©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Expression and Intracellular Distribution of the Heat-stable Protein Kinase Inhibitor Is Cell Cycle Regulated (*)

(Received for publication, August 29, 1994; and in revised form, November 18, 1994)

Wei Wen (1) Susan S. Taylor (1) Judy L. Meinkoth (2)

From the  (1)Departments of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093 and the (2)Department of Pharmacology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104-6084

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The heat-stable protein kinase inhibitor (PKI) is a potent and specific inhibitor of the catalytic (C) subunit of the cAMP-dependent protein kinase. We report the isolation of a polyclonal antibody raised to purified recombinant PKIalpha. Using this antibody, the intracellular distribution of endogenous PKIalpha was assessed by immunostaining. The PKIalpha expression and intracellular distribution varied as a function of cell cycle progression. PKIalpha expression appeared low in serum-starved cells and in cells in G(1) and increased as cells progressed through S phase. Its distribution became increasingly nuclear as cells entered G(2)/M. Nuclear levels of PKIalpha remained high through cell division and decreased again as cells reentered G(1). The cell cycle regulated expression and nuclear distribution suggests a specific role for PKIalpha in the nucleus during the G(2)/M phases of the cell cycle. Consistent with this, microinjection of PKIalpha antibody into serum-starved cells prevented their subsequent cell cycle progression. Similarly, overexpression of C subunit in cells arrested at the G(1)/S boundary prevented their subsequent division. Together these results support the idea that PKIalpha plays an important role in the inhibition of nuclear C subunit activity required for cell cycle progression, although a determination of the relative amounts of endogenous nuclear PKI and C-subunit will be required to substantiate this hypothesis.


INTRODUCTION

The heat-stable protein kinase inhibitor (PKI) (^1)is a highly specific inhibitor of the catalytic (C) subunit of cAMP-dependent protein kinase. PKI binds to the active site of C with high affinity specifically in the presence of cAMP when C is dissociated from the holoenzyme complex. The existence of an inhibitor protein was first suggested in 1964(1) , and PKI was subsequently purified (2) and studied extensively by a variety of techniques (for review, see Ref 3). Several different isoforms of PKI have been identified, including PKIalpha(4) , PKIbeta1, and PKIbeta2 (5, 6) . PKIbeta1 and PKIbeta2 result from alternative splicing of the RNA at the amino terminus(6) . Analysis of peptide analogs determined that high affinity binding of PKI to C-subunit could be attributed primarily to 20 residues near the amino terminus. This region contains a cluster of arginine residues that mimics the basic subsite present in peptide substrates and a second hydrophobic site required for high affinity binding of PKI to the C subunit.

In spite of extensive biochemical studies, the physiological roles of PKI remain unclear. It was originally proposed that PKI was simply a mechanism to dampen the kinase response by inactivating low levels of enzyme (for review, see Ref 3). However, the association of testicular PKI with microtubules (7) and the isolation of several PKI isoforms (4, 5, 6) that exhibit tissue- and developmental stage-specific expression suggest additional roles for these inhibitors(8) . Several studies have implicated a role for PKI in cell cycle progression. In amphibians (9) and mammals(10) , microinjection of PKI into quiescent oocytes resulted in the induction of mitosis. Similarly, injection of an amino-terminal PKI peptide (PKI) into mammalian cells induced chromatin condensation, microtubule reorganization, and changes in cell shape similar to those that occur just prior to mitosis(11) . Recent studies demonstrated that PKI entered the nucleus following its injection into the cytoplasm in REF52 cells(12) , suggesting a specific role for PKI in the regulation of nuclear stores of C subunit.

To better understand the physiological roles of PKI, an antibody was raised to purified recombinant PKIalpha and used to characterize the expression and distribution of endogenous PKIalpha.


MATERIALS AND METHODS

Cell Culture and Synchronization

REF52 cells were propagated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum essentially as described previously(13) . Wistar rat thyrocytes were grown in a six-hormone containing medium (6H) as reported earlier(14) . REF52 cells were arrested in G(0) by incubation in Dulbecco's modified Eagle's medium containing 0.05% fetal calf serum for 24-36 h and at the G(1)/S boundary by incubation in isoleucine-deficient Dulbecco's modified Eagle's medium for 48 h followed by treatment with aphidicolin (10 µg/ml, Sigma) in growth medium for 24 h. Cell synchrony was monitored by incorporation of bromodeoxyuridine and its detection in replicated DNA by indirect immunofluorescence with a monoclonal antibody raised against bromodeoxyuridine (Amersham Corp.)(15) .

