulation of adenylylcyclase raises cAMP levels and induces the exit of thyroid cells from G o into the cell cycle. TSH or cAMP depletion drives cells out of the cycle into quiescence (2,6).
There are multiple isoforms of PKA, typically determined by their specific regulatory subunit. We have been studying the PKAII isoform, which is abundantly expressed in neural and endocrine tissues. The regulatory subunits of this isozyme, RII␣ and RII␤, have low affinity to cAMP (7)(8)(9). The RII subunit binds to a PKA anchoring protein (AKAP), which localizes the PKAII in the Golgi-centrosome area and in the cytoskeleton (10 -12). Our evidence links intracellular targeting of PKAII to cAMP nuclear signaling and gene regulation. Overexpression of the bovine brain anchor protein AKAP75 restored cAMPinduced transcription in variant PC12 cells defective in cAMP nuclear signaling (13). Conversely, thyroid cell expression of a mutant derivative of AKAP45 that displaces membrane-bound PKAII depressed cAMP-induced thyroglobulin gene expression (8). Finally, AKAP75 expression increased the rate and the magnitude of c-fos expression induced by cAMP in human embryonic kidney cells and increased cAMP-response elementdirected gene transcription in cerebellar granule cells (14,15).
We describe here changes in the components of the cAMP signaling pathway during cycle progression in synchronized thyroid cells. We find that the G 0 and G 1 -S transitions are accompanied by significant modifications in the localization and activity of PKA. Cells entering G 1 display increased nuclear and cytoplasmic PKA activity. Later in G 1 , PKA activity is markedly down-regulated in the absence of significant variations of cAMP levels. Altering the activity of PKA affects the onset of S phase. Expression of a transdominant negative variant of a PKAII anchor protein, which translocates endogenous PKA from membranes to the cytosol, reduces PKA levels, downregulates nuclear cAMP signaling, and significantly shortens G 1 .

MATERIALS AND METHODS
Cell Lines, DNA Plasmids, Deletion Mutagenesis, and Transfections-The TL cell line is derived from the FRTL-5 thyroid cell line, which has been extensively characterized with respect to thyroglobulin expression and TSH-dependent growth (6,16). AKAP45 and AKAP75 plasmids contain the AKAP coding region under the control of the CMV promoter; the plasmids also carry the aminoglycoside transferase gene, which confers resistance to the neomycin analogue G-418 (8,14,17,18). RSV-NEO is a construct expressing the aminoglycoside transferase gene under the control of the long terminal repeat of Rous sarcoma virus. The AKAP45 mutant (AKAP45-RII*), which is deleted for the RII binding site of AKAP45, was generated by the polymerase chain reaction using specific oligonucleotide primers. The primers were designed to create NotI and XbaI restriction sites at their 5Ј-and -3Ј-ends, respectively. Polymerase chain reaction products were digested with NotI and XbaI restriction enzymes and cloned in the pRc/CMV vector. The correct AKAP45-RII* coding region was confirmed by DNA sequencing. DNA transfections in mammalian cells were carried out by the calcium phosphate procedure. We have used also an expression vector carrying the aminoglycoside transferase gene under the CMV promoter to control for promoter-dependent effects. Transfected cell lines (AKAP45 and control) were pools of at least 100 clones.
RNA Analysis-Total RNA was purified by homogenization in guanidium isothiocyanate and phenol-chloroform-isoamyl alcohol extraction (19). Probes were 0.35-kilobase PCR fragments corresponding to RI␣, RII␣, RII␤, and GAPDH labeled as follows. 10 ng of the amplified DNA fragment (purified by electroelution) were labeled via 10 PCR cycles (1 min at 95°C, 1 min at 60°C, and 3 min at 72°C) in a volume of 30 l of PCR buffer (see below) containing 50 Ci (3000 Ci/mmol) of [ 32 P]dGTP; 2 nmol of dATP, dTTP, and dCTP; 10 pmol of each of the appropriate oligonucleotide primers; and 0.75 units of Taq DNA polymerase.
Single strand cDNA synthesis was performed as previously described (8).
Semiquantitative reverse transcriptase-PCR was performed coamplifying RI␣, RII␣, RII␤, and GAPDH cDNAs (GAPDH primers were added after the first five cycles). 20 l of the PCR products were resolved on four different 1.5% agarose/TBE gels, blotted onto different nylon membranes (Amersham Pharmacia Biotech), and hybridized with specific probes (see above).
Total RNA (15 g) preparation and hybridization were as described previously (8,14,19). cDNA probes for 18 S RNA or GAPDH were used as internal control.