Preparation of Proteins

PKIalpha was expressed in Escherichia coli DE3(BL21) using a T7-7 expression vector and purified by chromatography on DEAE52 as described(16) . The mouse brain specific PKIbeta1 cDNA was subcloned into EcoRI site of RSET(B) expression vector where NdeI site was changed to EcoRI site. PKIbeta1 was also expressed in E. coli DE3(BL21) under the same condition as described for PKIalpha. It was purified by boiling supernatant at 95 °C for 5 min and further purified by Superdex 75 gel filtration.

Generation of the PKI Antibody

The purified recombinant PKIalpha was subjected to preparative SDS-polyacrylamide gel electrophoresis. The band corresponding to PKI was excised and extracted in 50 mM (NH(4))(2)CO(3) at 37 °C overnight. Extracted PKI was then emulsified with Freund's complete adjuvant, and 0.25-0.50 mg of protein was injected subcutaneously into one rabbit. Incomplete adjuvant was used in all subsequent injections. Serum was assayed by immunoblotting and immunoprecipitation of purified recombinant PKIalpha.

Affinity Purification

Recombinant PKIalpha was coupled to an Affi-Gel 15 column (Bio-Rad) according to manufacturer's instructions. Serum was applied to the column that was extensively washed with 10 mM Tris-HCl (pH 7.2), 0.5 M NaCl. The PKI antibody was eluted with 100 mM glycine (pH 2.7). SDS-polyacrylamide gel electrophoresis of the affinity purified antibody revealed only two bands that corresponded to IgG heavy and light chains.

Indirect Immunofluorescence

For PKI staining, cells were cultured on 1 times 12-mm circular glass coverslips (Fisher). Following fixation in 3.7% formaldehyde in phosphate-buffered saline, the cells were stained with the PKI antibody (10 µg/ml) followed by staining with a tetramethylrhodamine isothiocyanate-conjugated anti rabbit antibody (1:100) (Jackson ImmunoResearch Laboratories) in the presence of homologous cell extract. All antibodies were diluted in phosphate-buffered saline, 0.5% Nonidet P-40, 1 mg/ml bovine serum albumin and incubated with the cells for 1 h at 37 °C. Following washing in phosphate-buffered saline and finally in H(2)0, the cells were mounted and observed under a Zeiss axiophot fluorescence microscope with a times40 (1.3 numerical aperture) objective. Chromosomes were stained with Hoechst 33258 (10 µg/ml) for 5 min at 25 °C.


RESULTS

A polyclonal antibody was raised to purified recombinant PKIalpha and purified on a PKIalpha affinity column. Both serum and the affinity-purified PKIalpha antibody specifically recognized as little as 10 ng of purified recombinant PKIalpha on Western blots (Fig. 1, lane1). In contrast, neither reagent reacted detectably with purified recombinant PKIbeta1 (lanes2-4). Preimmune serum (not shown) and antibody preincubated with excess PKIalpha (lane5) failed to react with PKI.


Figure 1: Specificity of the PKIalpha antibody. Purified recombinant PKIalpha (10 ng, lanes1 and 5) and increasing amounts of purified recombinant PKIbeta1 (lanes2-4) were subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes, and probed with the affinity-purified PKIalpha antibody diluted 1:5000 (lanes1-4), and affinity-purified PKIalpha antibody was preincubated with an excess (0.5 µg/µl) of purified recombinant PKIalpha (lane5).



We previously reported that PKIalpha entered the nucleus following its microinjection into the cytoplasm of rat embryonic fibroblasts(12) . To assess if endogenous PKIalpha exhibited a similar intracellular distribution, exponentially growing cells were fixed and stained with the PKI antibody. Fig. 2demonstrates that endogenous PKIalpha could be detected in the nucleus of Wistar rat thyrocytes and rat embryonic fibroblasts (REF52). Similar results were observed in mouse 10T1/2 fibroblasts (not shown). The specificity of the immunostaining for PKIalpha was demonstrated in several ways. First, preincubation of the antibody with purified PKIalpha (Fig. 2c) or deletion of the primary antibody (not shown) completely abolished the staining pattern. Second, the distribution of microinjected PKIalpha or of a C:PKIalpha complex as detected by immunostaining with the PKIalpha antibody precisely mirrored that observed following the injection of fluorescently labeled proteins. Thus, similar to the results obtained following injection of FITC-labeled PKIalpha, microinjected PKIalpha was detected in the nucleus following immunostaining with the PKIalpha antibody (Fig. 3, a and c). We previously reported that a microinjected C:PKI complex was restricted to the cytoplasm(13) . Consistent with these results, the PKIalpha antibody reacted with an injected C:PKIalpha complex primarily in the cytoplasm (Fig. 3, b and d). Lastly, the PKI antibody failed to detect microinjected PKIbeta1 (not shown). Together, these results document the specificity of the antibody staining for PKIalpha.


Figure 2: Immunostaining with the PKIalpha antibody. Exponentially growing Wistar rat thyrocytes (a) and REF52 (b and c) cells were fixed in 3.7% formaldehyde in phosphate-buffered saline and stained with the affinity-purified PKI antibody as described under ``Materials and Methods.'' Panelc illustrates REF52 cells stained with affinity-purified PKI antibody that had been preincubated with an excess of purified PKIalpha prior to staining.




Figure 3: Immunostaining of microinjected PKIalpha and C:PKIalpha complex by PKIalpha antibody. PKIalpha (128 µM)(c) and C:PKIalpha (100 µM)(d) were injected into the cytoplasm. The distributions of the PKI either as a free protein or as a complex were analyzed by staining with affinity-purified PKIalpha antibody followed by a tetramethylrhodamine isothiocyanate-conjugated anti-rabbit antibody. In parallel, the FITC-labeled PKIalpha (128 µM) (a) and C:FITC-PKIalpha (100 µM) (b) were also injected into the cytoplasm.



In asynchronous cells, both the intensity and intracellular distribution of PKI staining was heterogeneous, suggesting that the expression or intracellular distribution of PKIalpha might vary as a function of cell cycle progression. To determine if this was the case, REF52 cells were synchronized in G(0) by serum deprivation and then stimulated to enter the cell cycle with serum-supplemented growth medium. Similar to other reports(17) , serum-starved REF52 cells synthesized DNA between 12 and 20 h (Fig. 4A) after serum stimulation and maximal numbers of mitotic cells were observed between 24-28 h after serum stimulation (not shown). The relative expression and distribution of PKIalpha in synchronized cells varied as a function of cell cycle progression. Serum-starved REF52 cells exhibited very low levels of PKIalpha staining that were uniformly distributed throughout the cell (Fig. 5a). The fluorescence was slightly more intense in the nuclei of a few of the starved cells (not shown). Cells in S phase (16 h after serum stimulation) exhibited a greater overall fluorescence intensity compared with starved cells (Fig. 5c). Increased fluorescence was found both in the cytoplasm and in the nucleus. As the cells proceeded through S phase, the distribution of PKIalpha became increasingly nuclear. Nuclear fluorescence was maximal in cells analyzed at 20-24 h after serum stimulation (Fig. 5e), a time that corresponds to late S or G(2).


Figure 4: DNA synthesis in synchronized REF52 cells. A, REF52 cells were synchronized in G(0) by serum deprivation or (B) at the G(1)/S boundary by incubation in isoleucine-deficient medium followed by aphidicolin treatment. The cells were subsequently washed and stimulated with Dulbecco's modified Eagle's medium containing 20% fetal calf serum and pulse labeled with bromodeoxyuridine at 4-h intervals. Following fixation and staining with a bromodeoxyuridine-specific antibody as described previously(15) , the number of labeled nuclei was scored.




Figure 5: PKIalpha expression in synchronized REF52 cells. Cells were synchronized by serum deprivation (panelsa, c,and e) or by isoleucine/aphidicolin treatment (panelsb, d, and f). Following fixation, the cells were stained with the PKIalpha antibody as described under ``Materials and Methods.'' PKIalpha expression was low in serum-starved cells (a) but increased in cells stimulated with serum for 16 h (corresponding to S phase) (c) or 24 h (corresponding to late G(2) or M) (e). PKIalpha expression appeared greater in cells arrested at the G(1)/S boundary (b) than in serum-starved cells (a). PKIalpha staining was further increased at 3 h (corresponding to S phase) (d) and 12 h (corresponding to late G(2) or M) (f) following removal of aphidicolin and stimulation with fresh medium.