DNA Synthesis-DNA synthesis in TL cells was monitored by [ 3 H]thymidine incorporation as described previously (16). Experiments were performed in triplicate, and the data represent a mean of four independent experiments, all which gave similar results. Cell cycle progression (G 0 , G 1 , and S phases) was monitored by fluorescenceactivated cell sorter analysis. Where indicated, the cells were stimulated with 8-bromo-cyclic AMP (8-Br-cAMP), 8-chlorophenylthio-cyclic AMP (CPT-cAMP), or forskolin. Quiescent (G 0 ) TL cells did not express early genes (c-myc and c-fos). These were induced by TSH (23). Cyclin D and cyclin A were induced 24 h after continuous treatment with TSH. PCNA analyzed by immunofluorescence appeared 36 h following continuous TSH treatment.
Antibodies and Immunoblot Analyses-Polyclonal antibodies against RII␤, RII␣, RI␣, and PKA catalytic subunits were a gift of C. Rubin (Albert Einstein College of Medicine, Bronx, NY). In addition, specific anti-RII␤ or anti-RII␣ antibodies were generated by immunizing rabbits with a synthetic RII␤ peptide (peptide 31-57 from the AUG of the rat sequence) or RII␣ (peptide containing residues 53-73 from the start codon of the rat protein), respectively, cross-linked to soybean trypsin inhibitor. Total IgGs were purified, and the specificity of each preparation was tested by immunoprecipitation, immunofluorescence, and immunoblot by preadsorbing the antibodies to the specific peptides or control peptides. Anti ␣-mannosidase antibodies were a gift of K. Moreman (University of Georgia, Athens, GA). Western blot analyses were carried out as described previously (8).
Nuclear and Cytoplasmic PKA Assay-Cells were lysed in AT buffer containing 0.1% Triton X-100 for 5 min on ice. The lysate was layered on 1 volume of a sucrose cushion (AT buffer containing 1 M sucrose) and centrifuged at 2500 ϫ g for 5 min. The pellet and the upper phase represent purified nuclei and cytoplasm, respectively. The nuclear fraction contained ϳ90% of the transcription factors TTF1, cAMP-response element-binding protein, and PAX8 (24). In addition, each preparation was stained with propidium iodide to check purity. Assays (final volume 25 l) were performed as described previously (8). Holoenzyme activity was calculated by subtracting values obtained in the absence of cAMP and in the presence of protein kinase inhibitor (ϩcAMP) from the values obtained in the presence of cAMP. Free C-PKA activity was evaluated by subtracting counts/min obtained in the absence of cAMP from the values obtained in the presence of protein kinase inhibitor. Data were expressed as pmol of [ 32 P]phosphate transferred to peptide substrate during a 10-min incubation in the presence (PKA holoenzyme) or absence (free C-PKA) of 10 M cAMP. At the concentrations used, protein kinase inhibitor did not inhibit the binding of phosphorylated kemptide to phosphocellulose filters (8).
Immunofluorescence-Cells were treated and analyzed as described previously (8).

RESULTS
Amplification of Nuclear cAMP Signaling in G 1 -TL thyroid cells forced into quiescence by TSH withdrawal were exposed to 10 milliunits/ml of TSH and 1 g/ml of insulin to induce cell cycle entry. The onset of DNA synthesis occurred 36 -48 h later ( Fig. 1, upper panels). TSH stimulates cAMP synthesis; cAMP or agents that induce cAMP accumulation substitute for TSH and move TL cells into the cell cycle (2). The timing of DNA synthesis was monitored by thymidine incorporation (left panels, broken line) and fluorescence-activated cell sorting analysis (right panels).
To determine how cAMP induces cell cycle entry, we analyzed several parameters in the cAMP transduction pathway. cAMP levels increased during the first 30 min after hormone addition, returned to base line 1-3 h later, and did not vary significantly after (data not shown; see Ref. 2). Nuclear C-PKA activity, a marker of nuclear response to cAMP, increased in early G 1 cells and decreased 36 h after TSH and insulin stimulation ( Fig. 1, upper panel, continuous line). Similarly, cytoplasmic C-PKA activity slowly decreased in late G 1 (Fig. 1, middle panel, continuous line). The reduction of PKA activity paralleled the onset of S phase (36 h) (Fig. 1, upper and middle panels). To determine whether PKA down-regulation was necessary for S phase onset, we stimulated cells in late G 1 (30 h, arrow) with 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX), a cAMP-phosphodiesterase inhibitor, to maintain high and constant cAMP levels. Persistent activation of PKA inhibited and delayed DNA synthesis ( Fig. 1, lower panels). Note that PKA activity declines in stimulated cells and becomes refractory to cAMP (25). Down-regulation of nuclear and cytoplasmic PKA in late G 1 was not affected by other inductive stimuli.