To discriminate between the effects of cell cycle progression and serum stimulation, the distribution of PKIalpha was assessed in cells arrested at the G(1)/S boundary by isoleucine deprivation followed by treatment with aphidicolin, a specific inhibitor of DNA polymerase alpha (18) . Both the isoleucine-deficient and aphidicolin-supplemented medium contained serum. Under these conditions, REF52 cells synthesized DNA between 0 and 8 h (Fig. 4B) and entered mitosis between 12 and 15 h following removal of aphidicolin and the addition of fresh growth medium. Compared with cells in G(0) (Fig. 5a), cells arrested at the G(1)/S boundary exhibited higher levels of PKIalpha expression (Fig. 5b). PKIalpha staining was further increased, especially in the cytoplasm, as cells entered S phase (3 h after refeeding growth medium) (Fig. 5d). As the cells proceeded through S phase, nuclear PKIalpha staining increased and reached maximal levels just before cell division (12 h after refeeding growth medium) (Fig. 5f).

PKIalpha expression was also analyzed as cells proceeded through mitosis. Nuclear PKIalpha staining (Fig. 6a) was always far greater in cells with condensed chromatin (Fig. 6b) than in interphase cells. This effect was not due to differences in cell volume or architecture as confocal microscopy also revealed a dramatic increase in PKIalpha staining in cells with condensed chromatin (not shown). As cells proceeded through mitosis, PKIalpha staining remained distinct and in close proximity to the chromosomes even after nuclear envelope breakdown although PKIalpha did not appear to be chromosome-associated (Fig. 6, c-f). Similar results were observed using confocal microscopy (not shown). PKIalpha expression remained high in cells following division and reformation of the nuclear envelope (Fig. 6, g and h) but eventually declined to the levels found in asynchronous cells.


Figure 6: PKIalpha expression during mitosis. Staining with the PKIalpha antibody was performed as described under ``Materials and Methods.'' Following PKIalpha staining, chromosomes were stained with Hoechst 33258 (10 µg/ml) for 5 min at 25 °C.



The cell cycle-dependent alterations in PKIalpha expression and intracellular distribution suggested a specific role for PKIalpha in cell cycle progression. To examine this further, the effects of the PKIalpha antibody on cell cycle progression were assessed. The antibody was injected into the cytoplasm of serum-starved REF52 cells, which express very low levels of PKIalpha so that the antibody would bind and sequester newly synthesized PKIalpha in the cytoplasm, functionally depleting nuclear stores of PKIalpha. Following injection, the cells were stimulated with growth medium and fixed 36 h later, a time at that most of the cells should have divided. The number of surviving injected cells was compared with the total number of cells injected. Injection of the PKIalpha antibody, but not of control IgG, significantly reduced the ability of the injected cells to divide (Fig. 7A). To determine if the effect of the PKIalpha antibody was related to increased levels of free C subunit, C subunit was injected into cells arrested at the G(1)/S boundary, and the ability of the injected cells to divide following stimulation was assessed. Similar to the results obtained with the PKI antibody, overexpression of C subunit also inhibited cell division (Fig. 7B).


Figure 7: Microinjection of the PKIalpha antibody and C-subunit into synchronized cells. A, affinity-purified PKIalpha antibody (15 mg/ml) and control rabbit IgG (15 mg/ml) were injected into the cytoplasm of serum-starved REF52 cells. Approximately 2 h after injection, the cells were stimulated with fresh growth medium and fixed 36 h following stimulation. After staining with an FITC-labeled anti-rabbit antibody, the number of surviving injected cells was scored and compared with the number of cells injected. Approximately 80% of REF52 cells injected into the cytoplasm survive microinjection. If every surviving cell divided, the cell number would increase by a factor of approximately 1.6. The number of cells injected with the PKIalpha antibody remained constant (injected = 301, FITC = 236), while the number of cells injected with control IgG increased by approximately 1.6-fold (injected = 306, FITC = 481). (B). Purified C subunit (2 mg/ml) was coinjected with rabbit IgG (5 mg/ml) into the cytoplasm of REF52 cells arrested at the G(1)/S boundary. Cytoplasmic injection was performed since C-subunit rapidly enters the nucleus following introduction into the cytoplasm (27) and higher numbers of cells survive cytoplasmic compared with nuclear injection. Cells were arrested at the G(1)/S boundary so that the biological effects of C subunit could be monitored in shorter times since injected C subunit is unstable (not shown). As a control, rabbit IgG (5 mg/ml) was injected alone. Following stimulation for 22 h, the cells were fixed and stained, and the number of surviving injected cells was scored (for C-subunit, injected = 460, FITC = 354; for IgG, injected = 427, FITC = 830). In the assays where the cells were not expected to divide, the survival of C-subunit injected cells was same as the survival of IgG-injected cells. Over 300 cells were analyzed for each sample in two independent experiments.