To determine if the sensitivity of TL cells to cAMP nuclear signaling changed during G 1 , we measured C-PKA levels ( Fig.  1A, insets) and activity ( Fig. 1A) in nuclei of hormone-treated cells exposed to short (40-min) pulses of exogenous cAMP. Nuclear C-PKA accumulation was relatively insensitive to cAMP in G 0 cells ( Fig. 2A). In contrast, cells in early G 1 (12-24 h after TSH and insulin treatment) responded efficiently to cAMP. At 36 -48 h, cAMP-induced accumulation of C-PKA in the nucleus was less sensitive. To monitor the transcription of cAMP-induced genes, we measured the accumulation of c-fos mRNA in G 0 , mid-G 1 , and late G 1 cells treated for 30 and 60 min with CPT-cAMP. c-fos mRNA accumulated in early G 1 cells 30 min earlier than in G 0 or in late G 1 cells, while GAPDH mRNA accumulation did not vary (Fig. 2B). C-PKA levels rose rapidly in nuclei of G 1 cells compared with G 0 and G 1 -S cells despite constant cAMP levels (Fig. 2C). Additionally, phosphorylated cAMP-response element-binding protein accumulated in G 1 and markedly decreased in G 0 and G 1 -S transition (data not shown). These data indicate that the ability of nuclei to take up and/or to retain C-PKA varies during the cycle. It is low in G 0 , increases in G 1 , and falls at the G 1 -S transition. Different cAMP analogues reproduced the results described above (data not shown).
The enhanced sensitivity to cAMP of G 1 cells relative to quiescent cells might explain why cyclin A gene transcription is efficiently induced by cAMP in G 1 but not in G 0 cells (26).

PKAII Is Diffuse in the Cytoplasm in Quiescent Cells and Concentrates in the Golgi-centrosome Area as Cells Enter the
Cycle-Efficient accumulation of nuclear C-PKA in G 1 cells without corresponding increases in cytoplasmic cAMP levels suggested to us that the location of PKAII, known to affect nuclear cAMP signaling, might vary during cell cycle (8,14).
PKAII localization as a function of time after hormone stimulation was determined by immunofluorescence analysis. In dividing thyroid cells, the RII␤ and RII␣ PKA regulatory subunits localized in a discrete area corresponding to the Golgicentrosome area, whereas RI␣ was diffuse in the cytosol ( Fig. 3; Ref. 12). This area stained with anti-RII␤/␣, ␣-mannosidase, and ␤-tubulin antibodies and appeared as an intense bright spot in proximity to the nucleus (8,11,27). Fig. 4 shows RII␤ staining at 0, 24, 36, and 48 h after stimulation with TSH and insulin. Anti ␣-mannosidase antibodies were used to monitor the position of the Golgi apparatus (28). In quiescent cells, the RII␤ signal was diffuse in the cytoplasm, quite distinct from the perinuclear Golgi signal (Fig. 4, a and b). As cells exited G 0 and progressed into G 1 , the RII␤ signal concentrated in the Golgi area (12 h, Fig. 4, c and d). Upon entry into S phase, both the RII␤ and Golgi signals became progressively perinuclear (36 -48 h; Fig. 4, e, f, g, and h). The staining pattern of RII␣ was similar to that of RII␤ during G 1 (data not shown).
To demonstrate the localization of RII␤ in the Golgi-centrosome area, we stained cells in G 1 with antibodies to p58 formiminotransferase cyclodeaminase, a protein that binds microtubule and is localized in the Golgi apparatus (Fig. 4). Panels i, j, and k show the staining with anti-RII␤, formiminotransferase cyclodeaminase, and the overlay of both signals, respectively.
During transition from quiescence into G 1 , RII accumulated in the Golgi-centrosome area. At the G 1 to S transition, both RII and the Golgi apparatus appeared in a perinuclear ring. We suggest that this movement of PKAII plays a significant role in the transmission of cAMP signals to the nucleus and in the control of cell cycle progression.
Down-regulation of PKA Regulatory Subunits during G 1 -Our data indicate that the intracellular location of RII changes after exit from G 0 . To monitor these movements more precisely, we prepared extracts from quiescent and cycling cells and measured R subunit concentrations in particulate and soluble fractions (P and S, respectively, in Figs. 5 and 10). Cells were synchronized by starving from hormones and serum for 3 days and then induced into cell cycle by the addition of TSH, insulin, and serum. The peak of DNA synthesis under these conditions invariably occurred 48 h after induction (Fig. 1).
FIG. 1. Oscillations of nuclear and cytoplasmic PKA during cell cycle progression. Cells were synchronized by hormone and serum starvation and induced with 10 milliunits/ml TSH and 1 g/ml insulin in F-12 medium containing 0.5% bovine serum albumin. [ 3 H]thymidine incorporation or fluorescence-activated cell sorting analysis was performed as described under "Materials and Methods." Left panels, on the right ordinate of the left panels is shown [ 3 H]thymidine incorporation as -fold induction over basal. Nuclear PKA activity was measured in purified nuclei as described under "Materials and Methods." Cytoplasmic PKA activity is represented as the ratio between C-PKA activity and total PKA as described under "Materials and Methods." 1 corresponds to complete dissociation of holoenzyme (see "Materials and Methods"). The data shown are a mean of three independent experiments. In the bottom left panel, 0.5 mM 8-Br-cAMP and 0.5 mM IBMX were added 30 h after the initial hormone treatment (arrow). Inhibition of DNA synthesis also occurred in the absence of IBMX, although less efficiently. Right panels, cycle progression in thyroid cells was monitored by a cell sorter, as described under "Materials and Methods." Results are shown as a percentage and represent a mean Ϯ S.E. of four independent experiments. cAMP effects on cell cycle progression were blocked by pretreating cells with the PKA-specific inhibitor, H89 (10 M).
The specificity of the anti-RII antibodies was determined by immunoblot analysis on recombinant RII-purified proteins (RII␣ and RII␤) or on total protein extract (ex) from TL cells (Fig. 5A). Fig. 5B shows that all types of R subunits decreased 12-25 h following hormone addition. The decline in RII␣ could be detected at 12 h, whereas RI␣ decreased later, between 12 and 24 h. Although less dramatic than the loss of RII␣ or RI␣, a decrease in RII␤ levels between 12 and 24 h could be observed. Both RII␣ and RII␤ concentrations returned to G 0 levels at 48 h, a point corresponding to the peak of S phase.
Cycle-dependent fluctuations in concentration of R subunits are not specific to TL cells. Cell cycle entry also induced changes in R subunit concentration in the PCcl3 thyroid cell line (29) with kinetics comparable with those of TL (data not shown).
The decrease in R subunits concentration in G 1 cannot be accounted for by down-regulation of the corresponding mRNAs. In fact, R subunit mRNA concentrations were markedly elevated by TSH or cAMP treatment in early G 1 (RI␣) and middlelate G 1 (RII␣ and RII␤) ( Fig. 6 and Ref. 30). To determine further the mechanism of the R subunit down-regulation, we measured the RII subunit synthesis during cell cycle progression. As shown in Fig. 7 (upper panel), the translation rate of the RII␤ subunit was similar in G 1 (12-24 h), G 0 (0 h) and G 1 -S (36 -48 h) phases. The band of ϳ40 kDa present in the immunoprecipitates of RII in G 0 phase and in G 1 -S transition (36 h) cells was C-PKA, as indicated by the in vitro kinase A assay (Fig. 7, lower panel). In contrast, anti-RII antibody did not coprecipitate the C-PKA subunit in early and middle G 1 . These data indicate that the association between RII and C-PKA fluctuates during cell cycle.
Taken together, these analyses indicate that 1) the decrease in R subunit concentration in G 1 is post-transcriptionally regulated; 2) R subunit levels reach a minimum at a time in G 1 that corresponds to the maximal activity of nuclear and cytoplasmic PKA (12-24 h; Fig. 1); 3) PKA activation stimulates R subunit mRNA accumulation (R subunit transcription is known to be induced by cAMP (30)); 4) protein and mRNA levels of RII␣ and, to a lesser extent RII␤, peaked at 36 -48 h, a time corresponding to the onset of S phase and the minimum activity of the PKA pathway (Fig. 1); and 5) C-PKA is tightly bound to RII subunits in G 0 and G 1 -S cells, thus reducing basal PKA activity.