DISCUSSION

We report the isolation of a rabbit polyclonal antibody raised to recombinant PKIalpha. This antibody specifically recognized purified recombinant PKIalpha on Western blots and both free PKIalpha and PKIalpha in the form of a C:PKIalpha complex following microinjection into REF52 cells. The ability of the antibody to react with a C:PKIalpha complex suggests that it recognizes determinants on PKI other than those involved in binding to C subunit. The PKIalpha antibody failed to recognize PKIbeta1 either on Western blots or following its injection into REF52 cells. These results are consistent with the recognition by the antibody of determinants in the carboxyl-terminal region of PKIalpha, which are not well conserved in PKIbeta(5, 6, 8) . In this regard, the PKIalpha antibody differs significantly from the antibody raised against the testis-specific form of PKI (PKIbeta) isolated by Tash et al.(7) .

Immunostaining experiments revealed that both the apparent level and intracellular distribution of PKIalpha varied in a cell cycle-dependent manner. PKIalpha expression appeared much lower in serum-starved cells than in cycling cells. Whether this is a consequence of PKI degradation or changes in conformation or binding to components that render it undetected by the antibody under these conditions remains to be determined. Compared with both cells arrested in G(0) and at the G(1)/S boundary, PKIalpha expression increased dramatically as cells proceeded through S phase. Quantitative measurements made using confocal microscopy revealed that total cellular fluorescence increased in cells in S phase compared with cells in G(0). As cells entered S phase, PKIalpha appeared first in the cytoplasm consistent with the new synthesis of PKIalpha. It remains possible, however, that the increased fluorescence is due to the release of PKIalpha, which was previously sequestered or not detected with the PKIalpha antibody. This seems unlikely since binding of PKIalpha to C subunit, its only known physiological binding protein, did not abolish antibody recognition. As cells progressed through S phase and into G(2) and M, the distribution of PKIalpha became increasingly nuclear. Quantitative confocal microscopy revealed that nuclear fluorescence increased without a concomitant change in total cellular fluorescence. As cells progressed through mitosis, PKIalpha expression remained high and decreased again only after cell division as cells reentered G(1). These results differ somewhat from those reported earlier for the testis-specific form of PKI (PKIbeta), which was found to be localized to cytoplasmic microtubules in interphase cells and to the spindle apparatus in mitotic cells(7) . Whether this is a consequence of cell type-specific differences or differences in the species of PKI recognized by the respective antibodies is unclear. In REF52 cells, the fluorescently labeled brain-specific form of PKI (PKIbeta1) could also be detected in the nucleus following microinjection in the cytoplasm. (^2)

The up-regulation of PKIalpha expression in S phase and its subsequent accumulation in the nucleus during G(2)/M support a specific role for PKI in the nucleus as cells enter mitosis. Numerous studies have suggested a role for PKI in the inhibition of C subunit activity required for mitotic entry. In several species, oocyte maturation is accompanied by decreased cAMP levels and cAMP-dependent protein kinase activity(19, 20) . Microinjection of C subunit inhibits oocyte maturation; conversely, microinjection of PKI induces maturation(9, 21) . In somatic cells, microinjection of PKI induced many of the cellular changes that accompany mitosis, including changes in cell shape and chromosome condensation(11) . Consistent with these results, microinjection of the PKIalpha antibody abolished the ability of injected cells to divide. As reported previously(11) , similar effects were observed following microinjection of free C subunit. Taken together, these results support a specific role for PKI in the inhibition of C subunit activity required for cell cycle progression, although at present there is no direct evidence that there is sufficient PKIalpha to inhibit nuclear C-subunit activity. Most of the quantitation studies so far were done with total cell extracts(3) . No values for PKI concentrations in synchronized cells or in subcellular compartments is yet available.