cAMP Reduces the Decline of p27 in G 1 Cells-We have shown that thyroid cells in G 1 are extremely sensitive to nuclear cAMP signals (Fig. 2) and that this phenotype is associated with a significant down-regulation of RI␣ and RII␣ subunits (Fig. 4) and a reduction in RII-bound C-PKA (Fig. 7). We also demonstrated that forced activation of cAMP-PKA signal-FIG. 2. Amplification of nuclear cAMP signaling in G 1 cells. A, G 0 , G 1 (24 h), and G 1 -S TL cells were incubated with 8-Br-cAMP at the indicated concentrations for 40 min. At the end of incubation, nuclei were purified and assayed for PKA activity, as described under "Materials and Methods." The graph shows nuclear C-PKA activity of stimulated cells expressed as pmol of [ 32 P]phosphate transferred to peptide substrate during a 10-min incubation. 50 g of nuclear extracts from starved or cycling TL cells, stimulated with 250 M 8-Br-cAMP for the indicated times, were immunoblotted with anti-C-PKA specific antibodies (insets). B, Northern blots of total RNA (20 g/lane) extracted from G 0 , G 1 , and G 1 -S cells stimulated with 250 M CPT-cAMP for 0, 30, and 60 min. The hybridization was carried out with rat c-fos or GAPDH cDNAs. The kinetics of c-fos induction by 8-Br cAMP or CPT-cAMP were similar. C, immunofluorescence analysis of quiescent (G0) or TSH/insulin-induced TL cells (G24 and G36) was performed using anti-C-PKA specific antibodies as described under "Materials and Methods." A representative of three independent experiments is shown.

FIG. 3. R subunit localization.
Growing TL cells were stained with specific anti-RI␣, anti-RII␣, or anti-RII␤ antibodies, and immunofluorescent analysis was performed as described under "Materials and Methods." The type of signal corresponding to RI␣ (diffuse) or RII␣ and RII␤ (concentrated in the perinuclear area) was found in ϳ90% of the cells and with antibodies from different sources. Scale bars, 15 m.
ing in late G 1 cells delayed the onset of S phase (Fig. 1). Since the Cdk inhibitor p27 regulates the timing of the G 1 -S phase transition, we asked if p27 levels might be influenced by cAMP. Fig. 8A indicates that the concentration of p27 in G 0 cells fell as cells entered and progressed through G 1 . A pulse of cAMP in middle G 1 (10 -24 h) significantly increased p27 levels, whereas a pulse in G 0 had no effect (Fig. 8 B ). p27 mRNA levels were not influenced by cAMP (data not shown). Our preliminary data suggest that the degradation of p27 protein is inhibited by cAMP.
Cytosolic Translocation of RII Subunits Impairs cAMP Signaling to the Nucleus and Shortens G 1 -We have described a number of cellular events related to the cAMP signal transduction pathway that accompany entry into cell cycle. The amplification of cAMP nuclear signaling and the up-regulation of p27 by cAMP in G 1 may be related to the targeting of PKAII in the Golgi-centrosome region (Fig. 4) or to the decrease in total R subunit concentration. To discriminate between these possibilities, we manipulated intracellular PKAII localization.
We overexpressed a defective RII anchor protein (AKAP45) that lacks the N-terminal membrane anchor domain. AKAP45 binds RII and translocates PKAII from membranes to the cytosol (10,17,18). Pools of clones expressing AKAP45 were isolated and characterized. Expression of AKAP45 has been shown to significantly increase cytosolic RII levels, accompanied by down-regulation of cAMP-response element-binding protein phosphorylation, nuclear C-PKA accumulation, and transcription of cAMP-regulated genes (8). We determined the effect of AKAP45 on the kinetics of c-fos mRNA induction by cAMP 24 h after entry into cell cycle. Fig. 9 shows that c-fos mRNA was induced significantly later in AKAP45-expressing (A45) cells than in control cells (TL), indicating that AKAP45 decreases sensitivity to cAMP. That AKAP45 delocalized RII␤ was confirmed by immunostaining and immunoblot analysis of RII subunits in the cytosol and membranes (S and P in Figs. 5 FIG. 4. RII␤ redistribution during cell cycle progression. Immunofluorescence analysis of TL cells progressing into cell cycle. Staining was performed with specific antibodies to RII␤ (a, c, e, and g) and ␣-mannosidase (b, d, f, and h) as described under "Materials and Methods." i and j show the staining of G 1 cells (24 h) with anti-RII␤, anti-p58 (G2404, Sigma), a specific Golgi protein (40). k shows the overlay of i and j. Cells were starved of hormones and serum, maintained in 0.5% bovine serum albumin for 2 days (a and b). TSH (10 milliunits/ml in 0.5% bovine serum albumin) and insulin (1 g/ml) were added for 12 (c and d), 36 (e and f), and 48 h (g and h). The peak of DNA synthesis under these conditions was 48 h after the initial hormone stimulation, assayed by fluorescence-activated cell sorting and [ 32 H]thymidine incorporation. The distribution of RII and mannosidase was identical to that shown above in cells progressing through the cycle, stimulated by TSH, cAMP, or forskolin in the absence of insulin.

FIG. 5. Down-regulation of PKA regulatory subunits during G 1 .
A, specificity of the polyclonal antibodies directed versus R subunits. 100 ng of recombinant affinity-purified RII␣ and RII␤ were 32 P-labeled in vitro with C-PKA and immunoprecipitated with the antibodies indicated. 100 g of total protein extract (ex) from TL cells were immunoprecipitated with the indicated anti-RII antibodies. The immunoprecipitates were 32 P-labeled with C-PKA and analyzed on SDSpolyacrylamide gels. B, immunoblot analysis of RI␣, RII␣, RII␤, and catalytic subunits of PKA in TL cells. Cells were synchronized and analyzed as described under "Materials and Methods," except that the cells were treated with 5% serum, 1 g/ml insulin, and 10 milliunits/ml TSH. Fractionation of cells in particulate (P) and cytosolic (S) fractions was performed as described under "Materials and Methods." 100 g of each fraction were fractionated by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with specific antibodies to the different PKA subunits as indicated on the left side of the blots (see "Materials and Methods"). and 10). Unlike control cells, RII␤ staining remained diffuse in the cytoplasm before and after entry into the cell cycle (Fig.  10B). In addition, AKAP45 raised the levels of cytosolic RII␣ and RII␤, although the total amount of RII␤ subunit was reduced ( Fig. 10A; Ref. 8).
The effects of PKAII delocalization on cycle progression were monitored by comparing the time course of DNA synthesis and the levels of p27 in A45 and control cells. Strikingly, A45 cells entered S phase 12 h earlier than controls (Fig. 10C). That this was the consequence of PKAII delocalization was confirmed by transfecting TL cells with an AKAP45 mutant (A45-RII*), deleted for the RII binding site (see "Materials and Methods"). The profile of DNA synthesis in cells expressing A45-RII* was comparable with control cells. As expected, A45 cells contained significantly less nuclear p27 at 24 h than control cells, as demonstrated by histochemical staining with p27 antibody (data not shown). The effect of AKAP45 overexpression on p27 levels was also shown by immunoblotting cell extracts with anti-p27 antibodies. AKAP45 induced a rapid cAMP-resistant down-regulation of p27 (Fig. 11). Thus, failure to localize PKAII in the membrane leads to an early decrease in p27 and a shorter G 1 phase. DISCUSSION Activation of the cAMP-PKA signal transduction pathway can have either mitogenic or antimitogenic effects in mammalian cells (31). These opposite responses might indicate cellspecific targets of the pathway. Alternatively, the intensity or the time of delivery of cAMP-PKA signal relative to the phase of cell cycle might critically influence the response of the cell to a The cells were synchronized by TSH starvation and stimulated with TSH (10 milliunits/ml) for various periods of time indicated in hours. b see Fig. 1. c Expressed as pmol/g of proteins of nuclear C-PKA induced by 15-min treatment with or without 0.1 mM 8-Br-cAMP. d See Fig. 4. e See Figs. 5 and 6. In TL cells regulatory subunits are in excess to catalytic subunits (ϳ3/1) as deduced by the inhibition of exogenous C-PKA by fixed amounts of cellular extracts. Inhibited C-PKA is reactivated by exogenous cAMP.
f DNA synthesis in cells treated with cAMP and IBMX at 24 h after initial TSH stimulation. g Expressed as indicated in Footnote e in A45 cells (see also Figs. 2 and 10).
FIG. 6. Protein and mRNA levels of R subunits during cycle progression. Left panels, mRNA analysis of R subunits in cycling cells. Total RNA was extracted from quiescent TL cells or cells stimulated with TSH for 12, 24, 36, and 48 h. Specific primers to the R subunits and GAPDH were used to quantitate the relative mRNA transcripts by RT-PCR as described under "Materials and Methods" (8). Right panels, quantification of mRNA and protein levels in TL cells progressing into cycle. RI␣, RII␣, RII␤ mRNA in quiescent or TSH-treated TL cells were quantified by densitometric scanning of blots like those shown in Fig. 5 and in the left panels. The numbers shown on the ordinates represent arbitrary units. The protein values in G 0 cells were set to 1.

FIG. 7. Cell cycle-dependent association of RII and C-PKA.