There is a burst of phosphorylation as cells enter mitosis and a corresponding decrease in phosphorylation as cells exit mitosis. Changes in phosphorylation are likely to mediate, in part, the multiple structural alterations that occur during mitosis including chromosome condensation, nuclear envelope breakdown, spindle formation, cytoskeletal changes and disassembly of organelles. p34 activity, which is maximal in late G(2), is responsible for a number of mitosis-specific phosphorylation events. Phosphorylation of histone H1 by p34 results in chromosome condensation, and p34-mediated phosphorylation of the nuclear lamins results in lamin disassembly. In contrast, cAMP-dependent protein kinase inhibits p34mediated lamin release(22) , and addition of cAMP to mitotic extracts results in the loss of p34 kinase activity(23) . Consistent with these results, PKI and p34 appear to function cooperatively to effect many of the cellular changes that occur at mitosis. Microinjection of either p34(11, 17) or PKI (11) alone, induced changes in cell shape, chromatin condensation, and alterations in actin and tubulin architecture. Coinjection of p34 and PKI together induced additional changes including nuclear envelope breakdown, suggesting that p34 and cAMP-dependent protein kinase perform distinct and complementary roles in regulating entry into mitosis. The roles of cAMP-dependent protein kinase may include the maintenance of higher order chromatin structure, nuclear envelope integrity, and microtubule architecture, all of which must be dismantled prior to mitosis. In addition, a number of cellular processes including transcription and endocytosis are specifically repressed during mitosis. The activity of the transcription factor cAMP response element binding protein is dependent upon phosphorylation by cAMP-dependent protein kinase (for review, see (24) ). It also needs to be turned off prior to mitosis.

The nuclear localization of PKI makes it ideally situated to inhibit nuclear C subunit, unlike regulatory subunits that appear to be localized in the cytoplasm in many (25, 26, 27, 28) but not all cell types(29, 30) . Intracellular cAMP levels have been reported to fluctuate as a function of cell cycle progression with a dramatic increase observed in cells in S phase and the lowest levels observed during mitosis(21) . This would result in holoenzyme dissociation during S phase and the accumulation of active C subunit in the nucleus as cells enter G(2). Up-regulation of nuclear PKI expression at this time would provide a mechanism for extinguishing the activity of C in the nucleus without affecting its activity in the cytoplasm. In an earlier report, we found that microinjection of a C:PKI complex enhanced the rate of export of C subunit from the nucleus(12) . These results suggest that there may be multiple mechanisms through which nuclear stores of C subunit could be inactivated.

A number of earlier studies provided evidence for PKI regulation (for review, see (3) ). For example, PKI activity increased following serum stimulation in starved Chinese hamster ovary cells (31) and follicle-stimulating hormone treatment in cultured Sertoli cells(32) . In chick kidney cells, vitamin D down-regulated both PKI activity (33, 34) and mRNA levels(35) . Additionally, the expression of the alpha and beta PKI mRNAs is both tissue- and developmental stage-specific(8) . Our results suggest that PKI is subject to both positive and negative regulation in a cell cycle-dependent fashion. It will be interesting to determine if PKI is regulated posttranscriptionally, as is the case for the type I regulatory subunit(35) , or is subject to regulation at the level of transcription. Phosphorylation of the single tyrosine residue in PKI reduced its inhibitory activity on C subunit(36) , providing a potential mechanism through which PKI activity could be regulated through the activity of other cellular signaling pathways. cAMP-dependent protein kinase functions in a diverse array of cellular processes including metabolism, secretion, differentiation, gene expression, proliferation, and growth inhibition. PKI may be part of a coordinated cellular control network that helps to modulate the activity of this important protein kinase.


FOOTNOTES

*
This work was supported by funding from the California Tobacco Related Disease Research Program (2RT0066 to S. S. T., and 2KT0030 to J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: PKI, heat-stable protein kinase inhibitor; C, catalytic subunit of cAMP-dependent protein kinase; R, regulatory subunit of cAMP-dependent protein kinase; FITC, fluorescein isothiocyanate.

(^2)
W. Wen, unpublished results.


ACKNOWLEDGEMENTS

We thank Tom Deerinck for confocal microscopy and Kristin Martin, Carolyn Huttenmaier, and Christina Chen for assistance in preparing the manuscript.


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