A, TL cells were labeled for 4 h with [ 35 S]methionine and harvested at the indicated times of cycle. Triton X-100-solubilized membranes (300 g) were immunoprecipitated with anti-RII␤ antibodies. The immunocomplexes were separated on a 10% SDS-polyacrylamide gel and analyzed by autoradiography. The arrows indicate RII␤ and C-PKA subunits. Similar results were obtained with anti RII␣ antibodies. Since the catalytic subunit of PKA and the heavy chain of IgG migrate with similar mobilities on SDS-polyacrylamide gel electrophoresis, it was not possible to perform Western blot analysis with anti-C-PKA antibodies.
Neosynthesized RII band appears diffuse, since it is heavily phosphorylated in G 0 and G 1 cells. In three independent experiments, we did not notice significant variations in the amount of neosynthesized RII. B, in vitro A kinase assay on the immunocomplexes of RII␤ from synchronized or cycling TL cells as in A. The results are presented as arbitrary units and represent a mean Ϯ S.E. of three independent experiments. cAMP. In this paper, we study the effects of cAMP-PKA on cell cycle progression in thyroid cells, which are exquisitely dependent on cAMP for growth. We show that cAMP is required to enter G 1 from G 0 . The cAMP-PKA pathway is initially upregulated early in G 1 and then down-regulated later in this phase. Down-regulation is critical for progression from G 1 to S. We propose that regulation of the cAMP-PKA pathway after entry into cell cycle does not primarily reflect changes in cAMP levels. Instead it results from changes in the subcellular location of PKAII isoenzymes, in the concentrations of the RII subunits, and in the capacity of these subunits to bind C-PKA. Our data linking cAMP signal transduction to cell cycle progression are summarized in Table I.

Entry in the Cycle of Thyroid Cells (G 0 -G 1 Transition)-
Stimulation of quiescent thyroid cells with cAMP or with agents, such as TSH or forskolin, that elevate cAMP levels induces cell cycle entry and DNA replication (2). cAMP upregulates the expression of immediate early genes (2,23) and prepares the cell for DNA synthesis (26,32,33).
To stimulate gene transcription, C-PKA released from PKA holoenzyme must enter the nucleus. Quiescent cells respond weakly to cAMP, showing little accumulation of nuclear C-PKA (Fig. 2). 12 h after hormone stimulation, however, early G 1 cells become highly responsive to cAMP. Increased cAMP signal transduction is associated with accumulation of PKAII in the Golgi-centrosome area (Fig. 4) and a decrease in R subunit  . 10. Shortening of G 1 phase in A45 cells. A, expression of the R subunits in control or in TL cells stably transfected with AKAP45. 50 g of supernatant (S) and 120 g of particulate (P) protein fractions were immunoblotted with RII-specific antibodies. B, immunofluorescence analysis of RII␤ in control cells or in A45 cells. Synchronized cells were stimulated with TSH for 0, 12, and 36 h. Experiments were carried out as described in the legend to Fig. 1. C, DNA synthesis in control cells (squares), A45 cells (circles), or cells expressing AKAP45-RII* (asterisks). S phase was determined by [ 3 H]thymidine pulse-chase experiments as described under "Materials and Methods." [ 3 H]thymidine incorporation is reported as -fold over the basal level. The RII partition between cytosolic and membrane fractions was not altered in cells expressing AKAP45-RII* (data not shown). concentrations ( Fig. 5; Ref. 34). Membrane anchoring of PKAII stimulates cAMP nuclear signaling (8,14).
Immunoblot analysis indicated that the levels of both RII␣ and RII␤ were reduced in G 1 cells. The immunostaining patterns of the remaining RII␣ and RII␤ were similar, however (Figs. [3][4][5]. The translocation of PKAII may reflect de novo synthesis of a PKA anchoring protein (AKAP). We recently cloned a gene in thyroid cells encoding an RII-binding protein induced by cAMP early in G 1 (35). We propose that this AKAP facilitates G 0 -G 1 transition by amplifying the cAMP pathway and, consequently, the transcription of cAMP-induced genes.
Progression in G 1 : Down-regulation of the PKA Pathway-By the end of G 1 (36 h), the PKA pathway is again down-regulated. The concentration of nuclear C-PKA drops significantly, and the movement of C-PKA into the nucleus becomes relatively resistant to cAMP treatment. At mid-S phase (48 h), Golgi and RII subunit signals are located in a perinuclear ring, and the concentration of RII subunits steadily increases. S phase, bromodeoxyuridine-positive nuclei do not stain with anti-C-PKA antibody (data not shown). 2 Although the decrease in R subunit content might facilitate PKA activation in early G 1 (see Ref. 34), it cannot, however, account for the inhibition of PKA seen in late G 1 , when R subunit levels are still low. In late G 1 , cellular PKA is mostly of type II. Because of its low affinity for cAMP and its membrane localization, PKAII might bind tightly and draw C-PKA from the nucleus at low cAMP concentrations. In this role, PKAII could function as a repressor, reducing basal cAMP signaling. At intermediate cAMP levels, PKAII efficiently facilitates C-PKA import to the nucleus (8). Taken together, these data might explain the down-regulation of cAMP-PKA signaling in G 1 -S transition. G 1 -S transition can be influenced by manipulating the PKA pathway. Thus, although thyroid cells are exquisitely dependent on cAMP for growth, cAMP treatment in late G 1 or excess exogenous C-PKA strongly inhibits DNA replication (Fig. 1). Conversely, expressing a mutant AKAP75 protein, AKAP45, which delocalizes PKAII and inhibits PKA signal transduction, significantly advances the time of G 1 -S transition. The cytosolic translocation and the decrease in RII subunits may both contribute to the down-regulation of cAMP signaling in cells expressing AKAP45. Note that in these cells c-fos is activated by PKC but not efficiently by cAMP (Fig. 9). 3 A potential target of nuclear C-PKA is p27, an inhibitor of Cdk4. Cdk4 activation is essential for G 1 -S transition. p27 decays during the first 24 h after exposure to TSH and insulin. Treatment with cAMP significantly increased the levels of p27; similar findings have been reported in other cell lines that are growth-inhibited by cAMP (36). Another Cdk inhibitor, p21, was not affected by PKA stimulation (data not shown). In contrast, p27 concentrations in A45 cells were lower than in control cells. Thus, the levels of p27 and the timing and extent of DNA synthesis are correlated. The addition of cAMP later in G 1 -S (36 h) had no effect on DNA synthesis or on the downregulation of p27 (data not shown).
How the PKA pathway interferes with p27 decay is not clear. p27 is degraded by the ubiquitin pathway, and it is possible that one or more components of this pathway are modified by PKA-dependent phosphorylation. Our preliminary evidence suggests that cAMP reduces p27 degradation. Note that R subunits are also degraded by the ubiquitin pathway and that the reductions in R subunit and p27 follow similar kinetics (Figs. 5, 6, and 8). Thus, it is possible that cAMP-PKA might regulate ubiquitin-mediated proteolysis of both p27 and R subunits. In yeast, some components of the proteasome are known to be controlled by the cAMP-PKA pathway (37).
Inhibition of S phase onset can be induced by cAMP in a wide variety of cell types, depending on the time of cAMP stimulation. cAMP advances S phase if added early in G 1 (NIH 3T3 fibroblasts), whereas it inhibits entry if added later (31).
We showed previously that cAMP levels oscillate during cell cycle in Xenopus egg extracts and that such oscillations are required for mitosis-interphase transition (38) and for the onset of S phase. 4 We described above how cAMP signaling may be amplified or down-regulated in G 1 thyroid cells without marked fluctuations in cAMP concentrations (see also 2). These latter events, which are essential for G 1 progression, are downstream to adenylylcyclase and amplify small changes in cAMP concentrations. In Xenopus oocytes, cAMP levels fluctuate in response to internal signals, whereas cAMP concentrations in thyroid cells respond to external stimulation by TSH. By adjusting PKAII activity, thyroid cells can modulate their response at different times during cycle despite a constant blood TSH concentration. Conversely, down-regulation of the PKA pathway in late G 1 may buffer the cells from surges in TSH levels, which would otherwise block G 1 -S transition.
In conclusion, we propose that the cAMP-PKA signal transduction pathway determines the length of G 1 phase by affecting the concentrations of p27 and possibly other regulators of cell cycle progression. We have demonstrated this control system in thyroid cells, which are dependent on cAMP for growth, but we suggest that it plays a role in cycle progression in all cells. Our hypothesis is supported by studies of cycle progression in Saccaromyces cerevisae, where cAMP-PKA signals have been demonstrated to regulate cell size sensors and the entry into S phase (39). FIG. 11. Rapid decay of p27 in A45 cells. p27 levels in TL and A45 cells are shown. The p27/MAPK ratio is shown on the ordinate, and the value in G 0 TL and A45 cells is set to 1. Control and A45 cells stimulated with hormones and serum (0, 6, 12, and 24 h) were treated 4 h with 50 M forskolin and 0.5 mM IBMX. The p27/Cdk4 ratio shows similar kinetics (data not shown).